111111111111111111111111111 ,.._____ 1293 10476 4034 1.33% RY Pu-..” “‘3 {State ; j ‘J'fli Vfluswaztlf I \ ‘9.“ 7-- This is to certify that the dissertation entitled AN IMMUNOTOXICOLOGICAL EVALUATION OF PENTACHLOROPHENOL IN DAIRY CATTLE presented by James Harold ForseTT has been accepted towards fulfillment of the requirements for Ph, D, degreein Environmental Toxicology/ Dairy Sc1ence 022227669? a) Major professor Date 2/10/84 MSU is an Affirmun'vt Action/Eq ual Opportunity Institution 0-12771 )V1531.J RETURNING MATERIALS: ‘ Piace in book drop to LJBRARJES remove this checkout from .—:_ your record. FINES will be charged if book is returned after the date stamped beiow. AN IMMUNOTOXICOLOGICAL EVALUATION OF PENTACHLOROPHENOL IN DAIRY CATTLE BY James Harold Forsell A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science/ Center for Environmental Toxicology 1984 ABSTRACT AN IMMUNOTOXICOLOGICAL EVALUATION OF PENTACHLOROPHENOL IN DAIRY CATTLE By JAMES HAROLD FORSELL PentachloroPhenol (PCP) is an antimicrobial agent widely used for the preservation of wood. The immunotoxicity of a technical grade of PCP was evaluated in eight lactating Holstein- Friesian cattle. These animals were randomly allotted in pairs to either a control or a treatment group. The treatment group was fed technical PCP for 135 days (0.2 mg/kg body wt/day for 75 - 84 days followed by 2.0 mg/kg body wt/day for 56 - 62 days. In a second study the immunotoxicologic and clinico- pathologic effects of analytical and technical grade PCP were compared in dairy calves. Fifteen Holstein-Friesian bull calves were randomly assigned to one of five treatment groups: control, 1.0 mg and 10 mg analytical PCP/kg body wt/day, and 1.0 mg and 10.0 mg technical PCP/kg body wt/day, for 43 days. Assays conducted to evaluate lymphocytes included: 1) quanti- tation of serum immunoglobulins; 2) induction of blastogenesis in zitgg using lymphocytes obtained from the blood and other lymphoid tissues; 3) enumeration of lymphocyte subp0pulations; 4) quantitation of antibody formation induced by foreign anti- gens i2 vivo; 5) skin testing as a measure of delayed hypersensitivity; and 6) histOpathologic evaluation of lym- phoid tissues. NeutrOphil function was evaluated using latex particle phagocytosis and chemiluminescence. The clinico- pathologic profile consisted of 43 tests in hematology, clini- cal chemistry and urinalysis. The results demonstrated no statistically significant differences between mature lactating control and penta-treated cattle; nor in calves fed either analytical or technical PCP at 1.0 mg/kg versus controls. Major findings in the calves exposed to 10 mg/kg technical PCP included marked thymic atrOphy, decreased serum total protein, and elevation in serum gamma-glutamyl transferase (GGT). A decreased lymphocyte blastogenic response was seen in calves given 10 mg/kg analytical PCP. A third aspect of this research dealt with establishing the conditions required by bovine neutrOphils to change shape from a round to a bipolar form. PCP eliminated the shape change response in zitrg at levels of 0.1 mg/ml. Lastly, a technique was established and validated for isolating viable hepatocytes from cattle. This procedure was designed as a xenobiotic metabolism and activation system for use in co-culture with bovine lymphoid cells for $2.!iEEQ immunotoxicity testing. To Harold and Irene, my parents ii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my wife, Ann, and daughter, Sara, for their support and patience during the course of my Ph.D. program. Appreciation is also extended to Dr. Lee R. Shull, Dr. Robert K. Ringer, Dr. Steven Bursian, Dr. Stuart Sleight and Dr. John R. Kateley, my committee members. Perhaps as important as anyone in a graduate program, is one's fellow graduate students. In this regard, I acknow- ledge Donald Kirsch, Brian J. Hughes and John H. Kinzell. I also acknowledge the assistance of Edward W. Sparrow Hospital's Immunology Laboratory in providing technical skills for many of the immunologic assays used in these studies. iii TABLE OF CONTENTS List of Tables . . . . . . . . . . . . . List of Figures . . . . . . . . . . . . Introduction . . . . . . . . . . . . . Literature Review . . . . . . . . . . . PentachlorOphenol . . . . . . . . Chemical and Physical Properties . . Exposure to Pentachlorophenol . . . Absorption of Pentachlorophenol . . Distribution of Pentachlorophenol . Metabolism of Pentachlorophenol . . Excretion of Pentachlorophenol . . Toxicodynamics of PentachlorOphenol Contaminants in Pentachlorophenol. . Immunotoxicology . . . . . . . . . . Introduction . . . . . . . . . . . Indirect Effects . . . . . . . . . Immunology and Drug Metabolism . . . Immunosuppressive Chemicals . . . Drugs . . . . . . . . . TCDD . . . . . . . . . . . . . . Pesticides . . . . . Heavy Metals . . . . . Organochlorines . . . . . . Polybrominated Biphenyls . . . Carcinogens . . . . . . . . . . Isolated Hepatocytes . . . . . . . . . Isolation of Hepatocytes . . . . . . Isolated Hepatocyte Function . Isolated Hepatocyte and Xenobiotic Function Future of Isolated Hepatocytes . . . Chapter 1. Immunotoxicologic Evaluation Introduction . . . . . . Materials and Methods . . Results . . . . . . . . . Discussion and Conclusions iv Subchronic Administration of Technical Pentachlorophenol to Lactating Page vi . viii Dairy Cattle: p... 73 74 77 85 100 Page Chapter 2. Technical and Analytical Pentachloro- phenol in Dairy Calves: A Clinico- pathological and Immunotoxicological Evaluation . . . . . . . . . . . . . . . . 105 Introduction . . . . . . . . . . . . 105 Materials and Methods . . . . . . . . . 103 Results . . . . . . . . . . . . . . . 114 Discussion and Conclusions . . . . . . 128 Chapter 3. Bovine Neutrophil Shape Change Kinetics and its Inhibition by Pentachlorophenol. 133 Introduction . . . . . . . 134 Materials and Methods . . . . . . . . . 135 ReSUJ-ts o o o o o o o o o o o o o o o 140 Discussion and Conclusions . . . . . . 151 Chapter 4. Isolated Bovine Hepatocytes: A Model for Xenobiotic Metabolism . . . . . . 155 Introduction . . . . . . . . . . . . . 157 Materials and Methods . . . . . . . . 159 Results . . . . . . . . . . . . . . . 159 Discussion and Conclusions . . . . . . 131 Summary . . . . . . . . . . . . . . . . . . . . . . . 135 List of References . . . . . . . . . . . . . . . . . . 188 LIST OF TABLES Page Component Analysis of Three Grades of Pentachlorophenol . . . . . . . . . . . . . . . . . 7 Solubility of Pentachlorophenol . . . . . . . . . . 10 Physical-Chemical Properties of Pentachlorophenol . 10 Minimum Screening Panel for Defining Immuno- modulation After Chemical Exposure in Rodents . . . 30 A Partial List of Procedures Available for Detecting Immunomodulation After Chemical Exposure. 31 Selected Immunosuppressive Effects of TCDD in Various Laboratory Species . . . . . . . . . . . . 48 Pesticides Claimed to Have Caused Allergic Responses in Humans . . . . . . . . . . . . . . . . 52 Examples of Chemical Carcinogens . . . . . . . . . 63 Xenobiotics Which Have Been Studied Using an Isolated Hepatocyte System . . . . . . . . . . . 70 In Vivo Cell-Mediated Immunity in Cattle Fed Technical PentachloroPhenol Subchronically . . . . 93 Antibody Formation in Response to Injected Sheep Red Blood Cells (SRBC) in Holstein Cattle Sub- chronically Exposed to Commercial Pentachlorophenol.96 Results of Hematologic Examination of Blood From Calves Fed Analytical or Technical Grade Pentach- lorophenol . . . . . . . . . . . . . . . . . . . 115 Results of Clinical Chemistry Analysis of Serum From Calves Fed Analytical or Technical Grade Pentachlorophenol . . . . . . . . . . . . . . . . 116 Results of Clinical Chemistry Analysis of Serum and Urine From Calves Fed Analytical or Technical Grade Pentachlorophenol . . . . . . . . . . . . . 117 vi Table 2.4 Peripheral Blood Lymphocyte Surface Markers in Calves Fed Analytical or Technical Grade Pentachlorophenol . . . . . . . . . . . . . . . . Lymphocyte Surface Markers in Lymphoid Tissues Obtained from Calves Fed Analytical or Technical Grade Pentachlorophenol . . . . . . . . . . . . . Lymphoblastogenic Response to an Optimal Concen- tration of Leukoagglutinin (0.5 mg) in Calves Fed Analytical or Technical Grade Pentachlorophenol . Organ Weights of Calves Fed Analytical or Techni- cal Grade Pentachlorophenol . . . . . . . . . . . Yields and Viabilities After Isolation of Bovine Hepatocytes . . . . . . . . . . . . . . . Comparison of Gluconeogenesis Between Bovine Isolated Hepatocytes and Liver Tissue Slices. . . Comparison of [1-14C] Palmitic Acid Oxidation by Bovine Liver Slices and Isolated Bovine Hepatocytes . . . . . . . . . . . . . . . . . Oxygen Utilization as an Indicator of Isolated Hepatocyte Viability . . . . . . . . . . . . . . Comparison of Cytochromes P450 and b Levels Between Fresh Bovine Liver Tissue and Isolated Hepatocytes . . . . . . . . . . . . . . . . . . . Epoxidation of Aldrin by Bovine Isolated Hepa- tocytes O O I O O O I O I I O O O O O I O O I O 0 vii Page 119 120 124 126 170 174 176 177 179 180 LIST OF FIGURES Figure l. Pentachlorophenol . . . . . . . . . . . . . . . . Metabolism of Pentachlorophenol . . . . . . . . . Numbering System for Dibenzo-p-dioxins and Dibenzofurans . . . . . . . . . . . . . . . . . . Reverse Purfusion Technique for Rat Liver . . . . Red Blood Cell Number (RBC x 106/ul),Hemoglobin Concentration (HGB g/dl),and Packed Red Blood Cell Volume (PCV %) in Control and Penta-treated Cattle. Each Graphed Point Represents the Mean and Standard Error of All Data Collected Between the Times In- dicated. Normal Limits are Those Reported by Duncan and Prasse (1977) . . . . . . . . . . . Number of White Blood Cells (wsc x 103/ul) in Con- trol and Penta-treated Cattle. Each Graphed Point Represents the Mean and Standard Error of All Data Collected Between the Times Indicated. Normal Limits are Those Reported by Duncan and Prasse (1977) . . . . . . . . . . . . . . . . . . . . . . Numbers of Polymorphonuclear Neutrophils (PMN x 10 3/ul) in Control and Penta- treated Cattle. Each Graphed Point Represents the Mean and Standard Error of all Data Collected Between the Times Indicated. Normal Limits are Those Reported by Duncan and Prasse (1977). . . . . . . . . . . . . Total Number of Counted Lymphocytes (Mean i SEM) per ul of Blood and Two Subpopulations of Lym- phocytes, 1) Those Possessing Surface Immuno- globulins (519) and 2) Those Which Form Rosettes With Neuraminidase-treated SRBCs (En-RFC), in Control (C) and Penta-treated (T) Cattle. The Number of sIg-bearing Lymphocytes is Superimposed Above the Number of En-RFC Lymphocytes; Subtract the Number of En-RFC From the Total of En-RFC and 819 to Derive the Number of 319 Cells. Each Bar Represents the Mean of all Data Collected Between the Times Indicated. The Normal Range of Total Lymphocyte Numbers per ul was Reported by Duncan and Prasse (1977) . . . . . . . . . . . . . . . . . viii Page 18 24 65 86 87 89 90 Figure 1.5. 1.6. 1.7. 2.1. Page Lymphoblastogenic Response to Optimal Concen- trations of LA and Con A in Control (C) and Penta-treated (T) Cattle. a) Data Expressed as an Isotope Incorporation Index (III = Mean Counts/minute in Mitogen Stimulated Cultures Divided by the Mean Counts/Minute in Control Cultures). . . . . . . . . . . . . . . . 91 b) Data Expressed as Net Counts/Minute (NCPM)= Counts/Minute in Mitogen Stimulated Cultures Minus the Mean Counts/Minute in Control Cultures. Each Bar is the Mean :SEM of all Data Collected Between the Times Indicated. . . . . . . . . . . 92 Serum Immunoglobulin Concentrations of IgG, IgM and 19A in Control and Penta-treated Cattle. Each Graphed Point Represents the Mean :SEM of All Data Collected Between the Times Indicated . . 95 Neutrophil (PMN) Function Expressed as a Percent of Cells Phagocytizing OpSOnized Latex Particles In Vitro. C = Neutrophils from Control Cattle Opsonized with Serum from Control Cattle. T = Neutrophils from Treated Cattle Opsonized with Serum from Treated Cattle. 0 = Neutr0phils from Treated Cattle Opsonized with Serum from Control Cattle. Each Bar Represents the Mean :SEM of All Data Collected Between the Times Indicated. . . . . . 97 In Vitro Light Emitting Profiles (Chemilumi- nescence) of Neutrophils (PMN) Phagocytizing Opsonized zymosan A. This Assay was Performed on the Last Day of Penta Exposure Using Cells from Both Control and Penta-treated Cattle. Each Point Represents the Mean :SEM . . . . . . . . . . . . . Net Change in Serum Concentrations of IgG, IgM, and IgA Over a Ten Day Period in Calves Fed Analytical or Technical Grade Pentachlorophenol. 121 Primary and Secondary Antibody Response to Injected Human Red Blood Cells in Calves Fed Analytical or Technical Grade Pentachlorophenol. . . . . . . . 122 In Vitro Light-Emission Profiles (Chemilumi- nescence) of Neutrophils (PMN) Phagocytizing Opson- ized Zymosan A. The Plots Show the Mean Response for Each Treatment Group Over an Hour Period . . 123 ix Figure 3.1a. 3.1c. 3.1d. 3.2. 3.4. 3.5. Page Bovine Neutrophils (N) and Eosinophils (E) (x 1,000 Phase Contrast) Fixed in 1% Glu- taraldehyde After a Five Minute Incubation at 37°C With No CF. The Bar at the Bottom Left Represents 10 Microns . . . . . . . . . . . . . 141 Bovine Neutophils (N) Showing Round Ruffled Features (Spherical Classification). Cells Were Fixed in 1% Glutaraldehyde and Photo- graphed at x 1,000 Using Phasc Contrast. One Nonstimulated Eosinophil (E) Is Present . . 141 Bovine Neutrophils (x 1,000 Phase Contrast) Showing Nonspherical (Bipolar) Forms With One Neutrophil Showing the Beginning of a Uropod (U). Cells Were Fixed With 1% Glutaraldehyde After Five Minutes Incubation at 370C in the Presence of 1% ZAS . . . . . . . . . . . . . . 142 Bovine Eosinophil (E) Showing a Typical Res- ponse to 1% ZAS (x 1,000 Phase Contrast). A Bipolar Neutrophil (N) is Also Present. . . . . 143 Change in Shape of Neutrophils Exposed to Various Levels of ZAS. "Percent Bipolar" Refers to the Percent of Cells in the Suspension with a Bipolar Shape (Bipolar Plus Uropod). Incubation Time was Five Minutes. Each Point is the Mean i of Four Separate Experiments. . . 144 Change in Shape in Neutrophils Exposed to 1% ZAS. "Percent Bipolar" Refers to the Percent of Cells in the Suspension with a Bipolar Shape (Bipolar Plus Uropod). "Time-minutes" Refers to the Time After the Addition of Cells. Each Point is the Mean iSE of Four Separate Exper- iments . . . . . . . . . . . . . . . . . . . . 146 Change in Shape of Neutrophils Exposed to Various Levels of Alpha-casein and Casein (Partially Dephosphorylated and Hydrolyzed). "Percent Bipolar" Refers to the Percent (Mean) of Neutrophils in the Suspension with a Bipolar Shape (Bipolar Plus Uropod). Incubation Time was 7.5 Minutes . . . . . . . . . . . . . . . . 147 Change in Shape of Neutrophils Exposed to 1% Zymosan Activated Serum in the Presence of Three Different Grades of PentachlorOphenol (PCP); Ana- lytical or aPCP, Purified or pPCP and Technical or tPCP. The Numbers Above the Bars Refer to mg PCP/ml Used. The Closed Bars Refer to Ethanol and the Open Figure Page Bars to Dimethyl Sulfoxide Used as Solvents to Deliver the PCPs. "Percent Bipolar" Re- fers to the Percent of Cells in the Suspension with a Bipolar Shape (Bipolar Plus Uropod). Incubation Time was 7.5 Minutes. The Controls Contained 1% Solvent, the Maximum Used to Deliver PCP . . . . . . . . . . . . . . . . . . .149 3.6. Bovine Neutrophils Showing the Unresponsive "Wrinkled" Appearance of Cells Incubated in the Presence of 0.10 mg/ml PCP and 1% ZAS for 7.5 Minutes at 370C (x 1,000 Phase Contrast). . . . 150 4.1. Isolated Bovine Hepatocyte. . . . . . . . . . . 171 4.2a. Total Glucose Production . . . . . . . . . . 172 4.2b. Rate of Glucose Production from Exogenous Substrate in Bovine Isolated Hepatocytes . . . 173 xi INTRODUCTION Pentachlorophenol is a broad-spectrum biocide, due to its ability to uncouple oxidative phoSphorylation in many species, including man (EPA, 1980). Additional toxicity may result from the dioxins and other contaminating chemicals pre- sent in pentachlorOphenol (EPA, 1980). Most of the greater than 40 million lbs/yr of pentae chlorOphenol produced in the Unites States is used as a wood preservative (EPA, 1980). PentachlorOphenol-treated wood is often used in construction of livestock housing and feeding facilities. Livestock housed in such facilities may become contaminated with pentachlorophenol by ingestion, dermal con- tact and inhalation. In Michigan, reports of poor health in dairy cattle have been attributed to exposure to pentachlorophenol (Thomas 23 nI., 1977). Among the clinical signs reported, are de- creased milk production, poor general appearance, skin lesions, increased incidence of mastitis, persistent infections, high calf mortality and death (Thomas 2E nI., 1977). Many of these signs suggest a suppression of immune function. Prior to this research, the question of immunomodulation as a consequence of pentachlorOphenol exposure in dairy cattle was not addressed. In fact, many of the methodologies needed to evaluate bovine immune function were undeveloped or unrefined prior to this study. This dissertation describes methods which were develOped and used for evaluation of immune function in cattle. These methods were then used to evaluate immune function in lactating dairy cattle and calves orally exposed to pentachlorOphenol. A xenobiotic metabolism system was also developed for use with chemicals needing activation before proper evaluation for toxicity. This system involved the isolation of bovine hepatocytes and their use in In_zIn£n_xenobiotic activation systems. LITERATURE REVIEW PENTACHLOROPHENOL In l930,chlorinated phenols were produced in order to test their efficacy as wood preserving agents. Pentachloro- phenol, tested two years later, was found to be a superior wood preservative (EPA, 1980). The effectiveness of penta- chlorophenol as a preservative resides in its ability to pre- vent wood biodeterioration caused by fungi, particularly Basidiomycetes, which have the ability to enzymatically degrade the structural cellulose and lignin in wood. In addition to its use as a wood preservative, pentachlorOphenol is useful in control of wood-destroying insects, as a herbicide defoliant, a mossicide and a mushroom house biocide. PentachlorOphenol is a toxicant in bacteria, fungi and insects because it uncouples oxidative phosphorylation in liv- ing cells. Since oxidative phosphorylation is similar for the aerobic generation of adenosine triphosphate in all biologic systems, pentachlorOphenol is a highly Operative broad- Spectrum biocide. In 1974, approximately 54 million pounds of pentachloro- phenol were produced in the United States (Fuller gn.nI,, 1977). In 1978 about 43.6 million pounds of pentachlorOphenol were used in the treatment of wood products. This level of produc- tion has been maintained. Commercial grade pentachlorOphenol is 80% pentachloro- phenol, 6% tetrachlorOphenol isomers, and 6% other chlorinated phenols. The remainder of the pentachlorOphenol is composed of other chlorinated compounds including dibenzo-p-dioxins and dibenzofurans. Before it is used, pentachlorophenol is dissolved in a petroleum solvent. A water soluble penta— chlorOphenol is available as the sodium or potassium salt. The presence of the dibenzo-p-dioxins and dibenzofurans in pentachloroPhenol has generated considerable interest due to the known toxicity of these chemicals and the concern for their contribution to overall pentachlorOphenol toxicity (Federal Register, 1978). It was because of the presence of dioxins in pentachlorOphenol and the extensive use of this pesticide which caused it to be placed on notice of "Rebuttable Presumption Against Registration" (RPAR) by the Office of Pesticide Programs of the Environmental Protection Agency (Fed- eral Register, 1978). Such a notice was issued on October 18, 1978, when the EPA determined there was evidence of sufficient risk in the use of a given pesticide to warrant a review of the advisability of its continued use. The RPAR review process starts only after the existence of a risk (trigger) has been demonstrated by the Environmental Protection Agency. In this case, the pentachlorophenol trigger was EPA's determination that pentachlorOphenol may exceed risk criteria relating to teratogenic and fetotoxic effects in mammalian tests Species (Federal Register, 1978). The information used as a basis for the trigger is compiled and published in the Federal Register which is the formal notice of RPAR. This document is referred to as a Position Document One or PDl. The public is then given a period of time whereby interested parties may rebut the risk data ad- dressed in the PDl. The outcome of the rebuttal determines whether the chemical is returned to registration, a move which results in the publishing of a Position Document Two (PD2) in the Fed- eral Register; or a risk-benefit analysis and a prOposed regu- latory position are published in what is referred to as a Po- sition Document 2/3 (PD2/3). The latter situation results when the rebuttal is not successful. A PD2/3 document has been pub- lished for pentachlorophenol (Federal Register,l981). The PD2/3 document is then reviewed by various agencies leading to the publication of a Position Document Four (PD4) which con- tains the final regulatory action to be taken on a chemical. The PD4 on pentachlorOphenol has not as yet been published. PentachlorOphenol is synthesized through the direct chlorination of phenol using catalysts such as FeCl3, A1C13, and SbCl3 with heat. The process is a two-step reaction in which the first stage involves raising the reaction temperature to 105°Ctx>form tri- and tetrachlorinated phenols. The subse- quent step involves increasing the reaction temperature to keep the mixture molten in order to chlorinate the lower chlorinated phenols to the fully chlorinated phenols (Cirelli, 1978). It is during the second, high temperature stage that various dioxin and furan ~ congeners found in pentachlorOphenol are formed. These impurities are responsible for the buff-colored crystals characteristic of the commercial or technical grade of pentachlorophenol (tPCP). PentachlorOphenol preparations are available with re- duced levels of dioxins and furans. Dow Chemical Co. develOped a procedure to produce pentachlorOphenol with significantly re- duced levels of impurities. Dow calls their improved pentach- lorOphenol Dowicide EC-7R. Analytical grade pentachlorOphenol (aPCP) is also available. Table 1 compares the composition of the different grades of pentachlorophenol. The final physical form of pentachlorOphenol after manu- facture includes pellets, prills, and one-half ton blocks. PentachlorOphenol treating plants receive pentachloroPhenol bags, bulk and solid blocks and cut the pentachlorOphenol with petroleum solvents to a 5-7% solution. Solvents used to dissolve pentachlorophenol vary; typically they include ker0* sene, mineral spirits, number 2 fuel oil, methylene chloride or liquefied petroleum gas (Cirelli, 1978). The wood preservation process is either a pressure or non-pressure process with 95% of the wood being treated with the pressure system. The pressure treatment of wood results in retentions of 20 to 30 pounds of preservative per cubic foot for the full-cell process and 6-12 pounds per cubic foot for the empty cell process (EPA, 1980). Non-pressure wood preservation methods include thermal, brush, dip, spray, diffusion, vacuum and cold soak methods. The estimated production of pentachlorOphenol-treated wood in 1978 was 80 million cu. ft. Most of the pentachloro- phenol-treated lumber (93%) was classified as poles, fence posts, Table 1 Component Analysis of Three Grades of PentachloroPhenol Chlorophenols (%) Commerciala Improvedb Analyticalb Pentachlorophenol 85-90 88-93 99.02 TetrachlorOphenol 4-8 7-12 .98 TrichlorOphenol 0.1 0.1 -- Other 2-6 0.1 -- Chlorodibenzo-p-dioxins (ppm) Octa 1000C 2-30 1.2 Hepta 378 2-3 1.8 Hexa 173 d 0.2-1 0 0.2 Tetra 0.035 -- -- Chlorodibenzofurans (ppm) Octa 54-182a Hepta 37—116 Hexa 6.8-7.1 Penta 0 9-1.1 Tetra 0.2-0.7 Tri 0 02-0.05 Hexachlorobenzene 30-80 ppm Polychlorinated Bi- phenyls 1-2 ppm Chlorinated diphenyl etherse 100-1000 ppm aAnalysis reported by Monsanto Chemical Company, St. Louis, MO, personal communication, Dr. Dan Roman, 1978. bAnalysis of Dow1cide EC-7(R) as reported by Dow Chemical Co., Midland, MI: personal communication, Dr. Robert L. Johnson, 1980. CAnalysis as reported by Kinzell gn_nI., 1981. dDoes not include 2, 3, 7, 8-TCDD. eNilsson 2E 31-: 1978. lumber and timbers. Little pentachloropheno1—treated wood (0.5%) is used for crossties, switch ties, and landscape ties, products which are traditionally treated with creosote solu- tions (EPA, 1980). The use of pentachlorOphenol (penta) treated lumber in farms in Michigan has been surveyed by Shull 2E nI.,(l981). Penta-treated wood was found in 47.2% of the main livestock buildings on the farms surveyed. In 21.3% of these buildings the extent of penta-treated wood use was classified as medium to extensive. The most prevalent use of penta-treated wood was for pole supports (45%) and splash boards (40.5%). How— ever, 4.8% of calf pens, 15.8% of feed bunks, 9.3% of corn cribs, 8.4% of bunker silos, 4.1% of hay racks. 0.9% of mineral feeders and 0.7% of feed bins were constructed of treated wood. Since treated wood is recommended for use only where wood is exposed to excessive moisture or soil, excluding feed contact surfaces, these authors concluded that treated wood is being used excessively and improperly on many Michigan dairy farms. Chemical and Physical Properties PentachlorOphenol (CGHClSO) is a fully chlorinated phenol (Figure 1). In its pure form,PCP is a white solid having a needlelike crystalline structure. PCP has a very pungent odor! especially when hot. It is almost insoluble in polar solvents but freely soluble in alcohol and ether. The solubil— ity of PCP in various solvents is shown in Table 2. PCP is a weak acid with pKa of 4.74 (EPA, 1980). It exists as a nonpolar molecule under acidic conditions and as a polar anion under basic conditions. Thus, PCP reacts with OH C1 C1 C1 C1 C1 Figure l. PentachloroPhenol strong alkali bases such as sodium hydroxide to form the water— soluble salt which is soluble at greater than 0.4g/lOOg water at pH 8.0 (Myeling and Pitchford, 1966). PentachlorOphenol is quite stable at elevated environmental temperatures and is rather chemically inert. It is not subject to the easy oxidative coupling or electr0phi1ic substitution reactions common to most phenols. However, photodegradation does occur, apparently, by free-radical pathways (Plimmer, 1970). Volatility of PCP is relatively low, but losses do occur from soil, water and treated wood. The physical-chemical pro- perties of PCP are given in Table 3. Exposure to Pentachlorgphenol PentachlorOphenol can be absorbed during oral, respiratory and dermal contact.' The data supporting the actual kine- tics involved with the different routes of PCP exposure are scarce, and subject to disagreement. Many factors are used in order to estimate eXposure levels. Thompson 2E nI.,(1979) found that the maximum expected concentration of PCP in air in a barn constructed in part from penta- treated wood was 0.02 mg/m3. Using estimated values for a 24 hour total air volume which could 10 Table 2 Solubility of Pentachlorophenol Solvent g/lOOg solvent (20-30°c1 Water 0.0014-0.0019 Carbon Tetrachloride 2-3 Benzene 11-14 Xylene 14—17 Acetone 21—33 Ethanol 47-52 Diethyl Ether 53-60 Methanol 57-65 Bevenue and Beckman, 1967. Table 3 Physical-Chemical Properties of PentachloroPhenol Color TWhite Molecular weight 266.26 0 Melting point l90-l9loC Boiling point 309-310 C decomposes 3 Density 0 o 1.978 g/cm at 22 C referged to water at 4 C Vapor pressure 20 C 0.00011 mmHg 100°C 0.12 mmHg Modified from Bevenue and Beckman, 1967. 11 be inhaled by a 514 kg cow and assuming a 100% retention of all PCP entering the lungs; a total exposure of 3.138 mg/cow or 0.006 mg/kg can be calculated (EPA, 1980). This level of ex- posure would result in an estimated blood level of 0.013 mg/ liter (Ppm), (Osweiler gn_nI., 1977). PCP exposure by the oral route would occur as a result of eating contaminated feed or by licking and chewing PCP- treated lumber. Penta-treated wood contains 10 to 16 grams of penta per board foot (bd. ft = 12 X 12 X l in) (EPA, 1980). A cow weighing 514 kg would have to consume an average of 0.039 bd.ft/day to receive a penta exposure of 1 mg/kg, 0.078 bd.ft/day for 2 mg/kg, 0.39 bd.ft/day for 10 mg/kg, and 0.78 bd.ft/day for 20 mg/kg. If 0.39 bd.ft was eaten/day,a daily exposure of 10 mg/kg would require the eating of 56 cubic inches of wood each day. The exposure which may result from licking the oozing (bleeding) material from freshly treated lumber or by eating feed into which this material has migrated can similarily be estimated. Wood preserving solutions usually contain 5 to 7% pentachlorophenol (EPA, 1980). At 6% PCP , it would require ingestion of 9 ml of the bleeding material to obtain a 1 mg/kg exposure level. Feeding adult lactating cattle 0.2 mg/kg or 2.0 mg/kg/day tPCP resulted in steady state blood levels of 2.9 and 12.5 ppm, respectively (Kinzell, 1981). Nonlactating heifers fed various grades of pentachlorophenol at 15 mg/kg had serum PCP concen- trations of 32.8 to 86.9 ppm (Parker gn_nI., 1980). Calves given 1.0 mg/kg or 10 mg/kg PCP, incorporated 12 in milk, had blood levels of 32 to 92 ppm (Hughes, 1982). The nonlinear relationship between the amounts of PCP ingested and the level of PCP in the blood indicates that absorption from oral exposure is not 100%, and varies considerably. PCP in the blood of dairy cattle housed in total confine- ment-free stall pole barns in which penta-treated wood was used extensively reached 0.004 to 0.033 mg/kg. In one barn where penta-treated wood was used extensively, including the feed- bunk, everage blood PCP levels reached 0.3 mg/kg (Van Gelder, 1977). The data above provides the best available information concerning actual levels of PCP exposure in cattle in a farm environment. Dermal absorption data are essentially non-existent for cattle and other food-producing animals. The implication that dermal exposure does occur in the farm environment comes from case histories of penta toxicosis in pigs where sows were farrowed on freshly treated lumber. Typically, the newborn pigs were affected and the sow was not. Volatilization of penta did not appear to be the problem, because covering the penta-treated floor resulted in the cessation of mortality (Schipper, 1961). A case history reported by Ryan (1983) recorded the death of piglets within 24-48 hours of birth on a wooden floor which was freshly treated with penta. Analysis of the floor wood showed 3100 ppm PCP present. PCP residue analysis in the livers of two pigs resulted in no PCP detected at their maximum limit of detection (0.02 ppm). Although it is highly unlikely, dermal absorption of dioxins was implicated by the author as the cause of death in this instance. 13 Chickens are also affected by penta, showing increases in liver fibrosis and morbidity when exposed to penta (Ryan and Pilon, 1982). In these cases penta is usually present in saw- dust or wood chips used for bedding (Curtis 23 nI., 1974). Determination of the level of animal exposure becomes a problem when a toxic chemical is common in the environment. Shull EE nI., (1981) revealed that penta-treated wood was present on nearly 50% of the dairy farms surveyed in Michigan. Penta was either used imprOperly or over-used in about 70% of these farms. It is clear that the potential for animal exposure and toxicosis is present, and that penta residue problems exist. Absorption of Pentachloronhenol Absorption of toxicants can take place along the entire length of the gastrointestinal tract from the mouth to the rectum. Most toxicants cross body membranes by simple diffusion. The rate of transfer is dependent on lipid solubility and con- centration gradient across the membrane (Klaassen, 1980). If a toxicant is a weak organic acid or base, the degree of its ionization in various portions of the gastrointestinal tract is important in determining the extent of absorption. The degree of ionization of a chemical is dependent upon the pKa of the chemical and the pH of the medium of each particular region of the gastrointestinal tract. PKa and pH are important because only the non-ionized (lipid soluble) form can be tranSported aeross biologic membranes by diffusion (Klaassen, 1980). Pentachlorophenol is classified as a weak acid with a pKa of 4.74 (EPA, 1980). Using the Henderson-Hasselbach equation for an acidic chemical (pH=pKa + log ionized / non-ionized), 14 the ratio of the non-ionized form of PCP to the ionized form can be calculated. The ratio thus calculated is l to 1000 in favor of the lipid-soluble (non-ionized) form. In an intestinal environment with a pH of 6, the ionized to non-ionized ratio would become 10 to 1 respectively. Under the latter conditions much less PCP will be absorbed, because only 10 percent of the PCP is in the absorbable, non-ionized form at any time. Thus, PCP is in the non-ionized lipid-sol- uble form in the stomach and subsequently tends to be absorbed very well. However, because of the very large surface area of the intestine, the overall capacity of the intestine for chemi— cal absorption is greater than would be expected based on common ion effects alone. The skin provides a relatively good barrier against many toxic agents. For a chemical to be absorbed by the percutan- eous route,it must pass through the outer densely packed layer of horny, keratinized epidermal cells (stratum corneum). This layer of the skin forms the rate-limiting barrier for the cut- aneous absorption of toxicants. Toxicants are absorbed by passive diffusion (Klaassen, 1980). Incidents where dermal absorption of PCP has caused death in humans have been recorded. Two cases of fatal poison- ing occurred in a hospital nursery where pentachlorophenol had been used as a fungicide in the laundry room. The infants contacted the PCP through their diapers (Armstrong 22 nI., 1969). In contrast to the skin, in which a chemical must pass through a large number of cells, toxicants absorbed by the lung may pass through only two cells. Toxicants that are absorbed by 15 the lung are usually gases, volatilized liquids and aerosols. Particles with sizes in excess of 5 um do not pass beyond the nasopharyngeal region. The vapor pressure of PCP is 0.00011 to 0.12 mmHg at 200 and 100°C, respectively (Benvenue and Beck- man, 1967). At 20°C the vapor pressure corresponds to a theo- retical air vapor density of 1.5 mg/m3. In a 514 kg cow, this exposure level translates to a maximal theoretical PCP ex— posure via the respiratory route of 0.46 mg/kg/day. As environ- mental temperature rises toward 100°C, the resulting increase in vapor pressure could substantially increase exposure. Radiotracer studies using carbon-l4-labe1ed pentachloro- phenol (14C-PCP), demonstrated that absorption of PCP is rapid and extensive regardless of the route of exposure. Braun 2E nI., (1977) gave single oral PCP doses of 10 and 1000 mg/kg to rats. Plasma levels of 14C increased rapidly, peaking within the first six hours. In Rhesus monkeys given a single oral dose of 14C-PCP, the l4C-PCP took 12 to 24 hours to reach maximum con- centrations (Braun and Sauerhoff, 1976). Harrison (1954) re- ported that when sheep were force-fed PCP-impregnated sawdust, they reached maximum levels of PCP in the blood within 3 to 6 hours. Kinzell (1981) showed that it takes about 10 hours for a single 0.2 mg/kg dose of l4C-PCP to reach a maximum serum concentration in a cow. Kinzell gave the cow 0.2 mg/kg/day of cold PCP for 95 days before administering the 14C-PCP dose; the cold-dosing was then resumed. The cattle given 0.2 mg/kg PCP/day required three days to reach steady state blood levels (Kinzell, 1981). Essentially, all PCP studies indicate rapid absorption of PCP into the bloodstream 16 after exposure from any route. Distribution of Pentachlorgphenol When PCP reaches the bloodstream it is bound to serum proteins, primarily albumin (Hoben gn‘nI3, 1976). The PCP is then circulated to the various body compartments. Al- though some Species variation exists, most studies show simi- lar results in the distribution of PCP in tissues. Disregard— ing serum and urine PCP values which fluctuate enormously de- pending on the length of time after exposure, the tissues which consistantly rank one and two for the highest residue levels of PCP are liver and kidney. Lower concentration of PCP occur in the gall bladder, lung, stomach and intestine. The lowest PCP levels are found in the fat, muscle and brain (Walters, 1952; Jakobsen and Yllner, 1971; Larsen 95 31" 1972; Braun and Sauerhoff, 1976; Braun 23 nI., 1977; Kinzell, 1981; Hughes, 1982). Since PCP is found in the gall bladder, liver and intes- tines, enterohepatic circulation of PCP is indicated. High levels in the stomach indicate that either gastric secretion of PCP is occurring, or the stomach is a major site of PCP ab— sorption. Placental transfer of PCP has been shown in cattle (Hughes, 1982). Hughes found that the serum level of PCP in the calf was 32 to 38% of the serum PCP level in the dam. Hinkle (1973) reported placental transfer of PCP in the hamster, and placental transfer of PCP has been noted in the rat (Larsen, 1975). Metabolism of PentachlorOphenol Pentachlorophenol metabolism studies in experimental animals have shown the following metabolites: PCP glucuronide 17 conjugate, tetrachlorohydroquinone (TCH) and the glucuronide conjugate of tetrachlorhydroquinone (Figure 2). A congener of PCP (2, 3, 4, 6 tetrachlorOphenol) is present in penta at up to 12% by weight, and has been shown to be excreted un‘ changed in rats (Ahlborg and Larsson, 1978). In most Species studied, PCP is excreted as the parent compound. No metabolism was evident in monkeys given a single 14C-PCP (Braun and Sauerhoff, 1976). In rats, oral dose of studies have shown PCP metabolites to break down this way; 43% unmetabolized, 49% PCP conjugate, 3% TCH and 3% the TCH conjugate (Ahlborg 2E nI.,l974) and in a study by Braun EE,El°r(1977)' 75% unmetabolized, 10% PCP conjugate and 15% TCH. In the mouse, Ahlborg 2E nI.,(l974) found 41% of the PCP to be unmetabolized, 13% converted to PCP conjugate, 24% to TCH and 22% to the TCH conjugate. Very little is known about the metabolism of PCP in humans and in large animals. It appears that the metabolism of PCP is inducible. The conversion of PCP to TCH is induced after treatment with phenobarbital (Ahlborg 2E nI., 1978). Also, the formation of the metabolite TCH is inhibited when SKF 525-A, a known inhibitor of several microsomal enzymes, is used. Excretion of Pentachlorophenol Urine is the main route of excretion for pentachloro- phenol. Feces and milk are minor routes of excretion. Only trace amounts of PCP, less than 0.05% of administered dose, was reported as expired in the air (Larsen g; nI., 1972; Jakobson and Yllner, 1971). 18 OH C1 C1 ’ PCP-glucuronide C1 C1 OH / C1 PCP PentachlorOphenol C1 Cl ‘4, TCH-glucuronide C1 C1 TCH Tetrachloro-p-hydroquinone OH Figure 2. Metabolism of Pentachlorophenol Typically, mice have been shown to excrete 68 to 83% of PCP administered either orally, subcutaneously, or intraperitoneally in the urine in 4 - 10 days. Another 8 to 21% was excreted in the feces (Larsen SE nI., 1972; Jakobson and Yllner, 1971). In rats, excretion of PCP in the urine ranges from 50% to 80%. Excretion of PCP in feces ranges from 19% to 43% (Braun SE nI., 1977; Ahlborg 2E nI., 1974). The ranges observed reflect differences in sex, route of administration, length of collection and dose of PCP. Rhesus monkeys given 10 mg/kg 14C-PCP (Braun and Sauerhoff, 1976) excreted 70 to 75% of the dose in urine and 12-18% in feces. The plasma half-life of penta in humans is given by Braun SE nI.,(1978) as 30 hours. Braun further stated that 86% of an oral dose of PCP is elimin- ated in urine. In the .cow, 75% of a 14C-PCP dose (0.2 mg/kg body wt/day) was excreted in the urine in 76 hours (Kinzell, 1981). An additional 5% was detected in the feces. Both l9 absorption and elimination followed first—order kinetics with half-lives of 4.28 and 42.8 hours, respectively. Excretion of PCP in the milk of cows has been reported by Firestone (1979), Kinzell (1981), and Hughes (1982). Kinzell determined that 5% of PCP was eliminated in the milk of the mature cow. Hughes (1982) found that PCP concentrations in milk were 7 to 10% of those found in bovine serum. It appears that PCP is primarily excreted in the urine. No significant secretion or reabsorption of PCP is believed to occur in the kidney (Braun and Sauerhoff, 1976). Enterohepatic circulation of PCP is indicated as pre- viously mentioned. Enterohepatic circulation of PCP would account for the constant low levels of PCP found in the feces of the different species studied; as unabsorbed PCP would be eliminated as a bolus in a shorter time period. Toxicodynamics of PentachloroPhenol As is typical of all uncouplers of oxidative phOSphorylation, the clinical signs of acute PCP intoxication are nausea, gastric upset, restlessness, sweating, rapid respiration, tachycardia, fever, cyanosis, thirst, loss of weight, and finally collapse and death associated with a rapid onset of rigor mortis (Murphy, 1980). Irritation of the skin, eyes, nose, throat, and respiratory tract may also occur. The severity of these symptoms is in- creased by environmental and physiological factors such as higher ambient temperature, decreased renal function and poor health. No specific treatment is available. Supportive ther- apy given concurrently with lowering of the body temperature 20 can be helpful. Prognosis is good if death does not occur in the first 24-48 hours. The oral LD of PCP is 146-175 mg/kg in rats, 120 mg/kg 50 in sheep, and 140 mg/kg in calves (Gaines, 1969). This would place PCP into the general category of a moderately toxic substance, based on its acute LD (Buck g; filo, 1976). 50 Chronic exposure to PCP has resulted in feed refusal, increased neonatal mortality and decreased body weights in pigs confined in penta-treated farrowing pens (Schipper, 1961), and decreased body weight and feed efficiency in heifers (McConnell SE nI., 1980). Pathologic findings in the above studies were,1esions on the mucosal surface of the stomach, mild emphysema, congestion in lungs, enlargement of lymph nodes, inflammation of small intestines, infarcted areas in the liver and spleen and hemorrhage in the kidneys of pigs. In the heifers there was an decrease in thymus weight, pro- gressive anemia, villous hyperplasia of the urinary mucosa, hyperplasia of the gall bladder as well as the bile ducts, and keratin deposition in the Meibomian gland of the eyelid. The level of exposure in the heifers (McConnell SE nI., 1980) was 15 mg PCP/kg/day. The treatment groups received various a- mounts of tPCP as a portion of the 15 mg/kg/day total. McConnell concluded that the lesions observed were due to and charac- teristic of dioxin toxicosis. In other studies which used lower amounts of PCP, few adverse effects were noted. Kinzell SE nI., (1982) observed no adverse effects on feed intake, body weight, and milk pro- duction in cows administered 2 mg/kg tPCP daily. However, 21 Kinzell did note enlarged liver, lungs, kidney and adrenals upon post-mortem examination. Herdt SE nI,, (1951) observed no effects on calf behavior or in hematological parameters in calves given 7.6 mg/kg sodium pentachlorophenate per day in water. Likewise, the post—mortem examination was normal. Deaths have occurred in humans as a result of acute PCP exposure. An incident in a hospital in St. Louis, Mo. in 1967 resulted in two infant deaths and several others tempor- arily affected when the hospital laundry used pentachlorophenol as a fungicide. The infants were contaminated by dermal exposure to nursery diapers and other linens (Armstrong gn_nI., 1969;, Barthel 2E nI., 1969; and Robson En nI., 1969). Residues in the linens ranged from 11.5 to 1,950 ppm. pcp in the tissue of one of the dead infants was 20-34 ppm in kidney, adrenal, heart, fat and connective tissue. One ill infant had a serum PCP level of 118 ppm. This was subsequently reduced to 31 ppm after an exchange transfusion. Serum PCP levels in nurses working during the time of the accident were 1 to 12 ppm. Chronic PCP exposure in humans also occurs. Survey data accumulated by Cranmer and Freal (1970) revealed that the general population had urinary PCP levels of 6 to 11 ppb. Occupational exposure to PCP by employees engaged in the treatment of wood has been monitored (Klemmer 2E nI., 1980). Mean serum PCP levels ob- served in the group with the highest exposure were 3.78 ppm. The only adverse effects observed were low-grade infections or inflammations of the skin and subcutaneous tissue, 22 of the protective membrane of the eye, and of the upper resPiratory tract's mucous membrane. However, no cause-and- effect could be established between these effects and PCP exposure. Also, there seemed to be no longrterm effects in the PCP exposed group. During the renal function testing the group of 22 workers had a mean of 2.3 ppm PCP in their urine for the year period. Renal function tests, including creatinine clearance and phOSphorus reabsorption, were depressed while the workers were on the job, but showed significant improvement following a non-exposure period (Begley EE.E£°' 1977). In cattle, Kinzell (1981) found chronic interstitial nephritis and subacute urocystitis as the major pathologic changes in penta- treated cattle. In XEEEQ testing of the kidney slices confirmed significant loss of renal function. However, Kinzell indicated that the relationship of lesions to the administration of penta was not clear. He stated that the effect could be related to the use of urethral catheters, causing an inadverdent introduction of infectious microorganisms into the bladder. Hepatic porphyria has also been attributed to technical pentachlorOphenol exposure (Goldstein SE nI., 1977). Goldstein fed tPCP at 20 ppm to rats and produced porphyria along with increased hepatic aryl hydrocarbon hydroxylase (AHH) and cyto- chrome P-450 activitygand increased liver weight. Be- cause analytical PCP did not produce these effects, they were attributed to the contaminants in the penta used. In contrast, McConnell EB nI., (1980) did not observe hepatic porphyrin 23 changes or changes in urinary excretion of uro- or c0propor- phyrin in cattle fed up to 15 mg/kg day tPCP. McConnell 23 nI. (1980) did, however, observe increased AHH activity as well as cytochrome P-450 levels. These changes were also attributed to the contaminants in penta. Contaminants in Penta The chlorinated dibenzo-p-dioxins (CDDs) and chlori— nated dibenzofurans (CDFs) in penta appear to be reSponsible for most of its toxicity. The major congeners of CDD found in penta include hexachlorodibenzo-p-dioxins (HxCDD), heptach- lorodibenzo—p-dioxins (HpCDD), and octachlorodibenzo-p-dioxin (OCDD). Tetrachlorodibenzo-p-dioxin (TCDD) does occur in penta (Kinzell, 1981), however the Specific congener 2, 3, 7, 8— TCDD does not occur. For the numbering system used for di- benzo-p-dioxins and dibenzofurans see Figure 3. The degree of toxicity associated with dioxins varies with the amount and position of chlorination on the molecule. Typically,the more a dioxin is chlorinated, the more "inert" and less toxic it becomes. The LD50 of OCDD has not been de- termined, but it is very high. Johnson (1973) gave female rats up to 4 g/kg' OCDD and, failed to kill any of the rats. In comparison, HxCDD has two less chlorines and has a LD50 in rats of around 100 mg/kg (Johnson, 1973) and TCDD has been re- ported to have LDsdsin rats in the range of 0.022 and 0.045 mg/kg. The absorption of the CDDs also varies with the degree of chlorination. Norback gI nI. (1973) fed rats OCDD at 100 mg/kg and found 95% of the chemical in the feces and 4% in the urine. 24 0 9 l 9 2 8 2 8 7 3 0 3 . 7 4 5 6 4 5 6 Dibenzo-p-dioxin Dibenzofuran Figure 3. Numbering System for Dibenzo-p-dioxins and Dibenzofurans. Tissue levels of OCDD were highest in fat, liver and skin. In cattle, OCDD is not absorbed very well, (Firestone BE nI., 1979) the OCDD which is absOrbed accumulates in the fat and liver (Firestone 2E nI., 1979; Parker 25 nI., 1980). Dioxin exposure causes chloracne in the skin of man, non-human primates, and rabbits (Nicholson and Moore, 1979). Chloracne is a rather severe type of dermatitis and appears as an acne-like eruption. Dioxins have also been implicated as carcinogens. TCDD has been reported to be carcinogenic when fed to rats at 0.1 ug/kg/day for two years (Kociba 23.21-I 1978). However, other studies with TCDD have not shown carcinogenic activity (Innes 2E nI., 1969); Berry 25 nI., 1978). The potency of TCDD as a carcinogen in rats is inter- esting in light of studies by Poland and Glover (1979) which failed to demonstrate any covalent binding of TCDD. Also, there is inconclusive evidence that TCDD is even a weak mutagen (Wasson SE nI., 1977). Since most chemical carcinogens are mutagenic and bind covalently to macromolecules, Pitot and co- workers (1980) suggested that the liver cancer associated with 25 chronic administration of TCDD might arise from the promoting activity of TCDD. Pitot ES nI., (1980) subsequently showed that TCDD is a potent tumor promotor in a multistage hepato- carcinogenesis system in the rat. Chlorinated dibenzo-p-dioxins and dibenzofurans are potent inducers of the cytochrome P-450-mediated microsomal monooxygenases, and in particular aryl hydrocarbon hydroxylase unnn (Nicholson and Moore, 1979). AHH activity is mediated by the reversible stereospecific binding of TCDD to a cytosol receptor protein. The presence of the receptor is determined by the Ah locus in mice. It appears that TCDD binding to the receptor is reSponsible for the induction of AHH activity, as well as mediating TCDD's toxic reSponses in mice; (i.e., thymic in— volition and cleft palate formation) (Poland gn_nI., 1979; and Poland and Clover, 1980). Residual CDDs in animals and animal products is a problem. Firestone EE.El° (1979) reported the occurrence of dioxins in cow's milk. The levels of dioxin in the milk and body fat were 1000 times those in blood during exposure. The average daily excretion of HxCDD, HpCDD and OCDD in the milk was 33%, 3% and 0.6%, respectively, of the daily intake of dioxins as part of a penta dose. The low level of OCDD excretion in the milk iS largely due to its low absorption from the rumen. One hundred days after penta feeding was stOpped (Fire- stone 2E.El'r 1979), appreciable levels of dioxin were still detected in the fat of cows. The levels of dioxin detected were comparable to that found in milk. The authors estimated that the half-life of the different dioxin congeners in milk fat ranged 26 from 41 - 50 days. In this same experiment, Firestone SE nI., (1979) obtained evidence suggestive of some placental transfer of dioxins in cattle. IMMUNOTOXICOLOGY Introduction At the present time, an acceptable protocol for immuno- toxicity testing is not available. Guidelines for a rudimen- tary immunotoxicologic test profile which should be incorporated into a total toxicity evaluation scheme‘was outlined by the EurOpean Economic Community (EEC) in 1977. The guidelines suggested by the EEC include protocols designed to evaluate the potential risk of test chemicals by determining the functional significance of any effects observed on lymphoid organs found in routine toxicological studies, and to obtain information on the target immune organ, cell type or function impaired. A paragraph taken from the EEC guidelines illustrates how the EEC suggests that the evaluation be per- formed. At the termination of a toxicity study, thymus, spleen, and lymph nodes should be weighed and examined micro- sc0pically. From these data and measurements of serum immunoglobulin fractions and counts of circulating lymphocytes, the conclusion should be reached whether the substance has an effect on the lymphoid system or whether or not Specific function tests have to be performed. Obviously, the intent of this statement is that immune function testing be done only if a problem is observed, and after the toxicity testing is completed. More Specifically, further evaluation is suggested only after hematologic and 27 28 histOpathologic studies have been completed. While it is true that all the procedures mentioned above are necessary, these tests may be incapable of detecting all but the most immuno- toxic chemicals. Generally, histopathologic changes as well as serum immunoglobulin levels alone are inadequate as indicators of immune function. This is eSpecially true at lower levels of chemical exposure. The circulating leucocyte count and differential count then become the only remaining tests of immune function on which to base a decision of immune toxicant. While leucocyte number and morphology are fairly sensitive indi— cators of host well—being, they may be difficult to interpret, eSpecially in light of normal serum immunoqlobulins and lymphoid organ morphology. Ultimately, this scheme can lead to one of three out- comes. First, the tests can give a false negative (immunotoxic) result for the chemical tested. Secondly, they can give a positive result, but provide little information as to mode of action. This outcome would necessitate setting up a whole new study based on minimal knowledge of the immune lesion. As a result the new experiment would be large and cumbersome and may only provide the investigator with the data which should have been obtained in a prOperly designed original experiment. The third outcome is relative. The chemical passes the tests and is characterized as a non-immunomodulating chemical. This outcome, however, depends on the profile used. An alternative to this scheme is to have immune function 29 tests which are incorporated into and run along with the toxicologic testing. These tests should be designed to evaluate all major functions of the immune system, be easy to perform, and be relatively inexpensive and reproducible among labs. One approach to immune function testing is being fos- tered by the National Institute of Environmental Health Science group at Research Triangle Park, NC (Dean SE nI,,l982). They pro— pose a minimum panel of In_nIXn and In_XIn£n assays to assess immunological competence and host resistance following chemical exposure. The assays described are designed to test cell- mediated immunity (CMI), humoral mediated immunity (HMI), and phagocytosis by macrOphages in rodents (Table 4). This assay panel is currently being evaluated by the National Toxicology Program in the chemical bioassay program. Another comprehensive panel of 12.2122 and In XEEEE assays is being evaluated by the National Institute of Environmental Health Sciences group. This panel of tests is to be used in detecting immunomodulation following chemical exposure and is outlined in Table 5. These procedures evaluate pathotoxicology, host resistance models, delayed-type hypersensitivity, CMI, HMI, macrophage function, and bone marrow progenitor cells in de- tecting immunotoxicity. While a standardized approach to the immunotoxicolOgic evaluation of chemicals in animals seems to be a practical goal, there are some negative aSpects. First, once a protocol is commonly accepted, it tends to inhibit introduction of new in- formation and procedures into the system. Also, there is a 30 Table 4 Minimum Screening Panel for Defining Immunomodulation After Chemical Exposure in Rodents Parameter Procedures Performed Clinical Pathology Hematology profile - complete blood count (CBC) and differential Pathotoxicology NecrOpsy - weights of body, spleen, thymus, liver, kidney, brain Host resistance Susceptibility to transplantable syngeneic tumor at LD 10-20 Delayed (cutaneous type) Skin testing with a T cell dependent hypersensitivity (DTH) antigen Cell-mediated immunity Induction of blastogenesis with (CMI) Concanavalin A (Con A), Phyto- hemagglutinin (PHA) or with allo- genic leucocytes in a one-way mixed lymphocyte culture (MLC) Humoral immunity (HMI) Immunoglobulin levels (IgG, IgM, IgA), antibody response to T cell dependent antigen and blastogenic response to lipOpolysaccharide (LPS) Modified from Dean 22 nI., 1982. 31 Table 5 A Partial List of Procedures Available for Detecting Immuno- modulation After Chemical Exposure Parameter Procedures Performed* Clinical Pathology Hematology profile - CBC and diff- erennial with morphology and plate- lets . Chemical Pathology - CK, HBD, ALT, BUN, creatinine ACP, ALP, LD, CHS, albumin (A), globulin (G), A/G, total protein and complement activity . Pathotoxicology NecrOpsy - weights of body, spleen, thymus, liver,kidney, brain Histology - liver, thymus, adrenal, lung, kidney, heart, spleen, lymph node+ and bursa+ (avian Species). Host resistance Tumor assays - tumor cell challenge TD 10-20, Endotoxin hypersensitivity LD 10-20, Resistance to parasites. Delayed hypersensitivity Skin testing with a T cell dependent antigen, T cell enumeration+. Lymphocyte proliferation One—way mixed leucocyte culture, (clonal expansion) Mitogens - PHA, Con A, LPS. Humoral immunity Immunoglobulin levels (IgG, IgM, IgA) Primary and Secondary anti- body response to T-dependent and T— independent antigens, B cell enumeration.+ MacrOphage function Resident peritoneal cell numbers and nonSpecific esterase staining, Phagocytosis Lysosomal enzymes - 5' - nucleotidase, acid phosphatase, leucine amino pep- tidase Cytostasis of tumor target cells Reticuloendothelial system clearance of foreign material. (cont'd) 32 Table 5 (Cont'd) Parameter Procedures Performed* Bone marrow colony forming CFU-S-multipotent, hemat0poietic units stem cells CFU—GM-granulocyte/macrophage progenitors Cellularity 59Iron incorporation in bone marrow and spleen. Autoimmune reSponse+ Antinuclear antibody determination. *Abbreviations used are: CBC, complete blood count which includes red blood cell count, hemoglobin, packed cell volume and white blood cell count; CK, creatine kinase; HBD, hydroxy- butyrate dehydrogenase; ALT, alinine aminotransferase; BUN, blood urea nitrogen; ACP, acid phOSphatase; ALP, alkaline phOSphatase; LD, lactate dehydrogenase; CHS, cholinesterase; PHA, phytohemagglutinin; Con A, concanavalin A; LPS, lipOpoly- saccharide; CFU, colony-forming unit. +Additions to the list by J. H. Forsell Modified from Dean 33 El-r 1982. For procedures, see Luster SE nI., 1981. and apprOpriate methods and materials section of this thesis. 33 tendency in those performing and interpreting the profile re- sults to feel too confident in the results. Especially since the scientific community has approved "this system" as the most apprOpriate. It is apparent that systems involved with host defense may be involved in decreasing drug biotransformation in the liver. Perhaps the two systems, drug metabolism of xenobiotics and the recognition of foreign antigens, evolved together. Both are systems which identify, manipulate and help rid the host of "foreign" substances. While the two systems seem to perform the same general functions, one system (immune) deals with molecular weight substances in excess of 10,000 and the other handles smaller molecular weight substances. If, in fact, the two systems are linked by some sort of feedback system, the question becomes why? Is the phenomenon (if real) bene- ficial to the host, does it work both ways, or is it just a nuisance biological artifact having no discernible benefit to the host? In order to reduce redundancy, the pertinent literature dealing with pentachlorophenol and the immune system and immunotoxicity in large animals appears in the introductions to the apprOpriate chapters in this thesis. Indirect Effects Whenever immunotoxicology studies are performed, the results must be interpreted carefully. There are a wide variety of physiological, pathological, and environmental factors which can act alone or in combination with other factors to 34 influence the immune system. Especially important are the hormones of the adenohypOphySis and in particular the ad- renal glucocorticoid hormones. These hormones can induce lymphopenia and cause depletion of lymphocytes from the thy— mus and other lymphoid tissues (Dougherty gn.nI., 1964; Claesson and Tjell, 1976). They may also affect the phagocytic activity of macrophages (White and Goldstein, 1972), and cause the sequestration of monocytes as well as their production and release (Van Furth, 1974). Nutritional factors can also cause immune deficiencies. It is known that children with protein calorie malnutrition have impaired cell-mediated immunity (Schonland En nI., 1972); this is also found in rats (Adlard and Smart, 1972). This immunosuppression due to moderate to severe undernutrition may be mediated by increased glucocorticoid levels. However, there are several specific nutritional deficiencies which can lead to decreased host defense. Among these are vitamin B (Robson 6 and Schwarz, 1975) and zinc (Andresen 2E nI., 1973). Natural, additives and inadvertent constituents in food may also cause immunomodulation. Among these are gallic acid (Archer 2E nI., 1977), a food additive carrageenan (Thomson SE nI., 1976), and bacterial toxins, such as staphylococcal enterotoxins A and B (Smith and Johnson, 1975). Pesticide residues on food can also cause problems. This subject will be discussed later. Minute quantities of an endogenous protein, alpha—fetOprotein (AFP) can cause a variety of noncytotoxic immunosuppressive effects (Murgita and Tomasi, 1975). Normally, AFP is 35 produced in the yolk sac of the fetal liver and disappears or reaches very low levels after birth. It is believed that AFP is able to protect the fetus from immunological attack by the mother (Mizejewski and Grimley, 1976). Alpha-fet0protein becomes elevated in animals with chemically induced liver tumors (Kroes EE.E£" 1975) or animals exposed to hepatocarcinogens (Kroes SE nI., 1973). When AFP is elevated in post-fetal life, it is often a tumor antigen. Its detection is being used in human medicine to help in diagnosis of hepatomas, germinal cell tumors and other con- ditions. Apart from these roles, AFP when induced, may then play a significant role in immunosuppression. Other possible effects on host defense may be mediated by chemicals through mechanisms not yet understood. Because of this, it is important that the researcher not overlook these possibilities and that the apprOpriate controls be run. Immunology and Drug Metabolism Disease therapy involving drugs which depend on cyto— chrome P-450 for biotransformation and elimination is often complicated by changes in the cytochrome P-450 steady state levels. Changes in drug biotransformation due to enzyme in- duction or inhibition account for a large number of observed drug reactions and interactions (Goldstein EEEl-v 1974). Recently, evidence of immunostimulants affecting drug biotransformation by inhibiting the activity of microsomal en- zymes have been reported in both animals and man. (Mosedale and Smith, 1975, Farquhar En nI., 1976; Soyka SE nI., 1976). 36 If these events prove to be more than idiosyncratic reactions, physicians will have to be concerned about possible inter- actions between immunotherapeutic agents and other drugs. Previously, immunopharmacology dealt only with the study of the effects of drugs on the immune system and the mechanisms of action of a myriad of endogenous substances capable of mod- ulating the immune response. The most often reported effect of various immunostimu- lants on the drug metabolizing system has been depression of biotransformation activity. Animal studies have Shown that Bacillus Calmette-Guerin (BCG) (Farquhar EE nI., 1976), as well as Corynebacterium parvum (Soyka EE nI., 1976), both stimulants of the cell-mediated immune system, can inhibit the activity of several liver microsomal enzymes. In man, mean antipyrine serum elimination time (t 1/2) increases from 11 to 14.4 hours five to seven days after patients with metastatic malignant melanoma had received C; parvum i.v. daily for ten days (Rios SE nI., 1977). Also, drug biotransformation is decreased when reticuloendothelial cell activity is either activated or depressed (Barnes SE nI., 1979). When reticuloendothelial system (RES) cells of the liver are loaded with carbon particles, the metabolism and hepato- toxicity of carbon tetrachloride (a chemical which needs to be activated to cause hepatic damage) is decreased (Stenger 2E nI., 1969). Heme-oxygenase could be responsible for this pheno- menon, since it can degrade the heme from cytochrome P-450 and is induced in liver reticuloendothelial cells following 37 treatment with zymosan (Tenhunen EE nI., 1970) or endotoxin (Gemsa §n_nI., 1974). Both of these agents are known to de- crease drug biotransformation (Gorodisches 23 nI., 1976). A mechanistic explanation for the decreased capacity of experimental animals to metabolize drugs following pretreat- ment with immunostimulants is yet to be provided. However, assuming that this phenomenon can occur in man, an immuno- modulator-induced depression of drug metabolism could result in raised serum concentrations of any concomitantly adminis- tered drugs which are primarily dependent on biotransformation in the liver for detoxification or for elimination. In the combined immunotherapeutic and chemotherapeutic approach to the treatment of malignant disease, where many anti-cancer agents are pro-drugs which must be metabolized into thera- peutically active compounds, immunotherapy-induced depression of drug biotransformation might weaken the effectiveness of the cancer chemotherapy. A cell—mediated hypothesis for inhibition of drug bio- transformation was based primarily on a remarkable hepato- splenomegaly in mice caused by Q. parvum. Mice treated with doses of C. parvum sufficient to cause hepatosplenomegaly were abnormally sensitive to pentobarbitol (Castro, 1974). They also had prolonged anesthesia after pentobarbital or tribromo— ethanol, but not diethyl ether, suggesting that the mechanism was interference with detoxification (Mosedale and Smith, 1975). This phenomenon was confirmed in rats by Schroeder in 1976. On histologic examination, evidence of the development of microscopic foci of mononuclear cells (not Kupffer cells) 38 adjacent to injured or necrotic hepatocytes was seen. The mononuclear cell aggregates became very evident five days after n. parvum administration and at day seven were small islands of cells. At this time, some hepatocytes were frankly necrotic, liver weight reached its peak, and MFO activity was at its lowest. An inverse relationship existed between the extent of splenomegaly and the degree of inhibition of MFO activity (Soyka, 1981). The accumulation of mononuclear cells at a site of necrosis in the body is normal. However, when a splenectomy was performed prior to C. parvum administration, it blocked the effect of g. parvum on drug metabolism in the liver. The same effect was observed when whole body irradiation was used in- stead of splenectomy. The only difference between the two procedures was that Splenectomy must be done prior to C. parvum administration while irradiation would work concomitantly or 24 hours after 9. parvum administration. This suggests that cells of the RES or circulating immune cells elsewhere in the body can inhibit MFO activity. Involvement of the RES, particularly the Spleen, indicates that at least some of the effects described are not simply due to liver injury. It is becoming increasingly apparent that most immuno- modulators effectively decrease cytochrome P—450 levels and the liver's capacity to metabolize drugs. This raises questions about the status of drug biotransformations during natural stimulation of the immune system by viral infections. A decrease in hexobarbital oxidase has 39 been seen in murine hepatitis and in man, hepatitis infections have been observed to decrease drug metabolism (Rowland SE nI., 1976). An interesting and relevant example of this interaction in man involves the drug theOphylline. Patients getting 200 mg oxtriphylline every six hours (equivalent to 128 mg of the0phy- lline) for seven days showed evelated serum levels of theOphy- lline with 12-24 hours of vaccination with influenza vaccine. In one patient, theophylline levels in the blood increased to the toxic range and were accompanied by several symptoms associa- ted with theOphylline toxicity. The duration of this effect is unknown, however the the0phylline levels remained high for at least 72 hours, at which time theOphylline administration was stopped because two patients were showing signs of a toxic reaction. The elimination rate of a single oral dose of theOphy- lline was also significantly decreased in four healthy volun- teers 24 hours after administration of influenza vaccine. The t 1/2 changed from 3.3 hours to 7.3 hours (Renton SE nI., 1980). This phenomenon was also reported by Chang nnan., in 1978. Renton (1978) also noted the decreased elimination of theOphylline in children during acute viral infections of the upper reSpiratory tract. The suggested cause was decreased hepatic biotransformation of theOphylline by the mixed- function oxidase, P-450 system during the acute phase of the viral infection. No dietary restrictions were imposed during these human studies, plus many other factors are known to alter theophylline clearance (Ogilvie, 19781 However, conditions in each patient appeared identical both before and after vaccination. 40 This is a controversial area because not all research supports an interaction between the immune system and the MFO system. Mice given C. parvum and phenytoin (which under- goes extensive metabolism), show no alteration in phenytoin metabolism as measured by t 1/2 or AUC (area under the serum phenytoin concentration — time curve). This occurred even though the liver weight was increased and the P-450 activity (expressed either as nMoles cytochrome P-450/mg protein or as nMoles cytochrome P-450/g tissue), was significantly de- creased (Mullen, 1981). When phenytoin was given to eight human volunteers (four healthy and four with disseminated malignancy, but all having normal liver profiles), both before and after immuno- therapeutic regimes of either ECG or C. parvum, no difference was seen for AUC, Km and Vmax. Also, no effect was seen on the urinary excretion of 5-(p-hydroxyphenyl) - 5 - phenyl- hydantoin, the major metabolite of phenytoin. However, the cancer patients as a group had significantly lower values for AUC and Km when compared to the healthy volunteer subjects (Wan SE nI,, 1979). Antipyrine, a drug not affected by serum proteins (as phenytoin is) was also unaffected by Q. parvum pretreat- ment in cancer patients (Hamilton SE nI., 1980). Also, no changes in MFO activity were found in ducks having hepatitis, nor in some cases of hepatitis in humans, in which drug meta- bolism remained unchanged or is actually increased (Rowland, gig—n” 1976). 41 The previous studies indicate that the relationship between MFO activity and immunostimulation of cell-mediated immunity and RES does not always exist. This statement must be tempered because these studies used single therapeutic doses of the immunostimulants, and perhaps repeated doses are required in some cases to observe these interactions. Also,only certain receptors and metabolic pathways may be affected by immunostimulation. This would result in a "drug- specific" reSponse, or "immunostimulant-specific8 response or combination of both. Another concern is the use of cancer patients in some of the studies, who could have altered bio- transformation capacity. Other substances which were found to decrease cyto- chrome P-450 levels (Renton, 1981) included fungal products (statalon), a virus (Mengo), lipOpolysaccharide (§° nnII endotoxin), double stranded nucleotides (poly rI. rC), malarial infections (Plasmodium berghi) (McCarthy 2E nI., 1970), tilo- rone (Renton and Mannering, 1976), and bacteria (pertusSis vaccine) (Renton, 1981). One common feature of all these bio- logicals was the ability of each to induce interferon. Per— haps interferon production is linked somehow to drug metabolism. Tilorone, an antiviral agent thought to stimulate interferon synthesis (Renton and Mannering, 1976), decreases P-450 in rats and mice. The effect is not immediate and no endoplasmic reticulum damage is apparent. Tilorone also doubled hexobarbital sleeping time and greatly diminished hexobarbital blood clearance rate in rats. Thus, tilorone has a depressive 42 effect on hepatic drug-metabolizing enzymes In nyn (Renton and Mannering, 1976). Regardless of the dose of tilorone used, no greater than 40 - 50% of P-450 activity was lost. This suggests either that certain of the P-450 cytochromes are not vulnerable to the action of tilorone or that tilorone evokes a new steady- state level of cytochrome P-450, either by increasing its degradation or depressing its synthesis. This last situation is unlikely because the rate of incorporation of 8-amino- levulinic acid and heme into P-450 was not affected by til- orone (Renton and Mannering, 1976). If tilorone produces a lowered steady-state level in the hepatic mono-oxygenase system, it is possible that it may affect the same site responsible for the maintenance of the system under normal conditions, and that this site may be affected in the Opposite manner by inducing agents such as phenobarbital. Phenobarbital and other inducing agents have enabled us to increase the activity of the hepatic mono- oxygenase system; perhaps tilorone will permit us to lower the activity of this system at will. Renton (1981) has prOposed that the ability to depress cytochrome P-450-dependent mono-oxygenase is a property of all interferon-inducing agents and may be related to the production of interferon itself. However, interferon-inducing agents affect more than one aspect of host defense, and bio- logic effects (other than antiviral) can be attributed to "contaminating" substances (lymphokines or other mediators of 43 cellular immunity) found in the crude or partially purified preparations used in experiments (Sonnefeld SE nI., 1980). Renton (1981), showed that dextran, dextran sulphate and latex beads, all of which can be phagocytosed by RES cells in the liver, also decrease cytochrome P-450 and related drug biotransformation. None of these agents stimulates the pro- duction of interferon. It is also curious that poly rI.rC, an interferon inducing agent, has no effect on drug bio- transformation in mice immunosuppressed with cyclophosphamide or in homozygous nu/nu nude athymic mice (Renton, 1981). Since interferon production has been reported in both cyclo- phosphamide-treated mice and in nude mice, a decreased P-450 may be related to other a5pects of the immune system rather than interferon production. Two broad categories of human interferon have been identified on the basis of physical prOperties and type of stimulus needed for induction (Valle SE nI., 1975). One interferonrdesignated as type I or "classical" interferon, is produced In yIInn by buffy coat leucocytes or by non- lymphoid cells in response to virus and is stable at low pH (pH 2) and to heat (1 hour at 56°C). Recently this interferon has been divided into two distinct types, "alpha" and "beta" (Baron, 1979). The other interferon, type II, is now called "gamma" immune interferon,a mediator of cellular immunity, and is produced In yIIIn by lymphocytes in reSponse to mito- gens or specific antigens, and is acid and heat labile. All of the interferon inducers to date, including 44 tiloronehydrochloride and polyriboinosinic—polyribocytidylic acid, are potent inducers of alpha and beta interferon. It is the gamma interferon, however, which has been shown to be . significantly more potent (as much as 100 times more potent) in regulation of immune response (Sonnefeld E£.El°r 1980). When passive transfer of eXOgenous gamma interferon preparations was performed in mice, a significantly depressed aminOpyrine N-demethylase and cytochrome P-450 resulted. Passive transfer of "mock interferon" (produced by inoculation of tuberculin into nonsensitized mice), normal serum, or equivalent amounts of type I interferon had no effect (Sonnen- feld SE nI, 1980). This observation may account for the varied findings in previously mentioned studies. The effects of interferon on the cytochrome P-450 system may not be a direct cause and effect relationship. It has been shown that interferon has several activities that are not directly anti- viral (Gresser, 1977). Among these are inhibition of cell division of both tumor and normal cells, and enhancement of cell function (i.e., phagocytosis by macrOphages, cytotoxicity for tumor target cells by sensitized lymphocytes, increased number of antibody-forming cells, and production of proteins including interferon by "priming"). An interesting example of protein priming is the increased amount of a specific enzyme, aryl hydrocarbon hydroxylase,synthesized in interferon- treated cells after induction with benzanthracene (Nebert and Friedman, 1973). One other type of interferon activity not classified as directly antiviral is its effect on cell surfaces. Interferon can drastically change the number of surface 45 histocompatibility (H—2) antigens or cause a marked accumu- lation of viral antigens at cell surfaces of virus—injected murine lymphocytes (Friedman SE nI., 1975; Pitha EE.El°' 1976). Until the effects of interferon are determined, researchers should be prepared to accept that interferon-treated cells may display activities other than antiviral which may be involved in drug metabolism. The amount of interferon produced appears to be under genetic control. DeMaeyer and DeMaeyer-Guignard (1980), des- cribed IF loci which direct either high or low interferon pro- duction when mice are exposed to different viruses. When challenged with Newcastle Disease Virus (NDVhronly a single autosomal locus (IF-l) reSponsible for interferon production was identified and this contained two alleles, "h" for high and "l" for low interferon production. In response to other viruses,at least three other separate loci could be identified. In mice containing the h allekaat IF-l cytocyrome, P-450 was decreased by 35% 24 hours following injection of NDV. In another strain of mice containing the l allekaat IF-l, cytochrome P-450 was not significantly changed compared to controls. This indicates that interferon production at the IF-l locus causes a decrease in cytochrome P-450. The experiments with gamma interferon and with inbred strains of mice indicate that only certain types of interferon, or interferon produced on certain gene loci can lead to a decrease in drug biotransformation. This decreased drug bio- transformation due to an "interferon" mechanism would not preclude 46 decreases caused by other mechanisms operating during the defense of a host against an invading organism. One of the interesting aSpects of interferon is the variety of infectious agents and chemical substances capable of inducing its synthesis. As with other mediators, there is the suggestion that interferon production is the cells' reSponse to an infectious agent or chemical that either dis- turbs the cell surface and/or stimulates cell division. Immunosuppressive Chemicals Drugs Most immunosuppressive chemicals are drugs. Most of these drugs were develOped as antineOplastic agents. Because of their mode of action, they are not selective against can- cerous tissue, affecting all rapidly dividing cells they are able to contact in sufficient quantity, including hematopoietic cells and lymphocytes. There are many reviews concerning drugs that are immunosuppressive (Hersh, 1974; Bach, 1975; Zschiesche, 1975), so these will not be discussed here. In view of the large number and diverse structure of immuno- suppressive drugs, it is not surprising that many immuno- suppressive chemicals exist in our environment. 3992 The compound 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and related congeners and isomers are highly toxic impurities present in some pesticides such as 2, 4, S-trichlorophen- oxyacetic acid or 2, 4, S-T (Elvidge, 1971). These chemicals are also produced as byproducts in a number of chemical processes 47 and in combustion (Woolson gt al., 1972). TCDD was toxic to all mammalian species investigated. The precise effects often depend on the Species studied with the guinea pig being the most sensitive. However, a consistent effect of TCDD is damage to the thymus gland, manifested by cortical atrophy and thymic involution. As might be expected with thymic atrOphy, cell-mediated immunity appears to be primarily effected by TCDD exposure. Table 6 summarized representative studies covering the range of immunosuppressive effects of TCDD in laboratory animals. Newborns' cell-mediated immunity can be suppressed by treatment of their mothers with TCDD during gestation and nursing (Vos and Moore, 1974). The suppression caused by TCDD in mice (10 ug/kg, single dose) is reversible (Sharma gt_al., 1979). All immune systems monitored in this study recovered by eight weeks post-TCDD, including partial recovery by the thymus. This recovery occurred while hepatic lesions per— sisted. In addition, the effects of TCDD were not prevented by adrenalectomy (Van Logten et al., 1980), indicating that TCDD effects are not mediated through this system. In humans, no known immunosuppressive effects of the types mentioned above have been discovered. Most human TCDD exposure information is a result of exposure due to the in- dustrial accident in Seveso, Italy in 1976 (Walsh, 1977). A reactor used for the manufacture of trichlorophenol exploded and contaminated 400 acres and its residents. Other TCDD data is from studies on exposure of Vietnam soldiers and _ 48 Table 6 Selected Immunosuppressive Effects of TCDD in Various Laboratory Species In vivo or In Vitro Observed Representative Species Treatment Effect Reference Guinea pigs t3 vivo -thymus atrOphy Vos EE.El°I 1973 " " —decreased lymphocyte count " " " -decreased delayed-type " hypersensitivity " " -decreased antibody reSponse " to tetanus toxoid Mice i2 vivo -thymus atrOphy,decreased lymphocyte count Vos gt gt., 1973 " " -reduced graft vs host " activity " " -reduced rejection of skin allografts Vos and Moore, 1974 " " -reduced lymphocyte trans- formation by PHA " " " -reduction in serum globulins Vos gt al., 1974 " " -increased susceptibility to ‘— Salmonella bern infection Thigpen et al., 1975 " " -increased sensitivity to Escherichia coli endotoxin Vos gt gt., 1978 " " -reduced antitetanus titers Sharma, 1981 " " -reduced antisheep erythrocyte titer Hinsdill gt gt., 1980 " " -decreased contact sensiti— vity to dinitrofluorobenzene " " " -reduced resistance to Listeria monocytogenes " " " -reduced antibody response Clark gt gt., to TNP-Brucella abortus 1981 " ta vitro -increased suppressor cell activity . " Rats £2 vivo -thymus atrophy Vos gt gt., 1973 (cont'd) -decreased delayed-type hypersensitivity -reduced graft vs host activity —reduced rejection of skin allografts Vos and Moore, 1974 Table 6 Species Rats Rabbits 49 (cont'd) It vivo or Efl XEEES Observed Treatment Effect i2 vivo -reduced lymphocyte trans- formation by PHA and Con A $2 vivo —reduced antitetanus titers -reduced skin reactivity -decreased plasma cells in lymph nodes Representative Reference Vbs and Moore, 1974 Sharma, 1981 50 civilians to 2, 4, S-T, (agent orange and agent purple) used as a jungle defoliant (Baughman and Meselson, 1973). In- vestigations into these and other incidents of human exposure to TCDD are ongoing. It now appears that the Ah (aromatic hydrocarbon) gene complex in mice is involved in some of the immunotoxic res- ponses observed in this species (Silkworth and Grabstein, 1982). The Ah gene complex was discovered after aromatic hydrocarbons were found to induce drug-metabolizing enzymes in C57BL/6 (Ah+) mice, but not in DBA/Z (Ah-) mice (Nerbert gt gl., 1972; Goujon gt gt., 1972; Nebert and Gilen, 1972; Robinson gt gt., 1974). Because of these findings (Poland gt_gl,, 1974; Nebert gt gt., 1975),the presence of a cytosolic receptor or re- ceptors controlled by the Ah complex which specifically binds some polycyclic aromatic hydrocarbons (PAH) was postulated. Not only is the presence of the cytosolic receptor required to produce toxicity, due to PAH exposure in the mouse, but a structural requirement exists for the polycyclic aromatic hydrocarbon. The relative binding affinities of some halo- genated aromatic hydrocarbons including TCDD is closely corre— lated with mixed function oxidase induction and the toxic responses observed (Poland and Glover, 1977; Poland and Glover, 1980). The planarity, size and shape of the PAH seem to be important for receptor binding. In C57BL/6 mice the planar molecules TCDD as well as 3, 4, S, 3', 4', 5'-hexabromobiphenyl bind strongly to the cytosolic receptor and produce thymic involution (Poland and Glover, 1980), whereas the nonplanar 51 2, 4, 5, 2', 4‘, 5'-hexabromobiphenyl does not bind to the receptor and does not cause thymic involution. However, the relationship between enzyme induction and toxicity is unclear. Pesticides Many pesticides are potentially toxic in man. However, the frequency of systemic poisoning or of other major chronic diseases resulting from pesticides is rare. This illustrates the effectiveness of education and legislation on public aware- ness in the handling and use of pesticides. Epidemiologic investigations presently indicate that effects on the immune system are probably the most frequent toxic manifestation of contact with pesticides at infrequent, low level, or chronic exposures. Pesticides usually affect the immune system by eliciting an allergic reSponse. The most common aller- gic reSponse observed is allergic dermatitis of the cell- mediated inflammatory-reSponse type. This type of allergic dermatitis involves mast cell degranulation and is character- ized by erythema, edema and eczema. Typically, there is no respiratory system involvement and IgE antibodies against pesticides, and serum IgG elevations are rare. Another form of allergic reSponse, photoallergy, has also been reported with esposure to several pesticides, including paraquat and captafol (Nomura gt El" 1976; Horiuchi and Ando, 1977; PeOples gt gt., 1978). A list of pesticides reported to cause allergic dermatitis in humans is given in Table 7. Some pesticides elicit the production of autoantibodies. However, the presence of autoantibodies does not necessarily correlate 52 Table 7 Pesticides Claimed to Have Caused Allergic ReSponse in Humans Insecticides BHC Butyphos Cartap Chlorfenvinphos Chlorobenzilate ChlorOphos Cyanox Cyanphenphos DDT Diazinon Dichlorvos Dicofol Dimite Dinofen Fenitrothion Formothion Karathane LeptOphOS Lindane Malathion Methomyl Methyl, mercaptOphos Naled Nicutine sulfate Nitrofen Omite Ovex Phenthoate Pirimiphus-methyl Salithion Schradan Thiometon ThiOphanate Fungicides Benomyl Captan Difolatan Dinofen Mancozeb Maneb Nematin PCNB Thiram Zineb Herbicides CDAA Chlorothalonil 2, 4-D Daconil Paraquat 2, 4, 5-T Trifluralin Rodenticides PhOSphine 53 with autoimmune disease. Many people have detectable auto- antibodies but are asymptomatic. The types of autoantibodies detected and believed to be pesticide—related are liver, red blood cell, thyroid, kidney, stomach, and intestine. The pesticides reportedly reSponsible for the deve10pment of autoantibodies are DDT, lindane, fenthion, 2, 4-D (Katsenovich and Usmanova, 1970), caprolactam, methen- ilene-diamine, polychlorOpinene, thiram, chlorOphos (Brusilouski gt gt., 1973), anthio, milbex (Aripdzhanov, 1973), hexachloro- eyclohexane, methylmercaptOphos (Omirov and Talan, 1970), phosphamide, aldrin, monuron (Nikolaev gt gt., 1970), and carbon tetrachloride (Dodson gt gt., 1965). Many pesticides have been shown to cause immunomodulation of both cell-mediated immunity and humoral immunity. An exten- sive list of pesticides and their effects on laboratory animals is given by Sharma (1981). Upon review of this list, it becomes apparent that the present understanding of immunomodulation due to pesticide exposure is largely at the descriptive level. Little or no research has been directed to the question of the mechanisms of immunotoxicity. In a few pesticides studied, one would question whether immunosuppression occurs at all, or only as an indirect effect. DDT is an apprOpriate example. In studies of immunosuppression after DDT exposure, often no immunological response was observed unless relatively high levels of DDT were given. These high DDT exposure levels resulted in other toxic effects and death of some exposed animals (Wassermann gt gt, 1971). 54 One interesting finding associated with DDT is its apparent ability to protect against anaphylactic shock. Evidently, guinea pigs pre-treated with DDT show less severe episodes of anaphylactic shock (Gabliks gt gl,, 1973). The mechanism for the shock protection appears to be due to the ability of DDT to decrease the amount of histamine available for release at the time shock is induced. DDT does this by in- ducing the release of histamine from mast cells or by re- ducing mast cell number (Askari and Gabliks, 1973). Heavy Metals (Lead, Cadmium, Arsenicals, and Organotin) The immunotoxicity of metals is best known. Selye gt gt. in 1966 observed that lead greatly in- creased the susceptibility of rats to bacterial endotoxin. Selye showed that the damage done in rats by 100 ug of Escherichia ggtt endotoxin could be reproduced by injecting one ng endotoxin in- to rats given a normally tolerated dose of lead intravenously (5 mg/100 g body weight) as lead acetate. The change observed represents a 100,000 fold increase in endotoxin sensitivity. Subsequently, this phenomenon has been repeated in mice (Rippe and Berry, 1973) and chickens (Truscott, 1970). Another metal, cadmium, is also able to induce similar changes in rats exposed to Salmonella enteriditis endotoxin (Cook gt gt., 1974). Lead and cadmium affect other systems of host defense. Lead decreased host resistance to viral infections (Gainer, 1974). It appears this is due to reduced interferon synthesis. Other studies have shown some decrease in humoral immune response (Koller, 1973) and in phagocytic activity (Trejo gt gt., 1972; 55 Filkins and Buchanan, 1973) due to lead exposure. T lymphocyte function is altered by a single exposure to lead (10 mg/kg i.p.) in mice. Antibody reSponse to T lymphocyte-dependent antigens is markedly depressed due to this treatment, but returns to normal in three weeks (Winchurch and Thomas, 1983). Meanwhile, the T independent antigen response was unaffected or raised in this study. The B cell mitogen response to LPS was in- creased after lead exposure and decreased linearly, falling to control values by three weeks, essentially mirroring the drOp in blood lead level in the mice. Follow-up studies £2 ztttg showed that T cells derived from mice treated with lead do not c00perate with normal B cells but that B cells from exposed mice can make antibody when mixed with normal T cells. The form in which lead or cadmium is used in host-defense studies makes little or no difference in the results obtained (Dr. Pierluigi Bigazzi, personnel communication). How, when, and how often the metal is administered is important (Vos, 1977). Arsenicals appear to affect host defense by increasing susceptibility to viral infection (Gainer and Pry, 1972). A possible explanation for this is the inhibition of both inter- feron synthesis and action seen with high level exposure to arsenicals. At low levels of arsenical exposure, the activity of interferon was actually increased (Gainer, 1972). Biphasic responses to xenobiotics are not uncommon in toxicology. Per— haps the beneficial growth-promoting effects seen when arsenicals are used as feed additives is through a reduction in disease which may be in part caused through the increased interferon activity. 56 Organotin compounds, especially DOTC (di-n-octyltin- dichloride) and DBTC (di-n-butyltindichloride) are very selective immunosuppressants. These chemicals can exert a selective cytotoxic action on T lymphocytes (Seinen and Willems, 1976; Seinen gt gt., 1977a; Seinen gt gt., 1977b; Seinen gt gt.,l979),which results in a T cell dependent immu- nity depression and an atrOphy of the lymphoid system. In fact, this immune suppression is the most sensitive parameter of any toxicity associated with these two chemicals. This makes DOTC and DBTC two of the few "pure" immunotoxicants identified. The immunotoxicity associated with DOTC and DBTC is more than just a scientific curiosity. It is estimated that more than 25,000 tons of organotins are produced yearly (Van der Kerk, 1978). Among the uses for these chemicals are: as heat stabili- zers of polyvinyl chloride plastics; as biocidal compounds for wood, paper, and textile preservation; as an agricultural fungi- cide, miticide, and acaricide; as a component of antifouling paints; and as catalytic agents in a variety of industrial pro- cesses (Ross, 1965; Luyten, 1972). Hence, there are many Oppor- tunities for these compounds to enter the environment. Organochlorines Polychlorinated biphenyls (PCB) are a class of industrial chemicals which are very stable and have low flammability. They contain from 12 to 68 percent chlorine and are exceptionally persistent in the environment. PCBs have been used for over 40 years in many applications. Among these uses are: as insulating materials in electrical capacitors and transformers, plasticizers, 57 in waxes; in paper manufacturing; and for a variety of other industrial purposes. The health effects of PCBs are well es- tablished. Observed effects in mammals and birds include micro— somal enzyme induction, porphyrogenic action, estrogenic acti- vity and immunosuppression (Bitman, 1972; Vos, 1972). There are many good reviews on the effects of PCBs ranging from a consideration of physiochemical properties (Nelson, 1972) to the effects of acute and chronic exposure in laboratory animals (Fishbein, 1974). PCB seems to cause an immunosuppression which results in an enhanced susceptibility to viral infection and thus appears to have a deleterious effect on cell-mediated immunity. Friend and Trainer (1970) found a slight to moderate suppressive effect due to PCP (Aroclor 1254) exposure in ducks challenged with duck hepatitis virus. In guinea pigs, PCB exposure has resulted in reduced delayed-type hypersensitivity (Vos and Van Driel - Grootonhuis, 1972). Humoral immunity also appears sensitive to PCBs. Loose and coworkers (1979) showed a decrease in antibody synthesis to the antigen sheep red blood cells, a reduced serum IgA concentration, an increased sensitivity to an endotoxin from Salmonella typhusa, and a decreased resistance to a malaria organism (Plasmodium berghei) in mice. Likewise, Thomas and Hinsdill (1978) showed both a higher mortality rate in mice in- fected with Salmonella typhimurium and an increased sensitivity to endotoxin. These researchers also found a slight lowering of antibody levels to sheep red blood cells and a reduced gamma- globulin fraction in monkeys. With the exception of the study in 58 monkeys in which 5 ppm Aroclor 1248 was given in feed for six months,all the above effects resulted from fairly high PCB exposure levels (range of 50-1000 ppm). Thus, some doubt remains as to the effects on the immune system of low level PCB exposure. Polychlorinated biphenyl-induced immunosuppression could be due to the presence of particular PCB isomers. The isomer 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl was the most toxic of the hexachlorobiphenyls isomers tested in the chicken, mouse (Biocca gt_gl., 1976) and the rat (Vos, 1977), and pro— duced the most severe thymic atrOphy in these Species. In addition, toxic effects of PCB could be due to chlorinated dibenzofurans present as impurities in some of the PCB pre- parations (Vos gt gt., 1970). One of these dibenzofurans, 2, 3, 7, 8-tetrachlorodibenzofuran, has been shown to cause severe thymic atrOphy in chickens, guinea pigs, and mice. (Moore gt gl., 1976). Recently, Silkworth and Grabstein (1982) have shown that polychlorinated biphenyl immunotoxicity segregates with the Ah gene complex in the mouse. Furthermore, they showed that the immunotoxic potential of PCB is dependent on isomer plana— rity. These studies were done with 3, 4, 3', 4'-tetrachloro- biphenyl which is planar and gives immunotoxic effects, and 2, 5, 2', 5'-tetrachlorobiphenyl which is nonplaner and does not result in immunotoxicity. The 3, 4, 3', 4'-tetrachloro- biphenyl was found to compete with TCDD for a cytosolic re- ceptor in B6 mice, and like TCDD is a strong aromatic hydro— carbon hydroxylase and cytochrome P-450 inducer (Poland and 59 Glover, 1977). Hence, PCB toxicity may manifest itself by a mechanism similar to TCDD. Hexachlorobenzene, like PCB, is also immunosuppressive. In fact, it appears that the actual immune lesions seen are quite similar to those observed with PCB (Loose gt gt., 1977; Loose gt El"1979)' Polybrominated Biphenyls (PBB) PBB became important as an environmental contaminant when in 1973 PBB accidentally entered the food chain in Michi- gan. PBB was a major component in FireMaster FF-l, a commercial flame-retardant chemical. The FireMaster FF-l was inadvertently substituted for NutriMaster, (magnesium oxide) used as a feed supplement for livestock (Jackson and Halbert, 1974; Dunckel, 1975). Following this accident, a large number of studies reported PBB-associated immunological effects in cattle and laboratory animals. Among the first were reports that Fire— Master BP-6 (FireMaster FF-l without a 2% anticaking agent, calcium polysilicate added) caused atrOphy of the bursa of Fabricius in chickens and depressed antibody responses in guinea pigs (Damstra gt gt., 1982). Cattle inadvertently exposed to PBB, and accumulating up to 30 ppm in body fatyshowed no altered immune function (Kateley and Bazzell, 1978). In another study involving 114 Holstein cattle, 58 of which had PBB body burdens ranging from 0.02 to 24 ppm for at least two years, no immunomodulation was seen (Kateley gt gt.,l982). Two additional cattle in the 60 Kateley study were fed 25 grams of PBB daily for 39 consecu- tive days. These animals had usual immune competence until they had been fed 500 g of PBB, which corresponded to a tissue concentration exceeding 1,000 ppm. Above 500 grams of PBB fed, the cattle became moribund and exhibited changes in neu- trophil function and serum antibody titers. Beagles fed up to 4.0 mg PBB/kg for 61 days had in- voluted thymuses and reduced hematOpoiesis (Farber gt gt., 1978). In Rhesus monkeys, alterations in B and T cell functions occurred after feeding PBB at 1.5 ppm for 5 months and 25 ppm for 10 weeks (Allen and Lambrecht, 1978). PBB has also been reported to affect the immune system in the rat and mouse (Luster gt gt., 1978; Wilson gt gt., 1979; Luster gt gt., 1980). Fraker (1980) showed that antibody re- sponses to sheep red blood cells in mice fed diets containing 1, 10, or 100 ppm PBB for 30 days were 80%, 30%,or 12%, res- pectively, of control values. Cell—mediated immunity was not affected in this study. Human epidemiologic studies (Bekesi gt gt., 1979) have indicated that some farm family members with PBB exposure had exhibited a decreased number of lymphocytes with concomitant increases of lymphocytes with no detectable surface markers, and decreased lymphocyte function as measured by blastogenesis. Bekesi (personal communication) has also indicated that exposed Michigan residents have increased IgG, IgA, and C complement 3 levels as well as increased incidence of neuplasia. Unfortunately, these studies have not been confirmed. 61 It appears that the dose of PBB required to cause immunosuppression in most animals is very near the limit of overt toxicity. In some animals,no effects were seen (Kateley and Bazzell, 1978; Mudzinski gt_gt., 1979; Kateley gt_gt., 1982), or the animals'response returned to normal, indicating immunological recovery (Wilson gt gl., 1979). In other studies (Luster gt gt., l980),no functional alterations (i.e., bacterial challenge) of the immune system were observed even though several immunological reSponses were depressed. It appears that the immunotoxicity of PBB, not unlike that of PCB and TCDD, may vary greatly from species to species (Vos, 1977). Carcinogens A detailed discussion of carcinogenesis is beyond the scope of this review, however,a few comments should be made since environmental chemicals are believed to be reSponsible for initiation of most human cancers. For a unified coverage of oncology,see Pitot, (1978). One of the characteristics of advanced cancer,and eSpe- cially of neOplasms of the immune system,is a decrease in the host resistance to various infectious agents. Specifically, this phenomenon appears to have at least four characteristics; a decrease in mature granulocytes, an impaired cell-mediated immunity, a decrease or alteration in serum gamma-globulin levels, and production of blocking factors such as "blocking" antibodies and soluble tumor-associated antigens. 62 Cancer patients are often infected with organisms that normally would not cause disease in the immunocompetent host. This is especially true of fungi and cytomegalovirus, which are ubiquitous in the human pOpulation but rarely cause disease in healthy peOple. So, in a sense, chemical carcinogens as well as radiation can be considered immunotoxicants, since in many cases it is the infectious sequel to the cancer that leads to the ultimate demise of the patient. A partial listing of some known chemical carcinogens is given in Table 8. 63 Table 8 Examples of Chemical Carcinogens Benzo(a)pyrene 3-Methylcholanthrene Benz (a)anthracene 2-Acetylaminofluorene Pyrrolizidine alkaloids Aflatoxin Bl Safrole 3-Hydroxyxanthine Dimethyl nitrosamine Acetamide Thioacetamide Ethionine 1,2-Dimethylhydrazine N-Methylnitrosourea Beta-prOpiolactone Dimethyl sulfate Bis (chloromethyl) ether Ethylene bromide Benzyl chloride Bis (2-Chloroethyl)sulfide O-Toluidine Vinyl chloride Carbon tetrachloride Thiouracil Acrylonitrile Tetrachloroethane Weisburger and Williams,*l980 ISOLATED HEPATOCYTES Isolation of Hepatocytes Berry and Friend (1969) developed the first procedure for the isolation of viable hepatocytes from rats. Their pro- cedure was an tg_gttg process using a solution of collagenase and hyaluronidase to separate the hepatocytes. In 1972 and 1973 Seglen published a detailed description of the factors required for Optimizing the isolation of rat hepatocytes. Seglen was the first to perform the two-step method of isola— tion which has become the basis for all subsequent isolation procedures. In the two-step procedure,ca1cium is removed be- fore enzymatic dispersion using collagenase. This procedure enhances enzymatic dispersion by first breaking desmosomes. The basic i2 ytzg perfusion technique presently used in dissociating rat hepatocytes involves the surgical cannulation of the inferior vena cava. This cannulation is done anterior to the kidneys. The thoracic portion of the inferior vena cava is clamped and the organ is bled out through the portal vein. Calcium-free media is pumped through the liver prior to the addition of the enzyme. Collagenase is perfused through the liver until the entire organ is visibly softened. The liver is removed, the capsule dissected away, and the cells are mechanically dispersed. The cells are then purified and used as is,or cultured: (See Figure 4). 64 65 Thoracic Inferior Vena Cava Surgical Tie Diaphragm Liver . g i : i Cannuia : (Influeni) Parial - Vein \LEfflueni) :1 \ Left J Kidney Perfusion Solution Inferior Vena Cava / Figure 4. Reverse Perfusion Technique for Rat Liver 66 Modifications of this system are many. Perhaps the most notable and most used modification is that of Fry gt gl.,(1976). Fry removes the liver immediately after the rat is killed. The liver is placed in Ca2+ and Mg2+ free buffer and then cut to obtain liver slices 0.5-1.0 mm in thickness. The slices are then placed in a flask and are shaken in a 37°C water bath. The slices go through a series of calcium—free medias, EGTA- supplemented medias and finally the digestion solution which contains collagenase and hyaluronidase. The flasks are in- cubated until the supernatant is cloudy. This supernatant con- taining the isolated cells is filtered to obtain the hepatocytes. The rat hepatocyte isolation procedure proved to be easy and very effective in obtaining large numbers of viable hepa- tocytes. A virtual quantitative recovery of viable rat paren- chymal cells was readily accomplished by the early 1970's (Seglen, 1973b). In recent years, isolation of cells from other species has been accomplished. Species in which attempts to isolate hepatocytes has been successful are sheep (Clark gt gt., 1976; Ash and Pogson, 1977), dogs (Reese and Byard, 1981), monkeys (Poole and Urwin, 1976) and humans (Nau gt gt., 1978; Reese and Byard, 1981). To accomplish isolation of hepatocytes from these larger Species, the procedures described above had to be modified. Most of the modification dealt with the methods used in tissue procurement and initial handling. Surgical remnants, biOpsies, liver obtained from aborted fetuses (Nau gt gt., 1980); Reese and Byard, 1981; Strom gt gt., 1982) or portions of the liver must be used (Clark gt gt., 1976). 67 Isolated Hepatocyte Function . Traditionally,the viability of isolated hepatocytes has been determined by histochemical means. The major method used is trypan blue dye exclusion. Trypan blue exclusion has been and Still is being used by almost all researchers requiring a viability determination. However, there is a problem with trypan blue dye exclusion in that it is not very sensitive (Krebs gt gt., 1979) and is likely to give false positive (viable) results. The procedure cannot distinguish between healthy cells and dam- aged and denenerating (but still "alive") cells. AS a result, many investigators have had to establish viability of isolated cells by means other than trypan blue. Biochemical and histo- logic evidence of viability have become acceptable means of establishing true cellular viability. Some of the biochemical tests used have been designed solely to provide information on the cell's viability before they are used in research projects. The more useful viability tests have been discovered secondary to their use in other re- search. Gluconeogenesis is a biochemical test used to establish cellular viability. It is especially valuable as an indicator of cellular viability if precursors, biochemically (reactions) far removed are used for its synthesis (Krebs gt gt,,l979). Another procedure which has proved useful in cellular viability testing is stimulation of cellular reSpiration by succinate. Only a damaged plasma membrane will allow low levels of succinate to permeate at a rate sufficient to stimulate cellular respiration 68 (Bauer gt gt., 1975). Other tests which have been used are: 1) intracellular potassium and adenine nucleotide concentration (Ash and Pogson, 1977); 2) retention of constitutive enzymes like lactate dehydrogenase which are released into the media when membrane damage occurs (Clark gt gt., l976);3) electron microsc0py of isolated cells (Berry and Friend, 1969); 4) the demonstration of a response to a hormone such as glucagon (Zahlten and Stratman, 1974); and the synthesis of urea (Krebs gt gt., 1979). Isolated ngatocytes and Xenobiotic Metabolism Isolated hepatocytes have been used for xenobiotic metabolism and cytotoxicity studies. The use of isolated hepa- tocytes to study xenobiotic metabolism has several advantages over use of the whole animal. Compared to the whole animal, cells are essentially free of hard-to-control variables such as hormone or nutrient levels (Fry and Bridges, 1977). Also, dosing,time of exposurepand metabolite generation can be more rigidly controlled (Fry and Bridges, 1977) and done more in- expensively than in a large animal. Another advantage of cells is that several variables can be tested using a single prepara- tion of cells. Other alternatives to the whole animal approach are it ztttg systems such as organ perfusion, tissue Slices, sub— cellular fractions and tissue maintenance cultures. Although all of these techniques have been successful in some studies, more and more researchers are using isolated cells in xeno- biotic metabolism Studies simply because the system seems to 69 mirror the i2 ytzg Situation more closely (Fry and Bridges, 1977; Menzer, 1979). Specifically, some of the advantages of the isolated cells system cited by researchers include: dead or deteriorated cells can be selectively removed from the system (Belleman gt_gt., 1977); suSpended or cultured cells are totally functional (Nau gt gt., 1979); isolated cells retain complete subcellular organization (Nau gt gl., 1978); cells remain viable for several hours (Nau gt_gt., 1978; Nau gt gt., 1979); and the environment of the cells can be manipulated (Holtz- man gt gt., 1972; Erickson and Holtzman, 1976) so that hormonal or other homeostatic mechanisms can be controlled. In addition, the requirements for Optimal metabolic rate can be established (Bissell and Guzelian, 1979); cofactor additions are not required (Holtzman gt gt., 1972; Nau gt gt., 1978); and the total metabolism of xenobiotic can be studied (Billings gt gt., 1977), including the interrelationships between metabolic path- ways (Wiebkin gt gt., 1976; Morello, and Agosin, 1979) and substrates (Hayes and Brendel, 1976). The full sequence of reactions (Moldeus gt gt., 1978) and the rate limiting steps (Shinkey gt gt., 1979) can be studied as can the further meta- bolism of primary metabolites (Wiebkin gt gt,, 1978). In iso- lated hepatocytes, conjugation pathways are fully functional (Jones gt gt., 1978) and the depletion of conjugating components like GSH can be monitored (Hirata gt gt., 1979). Moreover, cells lend themselves to the study of rapid reaction sequences and make quantitation of Short-term reactions possible (Moldeus gt_gt., 1974). Xenobiotics which have been studied using iso- lated hepatocytes are listed in Table 9. Table 9 Xenobiotics Which Have Been Studied Using an Isolated Hepatocyte System. Xenobiotic alpha-l-acetyl methadol propoxyhene butamoxane ethinimate methoxybutamoxane p-nitrophenol ethylmorphine aminOpyrine aniline benzoic acid phenol hexobarbital diphenylhydantoin benza (a) pyrene benzodiazepine drugs biphenyl ethoxycoumarin 7-hydroxycoumarin alprenolol phenOphthalein 4-methylumbelliferone 2-napthol harmine paracetamol quinine sulfate dichloro-p-nitrOphenol ethoxyresorufin thiabendazol chloramphenical amphetamine p-nitranisole sulfadimidine sulfanilamide p-aminobenzoic acid cocaine 2-acetylaminofluorene napthalene acetaminOphen harmol aflatoxin B1 phenyramidol para-chloro-N-methylaniline Reference Billings et al., 1977 -— w— Erickson and Holtzman, 1976 Poland and Kappas, 1971 Morello and Agosin, 1979 Nau gt., 1978 .e; 2.2 Nau al., 1979 Wiebkin et al., 1978 Fry gt gI:,—l976 Moldeus et al., 1977 Moldeus gt EI., 1974 Moldeus gt g%., 1978a Moldeus et al., 1978b Hayes and—Brendel, 1976 Hultmark et al., 1978 Burke and—firfenius, 1978 Gerayesh-Nejad et al.,l975 Siliciano gt gt77 I978 Billings 2E.%l" 1978 Morland and Olsen, 1977 Stewart gt gt., 1978 Leffert et al., 1977 Block gthtTT 1976 Andersson gt gt., 1978 Decad gt gl., 1977 Jones and Mason, 1978 Dougherty gt gt., 1980 71 Future of Isolated Hepatocytes The continued use of isolated hepatocytes in toxicology and pharmacology studies depends on the success of researchers in maintaining cytochrome P-450 levels over extended periods of time. P-450 deteriorates after about 3-5 hours of incu- bation (Michalopoulos gt gt., 1976; Decad gt gt., 1977). Recently, P-450 longevity has been increased by defining nutri- tional (Bissell and Guzelian, 1979; Paine and Hockin, 1980) and hormonal requirements (Michalopoulos gt gt., 1976; Decad gt gt., 1977). The induction of P-450 in culture is very difficult to accomplish and only a few researchers have been able to do so (Sinclair gt_gt, 1979). Higher t2 Xtttg levels of cytochrome P-450 are routinely obtained by first in- ducing it 2239, The future of isolated hepatocytes may be altered by the discovery of a method for division and multiplication of differentiated hepatocytes. Presently,hepatic function itself is lost in 3-7 days after isolation. The isolated hepatocytes dedifferentiate and lose hepatic function as evidenced by loss of albumin secretion. This dedifferentiation may be reSpon- sible for the loss of P—450. Although multiplication of hepa- tocytes t2 ytttg has not yet been achieved by the scientific community, there is hOpe that this will occur when all the necessary parameters are defined, because hepatocytes will regenerate it vtzg (Bucher, 1963). Maintenance of hepatocytes as differentiated cells has been accomplished for up to 3-5 months by improving support systems (Rojkind, 1980; Freeman, 1981; 72 and Enat, 1982). If the multiplication of differentiated hepatocytes becomes a reality, isolating hepatocytes will no longer be necessary. Chapter 1 SUBCHRONIC ADMINISTRATION OF TECHNICAL PENTACHLOROPHENOL TO LACTATING DAIRY CATTLE: IMMUNOTOXICOLOGIC EVALUATION 73 74 INTRODUCTION Technical pentachlorOphenol, commonly referred to as "penta", has a variety of commercial applications. Its effec- tiveness as a broad Spectrum biocide is due to the oxidative phosphorylation uncoupling ability of its major component, pentachlorOphenol (PCP). It is used commercially as the active ingredient in various molluscicides, herbicides, insecticides, fungicides, bactericides and slimacides. However, most of the penta produced commercially in the Unites States is used for wood preservation. Since many structural components of livestock facilities such as feed bunks, bunk silos, Splash boards, support poles, and free Stalls are constructed of penta-treated wood, live- stock may be exposed subchronically to preservative chemicals. The extent of usage of treated wood on dairy farms in Michigan was determined by a recent survey which found penta-treated wood on nearly 50% of the farms (Foss gt gt., 1980). On live- stock farms, exposure can result from: 1) ingestion of feeds stored in or fed from penta-treated structures, or licked off of treated wood); 2) cutaneous absorption by direct contact with treated wood; and 3) inhalation of air containing preservative chemicals, particularly the volatile chlorOphenols. An implication of an association between exposure to penta and poor health of dairy cattle was reported by Thomas gt 31., 1977). Clinical signs in these cattle included de- creased milk production, poor general appearance, skin lesions, increased mastitis, persistent infections, high calf mortality, 75 and death. Certain of these effects suggested a suppression of immune function. Moreover, 2, 3, 7, 8—tetrachlorodibenzo- p-dioxin (tetra-CDD) causes thymic atrophy in many Species (Gupta gt 21" 1973). Even though penta does not contain 2, 3, 7, 8—tetra CDD, it has been reported to contain a small concentration of other tetra-CDD isomers (Kinzell gt gt., 1981) as well as hexa-, hepta-and octa—CDD and dibenzofurans (Fire- stone gt gt., 1979; McConnell gt gt., 1980; Kinzell gt gt., 1981). Various other chlorinated hydrocarbons such as hexachlorobenzene and chlorinated diphenyl ethers (Firestone gt gl., 1972) also exist in penta. Hexachlorobenzene has been shown to be immuno- suppressive in mice (Loose gt gt., 1977) and rats (Vos gt 31°! 1979), but the immunomodulation of other components in penta has not been reported. In a study recently reported by McConnell gt gt. (1980» various defense mechanisms were evaluated in growing dairy heifers fed analytical pentachlorophenol, technical grade pentachlorophenol, or various mixtures of both. The chemicals were administered subchronically at exposure levels 1-2 orders of magnitude greater (20 mg/kg body wt/day for 42 days followed by 15 mg/kg/day for 118 days) than what has been esti- mated to occur on some dairy farms (Van Gelder, 1977). Toxic effects which included increased mitogen-induced lympho- proliferation and decreased thymus weight were more prevalent in heifers fed technical PCP. Comparatively, the present study,which utilizes a more extensive 76 immune profileywas designed to ascertain whether a level of exposure approximating a farm environment would modulate the immune system in older lactating dairy cattle. A previous report (Kinzell gt gl.,l981) contains performance, general health, and pathology findings. 77 MATERIALS AND METHODS Chemicals and Reagents Commercial pentachlorophenol (MB-528) was generously supplied by the American Wood Preservers Institute (AWPI) and represented an industry composite from the three major manu- facturers of penta. The chlorOphenol composition of this lot as reported by the Supplier (Roman, 1978) was 85-90% penta— chlor0phenol; 4-8% tetrachlorophenols; 0.1% trichlorophenols; and 2-6% other components. The chlorodibenzo-p-dioxins (CDDs) were determined by high pressure liquid chromatography with confirmation by gas chromatography/mass Spectrometry. CDD con- centrations in ppm were: octa-CDD, 1000; hetpa-CDDS, 378; hexa- CDDs, 173; and tetra—CDDS, 0.035. The tetra-CDD does not in- clude 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. The analytical methodology is described by Kinzell gt gt., (1981). Other chemicals and reagents included: Ficoll, Con A and LA, (Pharmacia Fine Chemicals, Piscataway, NJ) and Ca2+ and Mgz+-free Hanks balanced salt solution, (HBSS), Hanks balanced salt solution, sheep red blood cells (SRBCS,) fetal calf serum and RPMI 1640 with 25mM Hepes (Microbiological Associates, Walkersville, MD), Vibrio cholerae neuraminidase (VCN), Freunds complete adjuvant, and penicillin and streptomycin (Gibco, Grand Island, NY), fluorescein-labeled (FITC) rabbit anti-bovine Ig reagents and single radial immuno- diffusion agarose plates for serum immunoglobulin concentrations (Miles Laboratories Inc., Elkhart, INL,[;H] thymidine ( [3H] Tdr) , (New England Nuclear Corp., Boston, MA), Hypaque 78 (or sodium diatrizoate, Sterling Organics, New York), 1.091 micrometer polystyrene latex particles (Dow Chemical Co., Indianapolis, IN), zymosan A (Sigma Chemical Co., St. Louis, MO), BCG (Research Foundation, Chicago, IL) and PPD (Connaught Laboratories, Toronto, Ontario). Treatment of Cattle Eight non-pregnant mature Holstein-Friesian cattle, four penta-treated and four controls, were paired according to stage of lactation. During the study, each cow was periodically fitted with an indwelling urethral catheter for urine collec- tion. Also, rumen cannulas were installed in four cows (two pairs) for use in feed digestion trials. The cattle were housed in a cold enclosed barn and allowed a period of five to six weeks for environmental and feed adjustment prior to initiating penta administration. Each pair was started on a treatment 5-7 wks. postpartum. All cattle were milked and fed twice daily. Several additional measurements including serum chemistries, urinalysis and a complete physical examination by a veterinarian were made and reported by Kinzell gt gt., (1981). Penta was mixed with the concentrate part of the ration and fed in equal parts twice daily, at the morning and evening feed- ing times. Treated cattle received 0.2 mg/kg body wt./day for 75-84 days followed by 2.0 mg/kg body wt./day for 56-62 days. Additional details on treatment of cattle were previously re- ported (Kinzell gt g1., 1981). 79 Blood Specimens Blood for hematological and immunological measurements was periodically collected from each cow by venipuncture. Complete blood counts were performed using EDTA anticoagulated blood and immunological studies were performed with serum, or cells from heparinized blood. Hematology Hematological values including red blood cell (RBC) count, hemoglobin (Hgb) content, and packed cell volume (PCV) were determined using a Coulter Counter model S. White blood cell (WBC) differential analyses were performed using Wright's stained blood smears. Erythrocyte sedimentation rates (ESR) were determined using the modified Westergreen method (Dawson, 1960) and a 120 minute end point reading. Leucocyte Preparations Lymphocytes and neutrophils were separated from heparinized blood by Ficoll/Hypaque (F/H) gradient centrifugation (Boyum, 1966). A portion of heparinized blood was incubated with car- bonyl iron fillings to remove phagocytic cells (phagocyte-de- pleted leucocytes or PDL) prior to F/H centrifugation. Lym- phocytes and neutrOphils recovered from the F/H gradient were washed with Ca2+ and Mg2+ -free Hanks balanced salt solution by centrifugation at 200 x g for 7 min. Remaining erythrocytes were lysed by hypotonic shock using freshly prepared 0.85% NH4C1. Following erythrocyte lysis, leucocytes were washed at 350 x g for three minutes with Ca2+ and Mg2+ -free Hanks bal- anced salt solution followed by two additional washes at 80 200 x g for seven minutes in the same solution. For immuno- logic studies, the cells were resuspended in Hanks balanced salt solution and evaluated for viability using trypan blue dye exclusion. Cell Viabilities generally exceeded 95%. Lymphocyte cell suSpensionS were adjusted to the desired number of cells per ml using a hemocytometer. Membrane Receptor Assays Erythrocyte (E) binding (Bach, 1973; Grewal gt gt., 1976; Higgins and Stack, 1977) was determined by a rosette assay with sheep red blood cells (SRBCS) treated with VCN (Weiner gt gt., 1973; Reeves and Renshaw, 1978). Two m1 of 5% SRBC solution were mixed with 0.4 ml VCN (50 units/ml) for 60 min at 370C. The VCN treated-SRBC (En) were washed 3 x with Hanks balanced salt solution and adjusted to a 5% solution. For the assay, 0.1 m1 of 2 x 106 PDL were added to 0.1 m1 of 0.5% En in a 10 x 75 mm glass tube and centrifuged at 30 x g for 8 min to form a compact pellet. The cell suspension was incubated at Z-SOC for approximately 16 hr. To enumerate En rosette forming cells (En-RFC), the RBC-PDL pellet was gently resuSpended and stained with 0.1% Gentian violet. Lymphocytes with three or more attached En were identified as En-RFC. The percentage of such cell was determined in triplicate by analysis of 200 cells per tube. Peripheral blood lymphocytes with surface immunoglobulin were enumerated using a polySpecific fluorescein-labeled (FITC) rabbit antibovine Ig reagent (Musc0plat gt gt., 1974; Reeves and Renshaw, 1978). PDL recovered from F/H gradients were 81 initially washed with phosphate-buffered saline (PBS) con- taining 0.1% sodium azide and then incubated with the fluore- scent reagent for 35 min at 2-4OC. Following staining, PDL were washed 3 x in PBS and examined microscopically in a wet preparation using a Zeiss ultraviolet microsc0pe. Two to three hundred cells were counted and the percentage of cells with surface bound fluorescence recorded. Lymphocyte Function Tests Mitogen-induced blastogenesis of lymphocytes was per- formed in triplicate using a microculture procedure (Douglas, 1971). Two hundred thousand lymphocytes were cultured in RPMI-1640 media supplemented with 5% fetal calf serum and antibiotics, 100 units/ml penicillin and 100 ug/ml Streptomy- cin, in a microtest tray. Two mitogens, leucoagglutinin and concanavalin A,were used to stimulate the lymphocytes. Blastogenic responses to LA were studied over an BOO-fold dose range, while Con A mitogenesis was studied over a 100- fold dose range. The lymphocyte cultures were incubated at 37°C in a 5% co humidified atmosphere for 72 hr. At 48 hrs: 2 the cultures were pulsed with 25 ul tritiated thymidine( [3H] Tdr) containing 0.25 uCi (Specific activity of 6.7 Ci/mM). Cultures were harvested using a multiple analysis sample harvester (MASH-II) and the isotOpe incorporation evaluated by scintill- ation counting. The isotOpe incorporation index (III), the ratio of the radioactivity in the cells stimulated with mitogen to the radioactivity of cells cultured in [3H] Tdr containing media was determined. In addition, net counts per minute (NCPM) 82 i.e.,the difference between the radioactivity in cells stim- ulated with mitogen versus control cultures, was determined in cultures reSponding to Optimal concentrations of mitOgens. Serum immunologlobulin concentrations (IgG, IgM, IgA) were determined by radial immunodiffusion (Mancini, 1965; Butler, 1971) using commercial reagents. In addition, during the last month of penta exposure the antibody response was evaluated following intravenous injection of 2 ml of a 10% V/V solution of SRBCs in saline. Serum was collected prior to immunization as well as on days 4, 7, 10, 14, 21, 28 and 35 post-immunization. SRBC agglutinin titers were determined using heat inactivated (560C for 30 min ) serum. The highest serum dilution exhibiting macrOSCOpic agglutination was re- corded as the titer. Delayed type hypersensitivity (DTH) was evaluated i2 ztzg by skin testing during the last 30-35 days of the dosing period. Four penta-exposed cattle and three control cattle were injected. with Bacillus Calmette-Guérin (BCG) emulsified in Freunds. complete adjuvant which was given subcutaneously in the Sternal region (brisket). The other control cow served as a DTH testing control and was not given BCG. All 8 cattle were subsequently challenged intradermally 30 days later with purified protein derivative (PPD) and the area of the edematous skin reaction recorded 24 and 48 hours after challenge. Neutrophil Function Tests NeutrOphil phagocytosis was studied by $2 vitro challenge with 1.091 mm polystyrene latex particles. Neutrophils, 83 separated from blood on F/H gradients and purified by washings and hypotonic shock, were suspended to approximately 4 x 107 cells/ml. Two hundred ul of polymorphonuclear leucocytes (PMNS) were mixed with serum Opsonized latex particles and in- cubated at 37°C for 20 min. The phagocytic reaction was ter- minated by addition of 0.1 ml glutaraldehyde solution (3%). The PMN suspension was subsequently stained with Turk's solution and examined micrOSCOpically. The percentage of PMNS (200 total) phagocytizing at least three particles was deter- mined. Serum for particle opsonization was obtained from penta-treated and control cattle for comparison. NeutrOphil chemiluminescence studies (Allen gt gt.,l972; Rosen and Klebanoff, 1976; Andersen gt gl., 1977) was as- sessed on the day of necrOpsy. In the assay, 2 m1 of PMNS (l x 106 per ml) were added under red light to dark adapted scintillation vials containing 0.5 ml of Opsonized zymosan A (S. cerevisia). PMN luminescence was measured at 5 min inter- vals over a 1 hr period in a Searle Isocap 300 liquid scintill- ation counter adjusted to detect visible light (non-coincident mode). Histology For histologic evaluation the lymphoid tissues were fixed with formalin and stained with hematoxylin and eosin. Special stains used were periodic acid-Schiff (PAS) and Congo Red. A gross and histOpathOlogic examination was performed on'each animal and was reported pre~ viously (Kinzell gt g1., 1981). The histological 84 features of thymus, Spleen and lymph node tissue are presented herein. Statistical Analysis Statistical analysis was done using SAS (Statistical Analysis System) at Wayne State University, Detroit, Michigan. The data were analyzed using analysis of variance (F distri- bution) and multiple analysis of variances by the likelihood- ratio test (Gill, 1978). Using this approach,individual points in time were never analyzed individually. Due to the large variation involved in these types of biological tests statis- tical analyses were designed to examine the change in the difference between paired (penta-treated and control) cattle over time (trend). For some experiments, the Student's t-test for either paired or unpaired data was used. 85 RESULTS Hematology Venous blood, obtained prior to and during the feeding of penta, was evaluated for several hematologic parameters. The erythrocyte counts, hemoglobin concentration, and packed cell volume for control and penta-treated cattle are pre- sented in Figure 1.1. The increases seen in these parameters at the end of the trial are most likely due to hemoconcentra- tion caused by water deprivation prior to euthanasia. Ery- throcyte sedimentation rate, which is an indicator of in- flammation, ranged from 0 to 0.5 mm/hr throughout the study regardless of penta treatment or occurrence of mastitis. The results Show no significant changes in the magnitude of the differences between control and penta-treated cattle over time with respect to these erythrocyte parameters. The WBC counts for control and penta-treated cattle are presented in Figure 1.2. Again, there were no significant changes in the magnitude of the difference between the penta- fed and control cattle over time. One cow in the penta-treated group became clinically septic with mastitis near the end of the 0.2 mg/kg treatment period. On day 63, the WBC count in- creased to 24,000 ul. No association between dose and mastitis was made because this particular cow had mastitis before dosing commenced and mastitis occurred in control animals. A second subclinical infectious episode of unknown etiology also occurred in this and another treated cow during the 2.0 mg/kg treatment period. In the first episode, the cow Figure 1.1. Red Blood Cell Number (RBC x 106/ul) Hemoglobin Concentration (HGB g/dl) and Packed Red Blood Cell Volume (PCV%) in Control and Penta-treated Cattle. Each Graphed Point Represents the Mean and Standard Error of all Data Collected Between the Times.Indicated. Normal Limits are Those Reported by Duncan and Prasse (1977). 86 I l {—0.2 mg Penta/kg/day——-><-2.0 mg Penta/kg/da',I N IO 3 9 __ H Control 3 8 ._ O—-O Treated 9 . . x 7 ,— —— Normal lelTS 8 a: 6 5 ‘ O 2| 42 63 68 84 2| DAYS FED PENTA l J 42 56-65 Figure 1.2. Number of White Blood Cells (WBC x 103/ul) in Control and Penta-treated Cattle. Each Graphed Point Represents the Mean and Stand- ard Error of all Data Collected Between the Times Indicated. Normal Limits are Those Reported by Duncan and Prasse (1977). 87 5.2mm own. m> 0 LL 0: I 3 “SJ - 8 IO l——[ 7////, l—‘F l——L WW 0% > .g l——£ ///////////// l-N B, H ////////////////////% 0‘0 i‘ 2 H ( I-m 51$: l——L W 0'” U” E _i W l-co a l——-—L /////////////// 0‘“ H W 1"") |-——[ W 0‘ > l—-—r V W 8 H W E: \ l——f W .3. l—-L W o 0. on W (S Ff W o' l——F W l-—{ W I L l l 1 L0 V M I‘d/EDI x SElAOOHdWA‘l “F DAYS FED PENTA Figure 1.5. Lymphoblastogenic Response to Optimal Concentrations of LA and Con A in Control (C) and Penta-treated (T) Cattle. A) Data Expressed as an Iso- tope Incorporation Index (III = Mean Counts/Minute in Mitogen Stimulated Cultures Divided by the Mean Counts/ Minute in Control Cultures). B) Data Expressed as Net Counts/Minute (NCPM) = Counts/Minute in Mitogen Stimu- lated Cultures Minus the Mean Counts/ Minute in Control Cultures. Each Bar is the Mean + SEM of all Data Collected Between the Times Indicated. 9] Leucoogglu’rinin (0.5 ug /Cu|’rure) ISOTOPE INCORPORATION INDEX (III) 80 6O 4O 20 80 6O 4O 20 I‘- O.2 mg Penta/kg/day —> <— 2.0 mg Penta/kg/day-N l l 1| I _. Concanavalin A (0.4 (lg/culture) 'II' _.r l L I l P- ri I 1 LI CT CT CT CT CT CT CT O 2| 42 62-68 2| 42 56-65 DAYS FED PENTA NET COUNTS/ MINUTE ( NCPM)x I03 92 B Leucaagglutinin (0.5 ug / culture) IO—OZmQ Pa nto/kq/day —§ H—ZD mg Penta/kg/day-Dl .1 PI 0) O I A O '40 ' Concanavalin A (0.4 ug/culture) 1 1 IOO-l i1 1 80- 60 CT CT CT CT CT CT CT 0 2| 42 62-68 2| 42 56-65 DAYS FED PENTA 93 Table 1.1 I2_Vivo Cell-Mediated Immunity in Cattle Fed Technical Pentachlorophenol Subchronicallya Experimental 24 hour 48 hour Group Response ReSponse Control(3)b 42.9 i 17.5 54.8 i 28.8 Treated (4) 46.6 + 14.2 48.4 i 8.6 Values represent mean area (cm2) i SE of skin reaction at 24 and 48 hours following intradermal injection of PPD in BCG sensitized cattle. a = 0.2 mg Penta fed/kg body weight/day for 60 days followed by 2.0 mg Penta fed/kg body weight/day for 75 days. b = Number of cattle tested. 94 to BCG did not react to the PPD injection. The area of indur- ation was comparable between control and penta-treated cattle. A histOpathologic study of the PPD injection Site revealed a marked infiltration of mononuclear cells in all animals. Humoral immunity in control and penta-treated animals was tested by quantitating the serum immunoglobulins (IgG, IgM, IgA) and by evaluating antibody response following intravenous immunization with SRBC (Figure 1.6 and Table 1.2). The magnitude of the difference in serum IgG, IgM, and IgA, concen- trations was similar in the two animal groups over time (Figure 1.6). Additionally, the mean peak anti—SRBC titer and the kinetics of the hemagglutinating antibody response were Similar in both control and penta-fed cattle (Table 1.2). Neutrophil Function Tests Neutrophils isolated from peripheral blood were tested for their phagocytic and chemiluminescent responses following t2_ytttg challenge with Opsonized latex particles or Opsonized zymosan. The percentage of phagocytizing neutrOphils from control and penta-treated cattle are shown in Figure 1.7. Neutrophils from control cattle were incubated with latex par- ticles Opsonized with control sera, while neutrophils from penta- treated animals were challenged with latex particles Opsonized in sera from penta-treated cattle. Additionally, neutrOphils from penta-treated cattle were challenged with latex particles Opsonized with control sera. The difference in the phagocytic reSponse of neutrophils from control and penta-treated cattle was comparable over the duration of the dosing regime regardless Figure 1.6. Serum Immunoglobulin Concentrations of IgG, IgM and IgA in Control and Penta-treated Cattle. Each Graphed Point Represents the Mean + SEM of all Data Collected Between—the Times Indicated. 95 l:— O.2 mg Penta/kg /day—><- 2.0 mg Penta/kg/daybl SOOO 4oooL-i-x-L_i\ I,’L\‘L‘-l 30001 \6/ 0—0 Control O—-O Treated I\) O O O I MILLIGRAMS IMMUNOGLOBULIN /dI 7O *— 60 — I IgA 50%—$\~Q—— /Q‘- \ ( J I I l J I I O 2| 42 63 70-84 2| 42 56-65 DAYS FED PENTA 96 Table 1.2 Antibody Formation in Response to Injected Shee Red Blood Cellsa (SRBC) in Holstein Cattle Subchronically Exposed to Commercial PentachlorOphenol. SRBC Control Treated Pre-immunization 2.75 i 0.48c 2.25 .t 0.48 Day 4, post-immunization 4.00 i 0.70 4.25 i 0.63 Day 7, post-immunization 4.25 i 0.86 4.50 i 0.50 Day 10, post-immunization 4.00 i 0.70 4.50 i 0.29 Day 14, post-immunization 3.67 i 0.90 4.67 i 0.34 Day 35, post-immunization 3.50 i 0.64 3.50 i 0.50 2 ml of a 10% v/v SRBC in saline injected intravenously. 0.2 mg Penta fed/kg body weight/day for 60 days followed by 2.0 mg Penta fed/kg body weight for 75 days. c = Values represent mean titer (log ) :SE for 4 cattle except day 14 figures which represents 3 cattle. Figure 1.7. NeutrOphil (PMN) Function Expressed as a Percent of Cells Phagocytizing Opsonized It Vitro. Latex Particles C = NeutrOphils ized with Serum T = NeutrOphils ized with Serum 0 = NeutrOphils ized with Serum from from from from from from Control Control Treated Treated Treated Control Each Bar Represents the Mean _ Data Collected Between the Times Indicated. Cattle Opson- Cattle. Cattle Opson- Cattle. Cattle Opson- Cattle. + SEM of all %%%%%%%%%%%%%%%%ZEE U /0 CTO CT N———— 0.2 mg Penta/kg/day ——> 42.0 mg Penta/kg/day-N O ’— o O I.— o 7////////////////////// ////////// //O l.— O //////////////// /////// o [.— _ o 7//////////////////////////////////////////////// /S 0 ¥ J I I 7 O O O O O O 9 <1- rO (\l — % SISOIAOOOVHd ONIIISIHXB SWIHcIOtIlflEIN 42 56-65 2| 6?) 68-84 42 2| DAYS FED PENTA 98 of the source of opsonizing serum. The results in Figure 1.8 depict the chemiluminescent response of neutrophils from cattle on the day of necrOpsy. NeutrOphils from the penta- treated cattle exhibited a chemiluminescent reSponse Similar to neutrOphils from control cattle, both in peak height and in the reSponse kinetics. Histology Gross and histOpathological evaluation of the Spleen, thymus, and lymph nodes from control and penta-treated cattle revealed tissues which were normal in Size, appearance, and histological architecture. Spleen weights of treated and non-treated cattle had identical ranges (900-1120 9) and means (1000 g). The thymus from penta-treated cattle had some peripheral lobules which were replaced by variable amounts of adipose tissue; however, similar findings were observed in control cattle. During the course of the experiment some cattle in both the treatment and control groups periodically experienced an upper respiratory infection (URI) of unknown but consistent etiology, including elevated body temperature, duration of 1-2 days, and mild anorexia. The episodes of URI did not appear dose-related and reSponded to drug therapy. Figure 1.8. £2 Vitro Light Emitting Profiles (Chem- iluminescence) of Neutrophils (PMN) Phagocytizing Opsonized Zymosan A. This Assay Was Performed on the Last Day of Penta Exposure Using Cells from Both Control and Penta-treated Cattle. Each Point Represents the Mean : SEM. 99 GOF' 0.. 5 0—0 Control m ChdonOmd Q E I a. U‘“D_ if \T\ 5 I Z? / Ti\ 8 I T LIJ I \ Z 2 30"- ? T\ .3 V ‘4 if T 2 . 20 L l J L I | | 0 IO 20 3O 4O 50 60 TIME (minutes) 100 DISCUSSION AND CONCLUSIONS Immunodulation is the contemporary term ascribed to variation (either enhancement or suppression of the immune system) resulting from genetic, physiologic, or environ- mental influences. In this study, several immunologic paramaters were evaluated in lactating dairy cattle fed 0.2 mg technical pentachlorOphenol per kg body wt/day for 75-84 days, followed by 2.0 mg/kg body wt/day for 56-60 days. The major physiologic compartment used in these investigations was blood which contained a total (acid hydrolyzed) PCP con- centration at steady state of 2.9 ppm and 12.5 ppm during the 0.2 mg exposure period and 2.0 mg exposure period, res- pectively (Kinzell, 1981). Analysis of the bovine immune response included both quantitative and functional studies of lymphocytes and neutrOphils, as well as Serum-borne immuno- reactive substances. No penta-induced cytotoxicity, alteration of lymphocyte surface markers, or changes in DNA or protein snythesis in lymphocytes were observed. Although the immune competence values in penta—fed cattle were within normal limits, one of the treated cattle experienced clinical mastitis during the 0.2 mg/kg body wt/day exposure period. Laboratory studies during the infection demonstrated increases in several tg_ytyg host defense parameters, including WBCS, percentage of PMNs,and serum IgG. Also, two treated cattle with an upper respiratory infection of unknown etiology reSponded lOl rapidly to antibiotic chemotherapy. Subsequent infections were not evident, further supporting the maintenance of immuno- logic competency in these animals. Interestingly, the mitogenic response of lymphocytes at the time of infection was reduced, but not to the extent to be considered abnormal. Several re- ports have described a reduced blastogenic reSponse in experi- mental animals and in humans with concurrent infections (Oppenheim gt gt., 1975). Presumably, serum factors manifested during infectious events are contributory to the reduced lym- phoblastogenic response, since incubation of normal lympho- cytes with serum from infected animals can markedly reduce the lymphoblastogenic reSponse. In these cattle, the LA and Con A induced lymphocyte blastogenic reSponse increased when the infection cleared following antibiotic therapy. Histologic examination of lymphoid tissues revealed no lesions; however, it cannot be conclusively stated that the functional Status of these Specific lymphoid tissues was intact. Considerable evidence indicates that the chloro- phenols (i.e. pentachlorOphenol and tetrachlorOphenol) do not accumulate in bovine tissues whereas the polychlorinated dioxins and furans steadily accumulate primarily in the liver and body fat during continuous exposure (Firestone gt gt., 1979). In these cattle, PCP levels in the thymus and Spleen at terminus average 2.8 and 3.4 ppm, respectively (Kinzell, 1981). Analysis of the total dioxin (sum of octa-, hepta-, and hexa-dioxin) concentration in the livers of the four penta- fed cattle was 145 ppbn. Dioxin concentrations in various lymphoid tissues were not determined. 102 Whereas these data shed some light on the disposition of these chemicals, they are not adequate for establishing the level of eXposure of different immunologic reactive cells in these tissues. Such studies (see Chapter 2) provide insight into the time required for any one of these chemicals to reach a toxic threshhold concentration in the non-blood immune compartments. While these studies were in progress, McConnell gt gt. (1980) reported that in 10-14 month-old Holstein heifers fed for 160 days with approximately 10 times more penta than cattle in this experimental regime received,exhibited some hematolo- gic changes. A limited immunologic evaluation in these cattle also revealed a dose-related (attributed to technical pentach- lorOphenol) enhancement of mitogen-induced lymphoblastogenesis and a decrease in absolute and relative thymus weightl. How- ever, no additional alterations in immune competence as mea- sured by the SRBC hemagglutinin response, serum complement (C3) level, mixed lymphocyte culture response, immunoglobulin levels and presence of ANA were noted. It is interesting to note that at the lowest level of technical pentachlorOphenol used in the McConnell gt gt. (1980) study (a mixture of technical and analytical pentachlorOphenol in a ratio of 10:90), approximately the same total amount of technical pentachlorOphenol was administered per animal (84 grams) as in this study (68 grams). Although an additional 1Comparatively, it was not possible to record accurate thymus weights because of the difficulty in excising the whole gland from older cattle. 103 13.5 to 18 mg/kg body weight of analytical pentachlorophenol was also contained in the McConnell dosing regime per day, no immunomodulation or effect on hematology was seen above that which was observed in cattle which were dosed with 100% analytical pentachlorophenol alone. Immunotoxicologic studies are rapidly emerging as an approach in which variOus chemicals in toxicologic studies are being evaluated. At present, knowledge regarding mechan- isms of host defense, immunoregulatory events,and immune effector mechanisms in humans and rodents far exceeds knowledge of these same processes in other species, including food pro- ducing animals such as cattle. Other immunotoxicological studies involving cattle include studies by Kateley and Bazzell (1978) who investigated cattle subchronically exposed to poly- brominated biphenyls (PBBS), and McConnell gt gt. (1980) who have investigated the pathOphysiologic effects of both analytical and technical pentachlorOphenol in young cattle. In addition, Haggard gt gt. (1980) recently reported that the feeding of excessive iodine induced immunomodulation. However, many more chemicals have been identified in cattle tissues resulting from environmental exposure to pesticides, heavy metals, and solvents (EPA 1977) which need to be evaluated for immuno- toxicity. Unfortunately, establishing immunotoxicity is a difficult process primarily because of the multifaceted nature of the immune system. There currently is a lack of agreement as to the importance, relevance and interpretation of results derived from individual testing procedures. However, when in— dividual in vitro and tg vivo immunoassays are combined and 104 used as a profile, the interpretation of the data generally permits a more thorough evaluation of immune status. The present investigation evaluated several aspects of immunity in lactating cattle exposed subchronically to penta. Since there was no evidence for either a quantitative or functional immunologic defect in cattle with blood levels of 12.5 ppm PCP, it can be concluded that there is no penta-induced immune deficiency in cattle with this body burden. Chapter 2 TECHNICAL AND ANALYTICAL PENTACHLOROPHENOL IN DAIRY CALVES: A CLINICOPATHOLOGICAL AND IMMUNOTOXICOLOGICAL EVALUATION 105 106 INTRODUCTION Pentachlorophenol is a commonly used wood preservative with widespread uses on modern dairy farms (Shull gt_gt., 1981). Because of the high potential for exposure of confined cattle to the chemicals with are present in commercial preparations of pentachlorOphenol, several experimental investigations have been conducted to ascertain possible toxic effects (Firestone gt gt., 1979; McConnell gt_gt., 1980; Kinzell gt_gt., 1981; Hughes gt gt., 1982). Whether or not pentachlorOphenol is toxic to the various bovine host defense mechanisms has been considered in some of this research. McConnell gt gt. (1980) showed that contaminants in technical grade pentachlorOphenol (tPCP) fed at high levels are immunomodulating in yearling cattle. These contaminants include several chlorodibenzo-p- dioxin (CDD) and chlorodibenzofuran (CDF) copgeners and hexa- chlorobenzene (Firestone gt gt., 1972; Firestone gt gt., 1979; Kinzell gt gt., 1981). The well-known 2, 3, 7, 8-tetra CDD which is not present in tPCP preparations (Kinzell gt gt., 1981) is a known immunosuppressant (Gupta gt gt., 1973). Hexa- chlorobenzene has also been shown to be immunosuppressive (Loose gt gt., 1977). In a previous study in which lactating dairy cattle were fed for five months a level of tPCP intended to typify actual farm exposure (Kinzell gt gt., 1981), no apparent compromise of the immune system was observed (Chapter 1). To date, there is no information on the immunotoxicity of PCP or its contaminants in calves. Thomas gt gt., (1977) reported 107 high mortality rates of calves on a few farms where there appeared to be an overuse of chemically treated wood. For several reasons, calves could be more sensitive to these chemicals than their adult counterparts. First, young animals are generally more sensitive than adults to many toxicants. For example, piglets have been Shown to be more sensitive to PCP than adult swine (Schipper, 1961; Ryan, 1983). Second, the immune system in calves is not entirely develOped at birth (Wells gt gt., 1977; Jensen, 1978; Renshaw gt gt., 1978; Tizard, 1982) which could exacerbate the immunotoxic potential of toxicants such as those in tPCP. Third, the metabolic fate of PCP or the contaminants could be quite diff- erent in monogastric calves versus ruminant adults due to pre- absorption metabolism in the latter. It is well-known that the microflora of the GI tract, particularly the rumen, signifi- cantly affects the fate and toxicity of many xenobiotics (Williams, 1977). The results of immunologic and clinical testing are given in this chapter. Additional data collected from the calves in this study are reported elsewhere (Hughes, 1982). 108 MATERIALS AND METHODS Technical grade pentachlorOphenol (tPCP)6 was generously supplied by the American Wood Preservers Institute and repre- sented a commercial composite from the three major manufactur- ers of pentachlorophenol. Analytical grade pentachlorophenol (aPCP)7 was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin. The chlorophenol and chlorodibenzodioxin (CDD) compo— sition of the tPCP was previously reported (Kinzell gt gt., 1981). The aPCP contained 99% pentachlorophenol and 0.98% tetrachlorOphenol. The CDD concentrations (PPm) were: octa- CDD, 1.2; hepta-CDDS, 1.8; and hexa-CDDS, 0.2 (R. Johnson, Dow Chemical Company, personal communication). Other chemicals and reagents included: Ficoll, and leu- koagglutinin (LA); (Pharmacia Fine Chemicals, Piscataway, NJ), 2+ - and Mg 2+ -free Hanks balanced salt solution (HBSS), Ca HBSS, Sheep red blood cells (SRBS), fetal calf serum, and RPMI-1640 with 25 mM Hepes (Microbiological Associates, Walker- ville, MD), Vibrio cholerae neuraminidase (VCN), penicillin and streptomycin (Gibco,Grand Island, NY), fluroescein labeled (FITC) rabbit and antibovine-immunoglobulin (Ig) reagents and Single radial immunodiffusion agarose plates for serum 19 concentrations (Miles Laboratories Inc., Elkhart, IN), [3H] thymidine ( [IH] TdR), (New England Nuclear Corp., Boston, MS), Hypague (Sterling Organics, New York), and zymosan A (Sigma 6Lot no. MB-528, Vulcan Chemical Co., Birmingham, AL. 7Lot No. 032487 109 Chemical Co., St. Louis, MO). Treatment of Calves Fifteen Holstein-Friesian bull calves, ranging from three to eleven days of age were randomly assigned to the following treatment groups: control, 1.0 mg aPCP/kg body weight, 1.0 mg tPCPAqg, 10.0 mg aPCP/kg, and 10.0 mg tPCP/ kg per day. These levels of exposure were maintained for 43 days with the exception of the first five days when the calves received 2.0 and 20.0 mg PCP/kg. The level of exposure was cut back when two calves died, one each in the aPCP and tPCP groups as a result of acute pentachlorophenol toxicity. Two replacement calves were obtained, however the replacement calf in the aPCP group also succumbed. PentachlorOphenol, either analytical or technical grade, was dissolved in corn oil. The pentachlorOphenol in corn oil was then added to milk so that the calves were fed an amount of milk equivalent to 8% of each animal's body weight every day. The calves were dosed twice daily. Since tPCP is 85% PCP and aPCP is 99% PCP, the dose of PCP was adjusted so that equi- valent amounts of PCP per kg body weight was fed. The calves were weighed every five days and the dose adjusted accordingly. The calves were individually housed in pens in a warm enclosed barn. Water was provided at all times and Starting at day 20, the calves were fed a calf starter ration gg libitum. 110 Sample Specimens Blood for hematologic, clinical chemistry and immuno- logic measurements was collected from each calf during the last week of the trial. Complete blood counts were performed with EDTA-anticoagulated blood, clinical chemistry with serum and immunologic studies with serum or cells obtained from heparinized blood. Urine samples were obtained by subsampling urine collected over a 24-hour period while the calves were in metabolism cages. Hematology and Clinical Chemistry Hematologic values including red blood cell (RBC) count, hemoglobin (HGB) content, and white blood cell (WBC) count were determined with a Coulter counter (model S). Packed cell volumes (PCV) were determined by micro-hematocrit centrifu- gation. White blood cell differential analyses and platelet estimations were derived from Wright-stained blood smears. All clinical chemistry analyses with the exception of sorbitol dehydrogenase, gamma-glutamyl transferase (GGT) and cortisol were performed on a Computerized Sequential Multiple Analyser (SMAC, Technicon). Sorbital dehydrogenase and GGT were analysed using a Gemsaec centrifugal analyzer (Electro- Nucleonics, Inc). Cortisol levels were determined by radio- . 8 immunoassay . Leucocyte Preparations Lymphocytes and neutrOphils were separated from hepar- inized blood on Ficoll/Hypague (F/H) cushions (Boyum, 1968), 8Michigan State University Veterinary Diagnostic Lab 111 and processed according to the method described previously (see Methods, Chapter 1) with one change. Remaining erythro- cytes were removed by hypotonic shock utilizing sterile double distilled water instead of 0.85% NH4C1. Membrane Receptor Assays Three membrane receptors were assayed on lymphocytes obtained from the circulating blood and from spleen, thymus and lymph node tissue. Sheep erythrocyte receptors or erythro- cyte (B) binding was determined by a rosette assay using sheep red blood cells (SRBC) treated with (VCN) according to the procedure described previously (Chapter 1). Complement (C) bearing lymphocytes were detected by rosetting procedures with antigen-antibody-C complexes. The complexes consisted of human type 0 erythrocytes sensitized with a subagglutinating dilution of rabbit anti-human RBC antibody and a subhemolytic concentration of mouse complement (EAC) (Niblack and Gengozian, 1976) surrounding lymphocytes with complement receptors. Surface immunoglobulins (Ig) were enumerated with a polySpecific fluorescein-labeled (FITC) rabbit antibovine Ig reagent according to the procedure described previously (Chapter 1). tympgogyte and Neutrophil Function Tests Mitogen-induced blastogenesis of lymphocytes as measured by [3H] thymidine uptake, was performed in triplicate by a microculture procedure (Douglas, 1971). One mitogen, leukoagg- lutinin (LAh*was used to stimulate the lymphocytes at one optimal 112 concentration of LA (0.5 ug) and two suboptimal LA concen- trations. Otherwise, the procedure was as described pre- viously (Chapter 1). Serum IgG, IgM, and IgA were determined by radial immunodiffusion (Mancini gt gt.,l965; Butler, 1971) with commercial reagents. Each Ig was quantified three different times for each calf over the last ten days of the trial. During the last 30 days of PCP exposure, the primary and anamestic (secondary) reSponse to human red blood cells (HRBC) was evaluated,Three ml of 10% (V/V) HRBC in saline was injected IV followed by 1.5 ml of 10% (V/V) HRBC injected 15 days later. Serum was collected before inoculation- as well as on day 4, 9, l4, 19, 24, and 29 after the first inoculation.. HRBC agglutinin titers were determined with heat-inactivated (56°C for 30 min) serum. The highest serum dilution exhibit- ing macrOSCOpic agglutination was recorded as the titer. NeutrOphil chemiluminescence studies (Allen gt gt., 1972); Rosen and Klebanoff, 1976; Andersen gt gt., 1977) were per— formed on the day of necrOpsy. In the assay, 2 ml of poly- morphonuclear neutrOphils (PMN) containing 1 x 106 cells per ml were added under red light to dark-adapted scintill- ation vials containing 0.5 ml bovine serum Opsonized zymosan A (Saccharomypes cerevisiae). PMN luminescence was measured over a one hour period in a Searle Isocap 300 liquid scintillation counter adjusted to detect visible light (noncoincident mode). Histology At necropsy, liver, kidney, Spleen, thymus and brain 113 were removed intact, trimmed of excess fat and connective tissue, and weighed. Bone marrow and a representative mesen- teric lymph node were also taken for histopathologic examin- ation. All tissues were fixed in 10% buffered formalin. Paraffin embedded sections of these tissues were stained with hematoxylin and eosin before histologic examination. A gross and histoPathologic examination were performed on each animal. These results along with neonatal toxicity and thyro- toxicity data, as well as PCP residue analysis are reported by Hughes, (1982). Statistical Analysis Data were analyzed by regression analysis and analysis of variances with Genstat V system at Michigan State University, East Lansing, Michigan. For some analyses, Dunnett's test was used (Gill, 1978). 114 RESULTS Hematolggy and Clinical Chemistry Venous blood and urine obtained during the final week of PCP exposure were used for various hematologic and clinical chemistry measurements (Tables 2.1, 2.2 and 2.3). All of the hematologic values were either within the normal range of values for calves cited by Schalm gt gt.,(l975) or did not differ significantly from controls. Pentachlorophenol treatment decreased serum concentra- tions of total protein and albumin (Table 2.2) and increased the activity of gamma-glutamyl transferase (GGT)(Table 2.3). Compared to control values for all of these parameters, the greatest differences were found in the calves fed 10 mg tPCP/ kg. No other blood or urine parameter differed significantly from controls. Whereas the decreases in both Serum total protein and albumin were dose-dependent (P<.10) , only the pro- tein values were below the normal range for bovines as reported by Duncan and Prasse (1977). Although there appeared to be a trend of decreasing globulin concentrations with increasing PCP exposure, the differences were not statistically signifi- cant. The increased activity of serum GGT was dose-dependent (P‘<.10) and reached a level in the 10 mg/kg tPCP group that exceeded the normal range in cattle by 10 times. (Michigan State University Clinical Laboratory, personal communication). Membrane Receptors The percent and number of surface receptors present on 115 .A.6va powwouocH >HuanHm no Hp609 ox\mE o.Hm .p< .O< .C< .p< 6 Hm .umo OmumHoumHm m.o v.mm m.H m.mm m.H m.Hv o.o ~.ov m.H h.ov Hp\m Homuzv .ocoo b: HmHoowsmuoo cam: H.o o.~H e.o o.HH H.o o.HH m.o o.HH m.o ~.~H oe Amaze he umHoomseuoo com: m.c m.Hm ~.N m.om o.H O.m~ ~.H m.m~ m.o m.om Hm A>USV.HO> umHsomsauoo cam: >.~ m.m~ m.m o.m~ m.~ v.o~ v.H o.m~ m.H ¢.m~ w A>Umv OESHO> HHmo pmxomm o.o m.m o.~ o.o o.H m.m w.o o.m m.o H.0H Hp\q Hbzv choHUOEom Hm.c no.» vv.H mm.w on.o ma.o em.o mm.h cm.o mm.m HS\oOHx: Hummv mHHwo COOHQ Com o o c am we HH HH mm on HS\: mHHLOOmmm o o o mm mm NoH eHN mm ooH Hs\: mHHneoeHmom we me mv om , mmH wow mMH mow vmm moo HS\c . omwumoocoz NHm mHm OH OH o o HH om mH mm HS\c mHHsaouuomc poucmecmmIcoz moo emote com mmH.m mOH woo.v com mmmeH mvHH omm.v Ho\c OAZSAV mHHcoouusw: ummHoscocau0E>Hom MNVH mom.e mH NHo.m meH Ho~.m Hem emv.v OHNH mmo.v Ho\c mmu>oonoE>H mmoH omm.m omm omm.o moeH mmm.0H omm mma.o Omm mmm.m H:\c Aumzv mHHoo OOOHQ manz mm + x mm + x mm M x mm H x mm H x mic: unmsmuommmz .cowa QOHm “Hmecé con .2009 30m .>Hmc< 30m Houucow mHococaouoHnomucoa compo HmoHccowe no HCOHuaHmc< pom mo>HmU E0um poon mo coHumcHmem onoHoume: mo muHommm .H.N memB 116 mm Or-IIO '0‘ M V‘HVDI‘NVM 01H cm a HWOOOOONCV N I‘D 0 am 00“ a a @HVNHOflmHVP-INONH CHOI‘O‘NQOO‘N 01—4 0 mm H m .cooe roe: Hmochooe .Eouom mnu CH mcoHuco Ocm mcoHcm pwusmmmecs may cwm3uon OOSOHOMMHC on» mHo .AHo.oV mv Amoév E .m>m© me How .mmOC :oHc n unp\u3 apon ox\qE ob oi 940 -m G OOQM own: a c 0 V omNmr-i owcoaum -o M 00 N CONN O O NOOO ONCI-l omMNr-l LOO - HHmMNHNDHfl'r-INHH N mm C mm H m .sHmse :on Ho HmoHu>Hmc< pom mw>HmU Scum Esuom mo N.c m.o H.o o.H o.H o.m h.o b.@ 0.0 mm o.H am m.c MOH m.H vOH H.o m.v N.o m.v o.H oeH ¢.o NVH m.o m.o v.0 m.w N.o N.OH .o v.oH H.o N.N H.o H.~ H.o H.m o.o m.m N.o m.m H.o m.m Hm oHH MH hOH $.o h.h m.N o.m mm + x mm + x .5009 3mH .mHmc< 3mH 'N OCH 0 0 o Hm OHHVH 0m 0" 'CO‘F‘I ' o co CNN H 0N O‘C‘OMNHWHQ‘r—INHO v-INOOO OHOO I!) O mm H x nHouuch mmeHmc< xuumHEoco HMOHSHHU mo muHsmmm Houucoo Eonm ucmumwpr >HucmoHuwcmHm u Houucoo Eouu uSOHOMMHp >HucmonHcmHmm .AHo.ov.mv omcoamwu anon ucmonHcmHm v .AOH.ov.mv wmcoamou moon ucmoHuHcmHmo oH .omOU 30H Ho\os 0:0..— o\zs a\oms H\gms H\UuE Hoxoe Ho\oe Ho\o Hoxo Ho\o Ho\oe Ho\oe moHca smo\us soon ox\os o.H . u c m n M pHo< OHHD mama muaHouuome mponHo conumu mpHuoHno ESHmmmuom EsHpom mononmmonm EsHonu SHHSboHO OSHESQH< ckuoum Hobos omoosHu U Hzomv camouuwc mono poon mucweousmmmz cHococaouoHcomucmm mpmuo .N.N OHQMB 117 AHUNOE 0CHSH000H0 Enu0mv .vaflEHOHOU #02 H 02 ACHE\HE 3on 0SHHSO HHC\OE 0cHSHum0uo 0SHHSO u 0H5500h0 .HHo.ov.e. Hoeoeoo some ueouooeHo sHucmoHuHcono .HHO.OV.OO 0mcomm0u 0000 unmoHuHcmHmo .m u c .maoouo H0zuo HHm How .N u an .mxmp mv 00m .0mOO 20H: u 90p\u3 apon mx\OE OH .0mOC 30H u >MO\03 upon mx\OE O.H0 0SHE\©0H00H0 bmH SNH om «OH Om va NH mmH mH HvH oopo HEHHn vmv.um0H0 0chHum0HO bmm OOHH namm OO~H OOH HOvH mmH mmoH mMH OoOH mu: v~\OE 0cHCHum0H0 0SHHD meO0.0 ~HO.H bNOOO mHO.H HO0.0 MHO.H NOO. ~HO.H mO0.0 MHO.H 0co: >uH>mum 0HHH00Qm 0cHHD nmm moo anH mOm mm mom mmm Ohm mm one an: vm\OE :H0uouo 0SHHO H NH o Hm pH He oH mm m an Ho\oe olooosHoHee M0.0 H.O 0.0 H.O m0.0 H.O m0.0 H.O M0.0 ~.O Hp\oe CHQSHHHHQ H0009 Hmm vow Hm He 9 OOH Ox oOH mH no H\D quv 0mmcHx 0CHum0uU mH mHH he eoH mm HHN mm emm mo omm H\: Heads mmmumnemoce mcHHoxH< own mmVH mom boo om mom NH mom on mHm H\: Haas mmmemoouosnoo oumuomH m mm m mm m HH v oH H em H\O H9HHmc¢ O0m m0>Hmu Eoum 0cHHO pom Ecuwm mo mHmSHmc< >uumHE0no HaoHcHHO mo muHsm0m .m.m 0Hbm9 118 lymphocytes obtained from the blood, Spleen, thymus and lymph node tissue of calves in the various treatment groups are presented in Tables 2.4 and 2.5. The numbers of En rosette- forming cells, EAC complement-bearing cells and surface Ig- bearing lymphocytes were comparable in all groups for all tissues. tymphocyte and Neutrophil Function Tests One tg ytyg and three tg ytttg immunoassays were con- ducted to evaluate lymphocyte and neutrOphil functions (Figures 2.1, 2.2 and 2.3, and Table 2.6). The blastogenic reSponse of lymphocytes, which is an tg ytttg indicator of cell- mediated immunity, was measured in three lymphoid tissues (blood, spleen, and lymph node) from control and PCP-treated calves. The response was determined at three concentrations with the T cell dependent mitogen leukoagglutinin. IsotOpe incorporation ( [3H] TdR) by cultured lymphocytes indicated that DNA synthesis in both aPCP and tPCP-treated calves was neither impaired or enhanced relative to controls (Table 2.6). Also, no differences were detected at the two submitogenic levels used (data not shown). Because insufficient thymus tissue was recovered from the calves in the 10 mg/kg tPCP group due to atrOphy,no statistical analyses could be applied to the lym- phoblastogenesis data obtained for the thymus. However, it appears that the response was drastically decreased by in- creasing PCP dose. Humoral immunity was evaluated by quantitating serum levels of immunoglobulins G, M, and A and by evaluating the 119 Table 2.4. Peripheral Blood Lymphocyte Surface Markers in Calves Fed Analytical or Technical Grade PentachlorOphenola Experimental Enb Ly, EACC sIgd Group NumberE %I Number % Number % Control 496 16.5 266 5.9 657 17.5 :261 i 8.0 1123 11.1 :162 i 3.9 Low Analytical 444 11.6 327 7.8 962 21.3 i117 i 3.3 1114 12.9 1214 i 3.0 Low Technical 492 8.8 217 3.7 1359 23.0 1178 i 1.1 i109 11.0 :666 i 6.4 High Analyticalg 324 10.7 116 3.8 652 21.6 :285 i 9.4 i 26 10.8 :263 i 8.6 High Technical 650 12.6 277 4.1 517 11.2 i323 i 2.5 i233 +3.0 1152 i 0.4 a1.0 mg/kg body wt/day = low dose, 10 mg/kg body wt/day = high dose for 43 days. bEn-rosettes which form with some lymphocytes and neuraminidase-treated SRBCS. cEAC-rosettes which can be made to form with complement- bearing cells. dsIg-lymphocytes which have surface immunoglobulins. eNumber of lymphocytes having each surface marker/ul whole blood :SE. fPercent of lymphocytes having each surface marker :SE. 9n = 2; for all other groups, n = 3. No Significant differences found (Pj>0.05). 120 Table 2.5. Lymphocyte Surface Markers in Lymphoid Tissues Obtained from Calves Fed Analytical or Technical Grade PentachlorOphenola Experimental Lygph Node Sptgen Thymus Group En EACC SIg En EAC sIg En EAC sIg Control 28.4 27.2 17.2 39.0 22.3 17.5 30.3 -0 <1.2 i 3.4 i 2.0 i 2.4 :10.0 i 8.7 i 5.6 i 4.9 Low 37.6 25.8 34.8 31.5 19.8 22.1 36.8 0 <1.0 Analytical i12-7 i 3.1 i 3.0 112.3 1 6.2 1_5.l i 7.8 Low 36.5 18.2 24.6 39.3 13.9 32.2 37.3 0 <1.0 Technical :12.3 i 4.3 i 5.8 :11.4 i 2-4 i 5.5 i 5.5 High 6 44.6 14.6 30.8 23.7 20.9 27.8 31.6 0 <0 5 Analytical :12.3 i 7.4 +10.4 t 0.3 i 7.9 i 5.8 i 7.6 High 24.5 10 4 15.8f 23.3 13.8 22.2 27.6 1.4 <0.5 Technical : 1.6 i 2 0 i 3.4 i 3.3 i 3.4 :12.1 1.2 a1.0 mg/kg body wt/day dose, for 43 days. low dose, 10 mg/kg body wt/day high bPercent of viable lymphocytes which form rosettes with neuraminidase-treated SRBCS :SE. CPercent of viable lymphocytes which can be made to form erythrocyte amboceptor complement (EAC) rosettes with human RBCS :SE. dPercent of lymphocytes with surface immunoglobulins (sIg) :SE. e f11 = 1. No significant differences found (P>>0.05). n = 2; for all other groups, n = 3. Figure 2.1. Net Change in Serum Concentrations of IgG, IgM, and IgA Over a Ten Day Period in Calves Fed Analytical or Technical Grade Pentachlorophenol. 12] .335 a: 30.5.00... to... _ 3x38 0 S .8222... to... _ .338 s 30.5.00... 30.. _ 30:9... 3 .no_~>_oc< 30.. _ .9500 _ lgG IgA +600- +400- +200- .EESOOEOCSEE. «£052.: Figure 2.2. Primary and Secondary Antibody ReSponse to Injected Human Red Blood Cells in Calves Fed Analytical or Technical Grade Pentachlorophenol. Mean Titer (logz) 15- 13‘ .1. .5 I (D l N I q 5- 3- /o l // ./4 -M fo/A\ / Jé—-— // Primary ———)I£ Secondary ———-) Response Response ("l—l" [TUITIIIIIfilil1rruul1uu11 5 1 O 15 2O 25 30 Days Past Sensitization o-—-—o Control 0— — — 0 Low dose analytical 1 mg/kg body wt/day A—- —A Low dose technical 1 mg/kg body wt/day x——-x High dose analytical 10 mQ/kg body wt/day a— — —0 High dose technical 10 mg/kg body wt/day Figure 2.3. tg Vitro Light-emission Profiles (Chemi- luminescence) of Neutrophils (PMN) Phagocytizing Opsonized Zymosan A. The Plots Show the Mean Response for Each Treatment Group over an Hour Period. Luminescence cpm X 103 I23 50— X 4... / \ I‘ \ High Analytical (10 m9/k9) 309 Control fiLow Technical ‘ (1 Ins/ks) // \ ‘0 High Technical ‘ \ - g (10 mslks) ‘0 Low Analytical (1 mg/kg) 20- l I I I I I I O 1 O 20 3O 4O 50 60 Time (minutes) 124 .c00HQm no 0poc nmfihH .OooHn How .mO.Amv ocsom m00c0n0MMHO HSMOHMHcmHm oz .O0CHEH0H0O uoz n .D.z .HS0EH00HH 0» 0:0 O0H0>o00u 0DmmHu HS0H0HmmDmcH .m.z .H N c .mmH .HHH. x0ch coHumHomHoocH 0mouomH uc0m0um0n m0=Hm> .mmH .2002. 0HDSHE H0O mucsoo H0: Hc0m0um0u m0SHm> .nsmo me How onoo cpo u smo\03 soon orxos oH .onoo 30H u Hmo\03 soon ox\oa o.H (0.0060) oo.vH amm.e H or.e H er.cHH co.o~H Hma.-H Hmochooe 0.m.z .m.z eo.o amH.~H Hm.HH mmm.v~ mo.mm cme.oe bon om.oH Hoe H ro.~ H omo.o~H om.e H gem.oHH HoposHmce mo.o mom omm.m HHo.mH 6m.HH ood.mm me.m eoo.oH bon 6H.oH meo.H H Ho.oH omo.e H eo.m H moo.mHH ea.H H mrm.eHH Hmochooe rm.m wer.m oe.~ ooH.m r~.eH meo.em so.MH ~Hm.Hm 30H Ho.mH nee H em.r H omm.m H no.6 H omH.m H HoposHmcm .o.z .o.z e~.o eme.eH m~.mH meH.em mm.HH omo.om 30H me.HH rmH.~HH He.mHH Hmo.oHH oo.m H ve~.HHH mo.~ ome.~H on.~ mmm.oH me.m~ ~o~.ra ae.m mom.ee Homecoo HHH zooz HHH zaoz HHH zaoz oHHH :zmoz asoso moEmn9 c00HQm 0002 SQESH Ooon Hmuc0EHH0me mHoc0nQouoHnomuc0m 0Omuo H00HC£009 no HmoHuchcm 00m m0>Hmo CH .0: m.O. chHusHOOmoxs0H Ho coHumuuc00cou HmEHumo :0 O9 0mcomm0m 0Hc0moummeonQE>H .O.~ 0Hnm9 125 primary and secondary antibody response following IV immuni- zation with HRBC. Changes in immunoglobulin levels over the last 10 days of pentachlorophenol exposure are Shown in Figure 2.1. Although reductions in all three immunOglobulinS measured are readily apparent in the high technical group, no statistical differences (P >.05) could be detected. The primary and secondary tg'ytyg responses to HRBC (Figure 2.2) were similar for all groups in both mean peak titer and kine- tics as measured by hemagglutinating antibody. NeutrOphil function was elevated on the last day of PCP exposure by chemiluminescent reSponses after tg ytttg challenge with Opsonized zymosan (Figure 2.3). The chemilu- minescent responses of neutrophils from control and PCP- treated calves were not Significantly different in either peak height or kinetics. However, it is interesting to note that the peak response occurred five minutes earlier using control PMNS compared to PMNS from any of the PCP-treated groups. Histology Gross evaluation of spleen, liver, and kidney revealed a statistically Significant (P<.01) increase in liver weight in both technical pentachlorOphenol groups, and an increase (P‘<.05) in kidney size in the high dose tPCP group (Table 2.7). No functional impairment was evident in the kidneys as indi- cated by cliniCOpathologic results shown in Tables 2.2 and 2.3. The change in the thymus weights was most remarkable (Table 2.7). The mean calf thymus weight from the high dose 126 .Hm0.0vm. H00ucoo EO0w 0:000wpr >H0000HMHSOHm w .HH0.0vm. H00ucoo £000 0000000H0 SHucmoHuHcmHm0 .unOH03 .00. soon 00 0000000 no 0:0H03 00000 p .uan03 H00. :Hm0n mo 0000000 00 HSOH03 000000 .mmH mEm0m SH .03. uan03 cmm0o :00: 0 .nsmo 2 00.. .mnoo nod. u snobs soon 9.86 H: .326 30H u soon; soon 9.8546 v H ovH mm H mm H o H v H H00H0n009 av OONHO>.O oMH mom 0o.~ ONm mmMH mH.O om mm m0.0 OH ON LOH: 3H m H mm H NsNH 3H 3H HmoHnsHoEe om mam N0.0 HNH mom m.~ one OHNH NN.O we mmH mH.O mm Ho LOH: m H s H s H ooHH 3H 3H Hoochome mm mom M0.0 mmH mom 0o.~ mom OmoH mm.O Ho mmH 9H.O om mm 30H m H. hHH Hm H ONHH HNH mvH HmoHumec< om now mm.O sOH mmm H.m owe moHH ON.O mm HVH mm.O mm mmH 30H N H a H NH H 2.0. SH SH am new Hm.O OHH OOm O.N mmo mmHH Hm.O no mmH ON.O om me H000cou .8: .3 , soom ch0m omw 0mm Hmvuz omw 0mm Hcvuz omw 00w .mvuz omw 00mmnhovu3 c 05000 uan03 N000Hx 00>HH c00Hmw 0 mmaNn9 H0000EH00me mHoc0£QO0oHcoonc0c 00000 Hoocho09 0o HooHusHmc< O00 m0>HmO mo mnccH03 cmO0o .>.N 0wa9 127 technical group decreased to 17% of controls. HistolOgic examination of a thymus section from a calf in the high dose tPCP group (10 mg/kg/day) after 42 days of exposure revealed that the atrOphy in the tPCP exposed thymus was a result of an absence of cortical lymphocytes. Cortical lymphocytes were readily apparent in the control thymus. Spleen weights were reduced by PCP exposure, but not significantly. No other significant histological changes associated with penta- chlorOphenol exposure were observed. 128 DISCUSSION AND CONCLUSIONS Toxic effects from PCP were more evident at the higher of the two doses (10 mg/kg) and in the calves fed tPCP versus aPCP. The total steady state (acid hydrolyzed) penta- chlorOphenol concentration in the blood of the 1.0 mg/kg and 10.0 mg/kg groups was 32 ppm and 92 ppm, respectively. There were no Significant differences in blood levels reached be- tween the two grades of pentachlorOphenol used. The thymus, Spleen, bone marrow, lymph, liver and kidney contained 21.6, 7.4, 12.5, 13.4, 25.3, and 26.6 ppm PCP, respectively, in the 10 mg tPCP/kg treatment group. At the 1.0 mg tPCP/kg level, these tissues contained 3.5, 2.3, 1.6, 4.5, 6.2, and 7.2 ppm, respectively. AS was the case with the blood levels, PCP in these tissues after exposure to aPCP was roughly equivalent to those occurring after tPCP exposure. Total protein and albumin values dropped as a result of pentachlorOphenol exposure and were lowest in the 10 mg/kg tPCP group. These declines may not be directly associated with PCP exposure. The calves in each group were not pair fed (see Materials and Methods). During the time grain was fed, calves in the 10 mg tPCP/kg group consumed only 14% of the grain con- sumed by the control calves, and calves in the 10 mg aPCP/kg group consumed 67% of the control calf grain intake. All other groups were on a near equal grain consumption basis with the control calves. As a result, the calves in the 10 mg/kg tPCP group weighed an average of 10 kg or 17% less than control 129 calves at the end of the Six weeks. The poor grain intake could well have been the sole contributing factor responsible for the decreased total protein and albumin levels, particu- larly since protein was not lost in the urine. The substantially elevated gamma-glutamyl transferase (GGT) activity which increased in a dose-response fashion with pentachlorOphenol exposure, eSpecially with the technical grade PCP, is associated with the hepatomegaly observed in both the 1.0 mg/kg and 10 mg/kg tPCP groups. Typically, GGT is an indicator of hepatobiliary disease and its activity follows that of alkaline phOSphatase (ALP), or more specifically, the hepatic isoenzyme of ALP. In this case, no increase was seen in ALP or any of the other indicators of hepatobiliary disease. Since GGT is a microsomal enzyme and its level in- creases in reSponse to microsomal enzyme induction, and since it is known that contaminants in technical penta- chlorophenol are inducers of mixed function oxidases in cattle (McConnell gt gt., 1980); the rising GGT level correlated very well with microsomal enzyme induction and increased liver weights. The earliest clinical sign of pentachlorOphenol toxicity in cattle, as reported by McConnell gt gt.,(l980) is the presence of a progressive normocytic, normochromic anemia. Cattle in the McConnell study had been exposed to 20 mg/kg/day of pentachlorOphenol containing various levels of technical pentachlorOphenol. The anemia was first detected at week four and was attributable to the toxic impurities in 130 pentachlorophenol. In the present study, no anemia was evident at the end of the six weeks. Similarily, in the study with lactating dairy cows in which they were dosed with tPCP at 0.2 mg/kg/day for 75 to 84 days followed by 2.0 mg/kg for 56 to 62 days, no clinical signs of anemia were present. Evidentally, high levels of exposure to penta- chlorophenolanulits toxic contaminants over periods of many weeks is required in order to produce clinical Signs of anemia in cattle. The data is this study' show that thymic atrOphy occurs long before anemia would become evident and at much lower levels of tPCP exposure. The effect of 10 mg/kg tPCP treatment was so devastating on the thymus tissue of these calves that it was not possible to isolate adequate numbers of lymphocytes from these tissues for the immunologic profile. Interestingly, those lymphocytes that were obtained from thymic tissue contained En markers (T cell markers) in the same concentration as thymocytes from the control animals. Evidently, whatever is causing the cortical depletion of lymphocytes from the thymus is not selectively affecting either the En set or null set of lymphocytes present. The degree of thymic atrOphy in the 10 mg/kg aPCP group while dramatic, was not quite as marked at it was in the thymic tissue from calves fed 10 mg/kg tPCP. Yet, the blasto- genic response was already severely reduced in the 10 mg/kg aPCP calves. The reduced blastogenic response seems to pre- cede atrophy, but could not be detected in the blood stream, 131 spleen, or lymph node. Reduced responses by thymocytes to mitogens has previously been observed in rats (Vos and Moore, 1974) and mice (Vos gt gt., 1978) exposed to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Although tPCP does not contain 2, 3, 7, 8—TCDD, it does contain a small amount of other tetrachlorodibenzo-p-dioxin isomers (Kinzell gt gt., 1981) and a Significantly larger amount of other highly chlo- rinated congeners. These congeners can act within an animal species Similar to 2, 3, 7, 8-TCDD if a greater exposure is used (McConnell gt gt., 1978). Stress is known to cause thymic atrOphy mediated via increased production of adrenal glucocorticoid hormones (Dougherty gt gt., 1964). However, elevated cortisol (hydro- cortisone) levels did not cause thymic atrophy in these calves since all calves tested had low normal cortisol levels (Table 2.3). Other measurements of immune function resulted in no immunomodulation detected due to PCP treatment. These data in- dicate that no immunologic effect was detected in either the 1.0 mg/kg aPCP or tPCP exposed calves and is consistent with the findings in mature lactating dairy cattle in which no quantitative or functional immunologic defects were seen when these cattle were exposed to up to 2.0 mg/kg tPCP (See Chapter 1). This is also consistent with the results re- ported by McConnell gt gt. (1980) in which no changes were seen in the immune system of non—lactating heifers given 1.5 mg/kg tPCP plus 13.5 mg aPCP/kg/day;beyond that which was 132 observed with 15 mg/kg aPCP. In this study, calf performance was poor at the 10 mg/kg level yet effects on the immuns system were minimal. Quantitative effects were limited to thymic atrOphy. The only functional impairment was the decreased mito- gen-induced blastogenic response in thymocytes. In comparison, McConnell gt_gt., (1980) reported a decrease in thymus weight and an enhancement of blastogenesis in blood derived lymphocytes as the only immune related changes attributable to exposure to the contaminants in 5.25 and 15 mg tPCP/kg/day. In summary, immunomodulation did not occur at levels of PCP exposure (1.0 mg/kg/day) which resulted in blood PCP levels of 32 ppm. At higher levels of exposure (10 mg/kg/day), PCP is immunomodulating as evidenced by the effects on the thymus. However, most of the effects are seen in and are associated with the contaminants present in the technical grade PCP. Chapter 3 BOVINE NEUTROPHIL SHAPE CHANGE KINETICS AND ITS INHIBITION BY PENTACHLOROPHENOL 133 134 INTRODUCTION Polymorphonuclear leucocytes (PMNs) are phagocytic cells intricately involved in host defense. In recent years, the identification of cell surface receptors on PMNS for immuno- globulins, complement components, and other chemotactic factors have advanced our understanding of the cellular signals which influence the chemotactic and phagocytic activities of these cells. Many additional chemotactic factors are known (Wilkinson, 1974). In 1979, Smith gt gt. studied the initial morphological changes in neutrOphils immediately after exposure to chemotactic factors. These investigators noted that chemo- tactic factors induce a characteristic morphologic change--a bipolar form. Agents which have no chemotactic activity do not alter the morphologic features of neutrOphils. Information regarding the chemotactic reSponse of bovine neutrophils is limited. Only two techniques have been used to evaluate neutrophil chemotaxis in cattle, the Boyden chamber and migration under agarose techniques (Roth and Kaeberle, 1981a, 1981b, and Markham gt gt., 1982). This report describes a rapid, inexpensive assay to evaluate chemotaxis in bovine neutrOphils which utilizes the change in neutrophil shape from spherical to bipolar forms. Several agents which are chemo- tactic for human neutrophils were also chemotactic for bovine neutrophils. Interestingly, however, f-Met-Leu-Bhe, a potent chemotactic agent for human neutrOphils, was totally inactive for bovine neutrOphils. 135 In addition, three different grades of pentachlorophenol were tested for their influence on neutrophil chemotaxis. These studies indicate that PCP could inhibit the neutrOphil Shape change response at concentrations of 0.1 mg/ml. Perhaps the neutrOphil shape change response may be a useful assay in screening agents of environmental concern for their influence on a primary host defense system. 136 METHODS AND MATERIALS Isolation of Bovine Neutrophils Blood samples from healthy, mature and lactating Holstein-Friesian cattle were collected in sodium citrate. Neutrophils were obtained by either centrifugation of whole anticoagulated blood or centrifugation on Ficoll-Hypaque (Ficoll Pharmacia Fine Chemicals Inc., Piscataway, NJ; Hypaque- Sterling Organics, NY) cushions (Boyum, 1968). The neutro- phils recovered from anticoagulated whole blood were isolated after centrifugation at 600xg for 20 minutes. The plasma and one-third of the packed red blood cell (RBC) volume were removed and discarded. To the remaining RBC pellet, two vol- umes of sterile, cold, double—distilled water (DDW) were added. The hypotonic lysis of erythrocytes continued for one minute, and was stOpped by the addition of one volume of three times the physiologic concentration of NaCl (2.7%) in sterile DDW. After erythrocyte lysis, leucocytes were washed three times at 200 x 9 2+ for seven minutes with Ca -Mg2+-free Hanks balanced salt solution (HBSS). A second hypotonic shock was required to remove resi- dual RBCS in some leucocyte preparations. The leucocytes obtained were at least 99% granulocytes. Of these, approximately 80-95% were neutrOphils (PMN) and the remainder were eosino- phils. Red blood cells were absent and platelets were few in these preparations. Polymorphonuclear leucocytes isolated from Ficoll-Hypaque (F/H) cushions were obtained following centrifugation at 900 X*g for 40 minutes. The plasma, mononuclear cells and all but 137 the packed RBC layer were removed and discarded. The cell pellet was then treated by hypotonic shock as described above. Yields were slightly lower than those obtained by the centri— fugation of whole blood. The viability of all neutrOphil suspensions was at least 99% as determined by trypan blue dye exclusion. Neu- trOphil suspensions were adjusted to 107 cells/ml in HBSS for shape change studies. Assessment of Neutgophil Shape Changes All glassware for these studies was treated with a mix- ture of three parts PBS and one part autologous serum. A 0.1 ml aliquot of the neutrOphil suspension was diluted in 0.9 m1 of phosphate buffered saline (PBS). The cells were then exposed to various conditions and biological and chemical substances to evaluate neutrOphil shape change. NeutrOphils undergoing shape changes were terminated by the dropwise addition of 2-3 ml of 1% glutaraldehyde (Sigma Chemical Co.) in PBS. The neutrophil suspensions were mixed constantly while glutaraldehyde was added. The PMN glutaraldehyde mixture was then allowed to stand overnight at 4°C. Cells were examined using 1000X phase- contrast micrOSCOpy and classified according to Shape using the classification system of Smith gt gt.,(l979). Kinetic Studies were performed at 37°C using 0.1 m1 of 107 PMN/ml added to 0.9 ml PBS containing zymosan-activated serum (ZAS). The ZAS was prepared by incubating 10 mg zymosan (Sigma Chemical Co.) in 1.0 ml fresh bovine serum for 45 minutes 138 at 370C. The serum was cleared by centrifugation and diluted with PBS before addition of PMNS. Once shape change parameters were standardized using ZAS, other biological and chemical agents reported to cause Shape change in human PMNS were investigated using bovine neutrOphils. The agents tested included N-formyl-L-methionyl- L-leucinyl-L-phenylalanine (f-Met-Leu-Phe)(Andrulis Research Corp., Bethesda, MD)(Schiffmann gt gt., l975),alpha-casein, and hydrolyzed and partially dephosphorylated casein (Sigma Chemical Co) (Wilkinson, 1972) and bacterial cell filtrate (BCF) (Keller and Sorkin, 1967). F-Met-Leu-Phe was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10"3 molar (M), then diluted to concentrations of 10's, 10'7 9 , and 10- M in PBS. Casein and alpha-casein were dissolved in HBSS and adjusted to pH 7.2. Final concentrations of these caseins in cell suspensions ranged from 0.1 to 25 mg/ml. Bacterial cell filtrates from g. ggtt ATCC #25922 (Difco Laboratories, Detroit, MI) were prepared in HBSS. Bacteria were cultured for 24 to 48 hours at 370C. The cell suspension was then filtered through a 0.22-um Millipore filter and the pH of the culture was adjusted to 7.4. The filtrate (BCF) was tested at concentrations ranging from full strength to 0.1% in HBSS. Influence of Pentachlorophenol (PCP) on NeutrOphil Shape Changg These studies utilized three grades of PCP: analytical grade PCP (aPCP) (Aldrich Chem. Co.,Milwaukee,WI) purified PCP(pPCP) (Dow Chem. Co., Midland, MI) and technical PCP(tPCP)(a commercial pentachlorOphenol which was generously provided by the American 139 Wood Preservers Institute L The tPCP represented an industry composite from the three major manufacturers of pentachloro- phenol. Each grade of PCP was dissolved in either ethyl alcohol (ETOH) or DMSO to a concentration of 20 mg/ml. Various con- centrations of the PCP solutions (0.2 mg/ml to 0.02 mg/ml) were used in the neutrOphil shape change studies. (In some ex- periments, PCP solutions were added to neutrOphil suspensions incubated in 1% ZAS. The PMN shape changes were assessed after a seven minute reaction time. 140 RESULTS Effects of Chemotactu:Factors on Cell Shape Change The appearance of unstimulated PMN and eosinOphils separated from anticoagulated blood is shown in Figure 3.1a. The cells appear round, have a centrally located nucleus and have a slightly ruffled membrane on a small portion of the surface. Incubation of neutrOphil suSpensionS in 1% ZAS caused significant Shape changes within two to three minutes. At least 50% of the PMN were elongated with a few Showing urOpod formation. To quantify the Shape change reSponse, the cells were categorized as Spherical or nonSpherical. The spherical classification included round cells and cells whose shape was generally round with a ruffled membrane (Figure 3.1b). The nonspherical category included cells whose overall form was elongated (bipolar) with, or without urOpods (Figure 3.1c). The neutrophil shape change response to various doses of ZAS is presented in Figure 3.2. The time course for shape change after addition of 1% ZAS in PMN suspensions is presented in Figure 3.3. NeutrOphils isolated from Ficoll/Hypaque cushions also changed Shape in response to ZAS. However, this PMN isolation procedure produced a higher percentage of dead cells, as well as cells showing "abnormal" shapes and excessive membrane ruffling in unstimulated control experiments. NeutrOphils recovered from Ficoll-Hypaque cushions were less responsive to 1% ZAS in that only 25 to 40% of the cells exhibited chemotactic factor-induced Shape changes. Figure 3.1a. Bovine NeutrOphils (N) and EosinOphils (E) (x 1,000 Phase Contrast) Fixed in 1% Glutaraldehyde After a Five Minute Incu- bation at 37°C with no CF. The Bar at the Bottom Left Represents 10 Microns. Figure 3.1b. Bovine NeutrOphils (N) Showing Round Ruffled Features (Spherical Classification). Cells Were Fixed in 1% Glutaraldehyde and Photographed at x 1,000 Using Phase Contrast. One Nonstimulated Eosinophil (E) is present. Figure 3.1c. Bovine NeutrOphils (x 1,000 Phase Con- trast) Showing Nonspherical (Bipolar) Forms With One Neutrophil Showing the Beginning of a Uropod (U). Cells Were Fixed With 1% Glutaraldehyde After Five Minutes Incubation at 370C in the Presence of 1% ZAS. Figure 3.1d. Bovine Eosinophil (E) Showing a Typical ReSponse to 1% ZAS (x 1,000 Phase Contrast). A Bipolar NeutrOphil (N) is Also Present. 153 Figure 3.2. Change in Shape of Neutrophils Exposed to Various Levels of ZAS. "Percent Bipolar" Refers to the Percent of Cells in the SuSpension with a Bipolar Shape (Bipolar Plus UrOpod). Incubation Time was Five Minutes. Each Point is the Mean + of Four Separate Experiments. - I44 .940. Eaton oon>=o< coonsN 6.. o.m O.N O._. m5 v.0 mod Pod H H _ o H H H L l O N O V 0 to O 00 00.. le|odga luemad 145 NeutrOphils incubated with casein and three casein-der- ivatives were evaluated for Shape changes over a ten minute interval. The casein and alpha-casein response is Shown in Figure 3.4. The response to casein is less dramatic than the response to ZAS. Also, the maximum shape change caused by casein was often observed to occur together with cell morpholo- gic alterations similar to those preceding cell death. Acid hydrolysate type I casein and a vitamin free casein were able to stimulate shape change, but to a much lesser extent than either the alpha-casein or the partially dephosphorylated and hydrolyzed casein. Filtrates of E. ggtt cultures (BCF) also induced shape changes in bovine neutrOphils. Undiluted BCF was most active, while BCF diluted 1:4 in HBSS was ineffective in inducing shape changes. It is noteworthy that BCF preparations varied Signi- ficantly. Some preparations stimulated shape changes in at least 60% of PMN while other preparations only induced Shape change in 25% of PMN. The peptide f-Met-Leu-Phe was ineffective in causing shape changes in bovine neutrophils over a wide concentration range. At the "most Optimum" concentration of 10-7M, f-Met-Leu- Phe stimulated only 1.5-3.5% of PMN into the nonspherical classification. Eosinophils contaminating neutrOphil preparations also reSponsed to ZAS. They could be stimulated to a much greater extent than were PMN when incubated with 1% ZAS (Figure 3.1d). Figure 3.3. Change in Shape in Neutrophils Exposed to 1% ZAS. "Percent Bipolar" Refers to the Percent of Cells in the Suspension with a Bipolar Shape (Bipolar Plus UrOpod). "Time-minutes" Refers to the Time After the Addition of Cells. Each Point is the Mean 1 SE for Four Separate Experiments. I46 30:55. . 0.5... mp 8 Or 0 I N —. . H2050 WI . . . . h 4 . . .H\.\n\$ \. - \0 H 05...; rI |0| L O N O V O (D O 00 00—. le|odgg wealad Figure 3.4. Change in Shape of NeutrOphils Exposed to Various Levels of Alpha-casein and Casein (Partially Dephosphorylated and Hydrolyzed). "Percent Bipolar" Refers to the Percent (Mean) of NeutrOphils in the Suspension with a Bipolar Shape (Bipolar Plus Uropod). Incubation Time was 7.5 Minutes. I47 50000 .835 0.0.. 0.... md O... _ no On. I \ no . IfiI O F O N O (O O V O In JBIOdIQ weaned 148 It is noteworthy, however, that eosinOphilS did not change shape when exposed to either BCF or casein. Effects of PentachlorOphenol on Cell Shape Change Results of studies evaluating the influence of aPCP, tPCP, and pPCP on bovine neutrophil Shape changes are presen- ted in Figure 3.5. All three grades of PCP significantly re- duced shape-change responsestIPMNS exposed to 1% ZAS. The shape change response could be eliminated at 0.1 mg/ml PCP. No Shape changes were induced in neutrOphils incubated with any of the three grades of PCP, or the two solvents, DMSO or ETOH. Neither was DMSO or ETOH able to inhibit Shape change when PMN were incubated with ZAS in the absense of PCP. A representative example of ZAS stimulated PMNS exposed to PCP is presented in Figure 3.6. These "wrinkled" neutro— phils were able to exclude trypan blue for ninety minutes while at 37°C and in the presence of any of the three PCPS at 0.2 mg/ml. Significant numbers of dead cells began to appear at two hours. Solvent controls Showed at least 98% viability at two and one-half hours. Figure 3.5. Change in Shape of NeutrOphils Exposed to 1% Zymosan Activated Serum in the Presence of Three Different Grades of Pentachlorophenol (PCP); Analytical or aPCP, Purified or pPCP and Technical or tPCP. The Numbers Above the Bars Refer to mg PCP/ml Used. The Closed Bars Refer to Ethanol and the Open Bars to Dimethyl Sulfoxide Used as Solvents to Deliver the PCPS. "Per- cent Bipolar" Refers to the Percent of Cells in the Suspension with a Bipolar Shape (Bipolar Plus UrOpod). Incubation Time was 7.5 Minutes. The Controls Contained 1% Solvent, the Maximum Used to Deliver PCP. I49 .ozcoo moan 0N6 09.0 - 00.0 «0.0 0000 0N6 09.0- 60.0 No.0 mono 0N6 O...O No.0 O N O V O (D O Q GOP .IeIodIg weaned Figure 3.6. Bovine NeutrOphils Showing the Unrespon- Sive "Wrinkled" Appearance of Cells Incubated in the Presence of 0.10 mg/ml PCP and 1% ZAS for 7.5 Minutes at 37°C (x 1,000 Phase Contrast). 151 DISCUSSION AND CONCLUSIONS This study shows that bovine neutrOphils reSpond to many of the same chemotactic factors as human neutrOphils. However, bovine PMN were totally unresponsive to f-Met-Leu- Phe, which is a potent chemotactic factor for human neutro- phils (Schiffmann gt gt., 1975). One percent ZAS was found to be the optimum concentration for neutrOphils to change shape. It is noteworthy that a ZAS concentration greater than two percent inhibited the bipolar response, a phenomenon that has been reported in human PMN (Keller and Sorkin, 1966). Bovine neutrOphils, however, appear to benmnmasensitive since they are inhibited at a lower ZAS concentration than that required for the inhibition of human neutrOphil chemotaxis (greater than 10%) (Smith gt gt, 1979). Casein and bacterial metabolites (Keller and Sorkin, 1965) are known to be chemotactic for human neutrOphils. Similar results were noted for bovine neutrOphils. Russell and Reiter (1975 and 1976), however, have reported that casein inhibits both ingestion and intracellular killing of bacteria by bovine neutrOphils. The inhibition occurred at physiological concen- trations of native casein micelles, and by the addition of puri- fied casein as sodium caseinate. These data indicate that casein may have significant effects on neutrOphil function which occur below the physiologic range of casein in milk (25-40 mg/ml). Alpha-casein at 5 mg/ml and hydrolysed casein at 10 to 25 mg/ml caused a significant decrease in the neutrophil shape change response. This impairment in neutrOphil Shape change probably precedes and is related to the casein-induced decrease in 152 PMN phagocytosis and bactericidal activity observed by Russell and Teiter (1975). Such impairment in neutrOphil function by casein may enhance the susceptibility of the udder to bacterial infection. It is interesting that casein and BCF will not cause appreciable Shape change in the bovine eosinophil. In contrast, the eosinOphil appears to respond faster, and to a greater de- gree than PMN to ZAS. These date suggest differences in re— ceptors or receptor density on their cell surfaces. Of particular interest was the significant effect that Ficoll-Hypaque gradient separation had on the bipolar Shape change response. Whereas Ficoll-Hypaque cushions and heparin- ized blood appear satisfactory for the isolation of bovine lym— phocytes for subp0pulation enumeration and function studies, (see previous chapters), their use markedly inhibits shape change in neutrOphils. Heparinized blood caused neutrOphil clumping, and the preparations were significantly comtaminated with platelets. Others have noted difficulties associated with the use of F/H cushions for separating bovine neutrophils for use in $2,!iEEE studies. Roth and Kaeberle (1981) reported that granulocytes exposed to F/H had Signi- ficantly reduced random migration under agarose while other granulocyte function tests such as chemiluminescence were not significantly altered. Lichtman gt gt., (1976) reported the presence of stimulated neutrOphil shapes when human cells were maintained in heparinized plasma. In addition, use of F/H cushions with or without heparinized samples resulted in less 153 PMN shape change in response to CF. Thus, neutrOphils from both humans and Cattle may be functionally impaired due to con- tact with Ficoll/Hypaque gradients and heparinized blood. An implied association between exposure to penta and poor health in dairy cattle was reported by Thomas gt gt., (1975). Clinical signs in the cattle included decreased milk production, poor general appearance, skin lesions, increased mastitis, persistent infections, high calf mortality and death. Some of these effects are suggestive of a suppression in host defense. In a previOus study, the effect of technical grade penta- chlorOphenol upon lactating dairy cattle performance, general health, histOIOgy (Kinzell gt gt., 1981) and immune function was evaluated. The level of technical pentachlorOphenol exposure used in that study was 0.2 mg/kg/day for 75-84 days followed by 2.0 mg/kg/day for an additional 56-62 days. The maximum level of PCP reached in blood during these studies was 0.014 mg/ml. This level is Similar to the lowest dose used in the present tg_ytttg study, a level of PCP in which no significant impairment in shape change reSponse was seen.. The importance of this finding is that technical pentachlorOphenol administered subchronically in the previous study was designed to approximate a level of exposure which dairy cattle might be expected to con- tact in a farm environment (Van Gelder, 1977). No immuno- modulation could be detected resulting from technical penta- chlorOphenol exposure in that study (Chapter 1). 154 The observation that chemiluminescence by neutrOphils exposed to PCP always peaked five minutes later than control neutrOphils was puzzling (Chapter 1), but was regarded as not important at that time. Since that study, I have made this observation again in calves exposed to PCP (Chapter 2). It is interesting to speculate whether this phenomenon is related to the inhibition of shape change seen tg 22359! At levels one to two orders of magnitude higher, pentachloro— phenol in the blood can reach 0.1 mg/ml (Parker gt gt. 1980). This blood level of PCP has been reached in a study done in nonlactating cattle (McConnell gt gt., 1980). It showed (among a lot of negative findings) an enhancement of mitogen— induced lymphoblastogenesis and a decrease in absolute and relative thymus weight, both of which were attributed to the amount of contaminants in the pentachlorophenol fed. No effect was seen on white blood cell number or differential leucocyte count. At the level of PCP exposure used in that study, a level which could be reached in an acute exposure, there data in— dicate that an effect on bovine neutrOphils could occur. That an effect could occur at this level is also supported by the fact that locomotion and perhaps Shape change requires some oxidative energy (Carruthers, 1967), and pentachlorOphenol is an uncoupler of oxidative phOSphorylation. Carruthers found that dinitrophenol, an uncoupler of oxidative phosphorylation, at a 3M or 0.184 mg/ml had a partial inhibitory concentration of 1 x 10- effect on both random and directed motility of human leucocytes tg vitro. tg vivo investigation of this phenomenon should reveal 155 interesting results. The data presented above provide the framework for further studies involving many aspects of bovine eosinOphil and neutrOphil investigations; eSpecially in the areas of chemotaxis, shape change, adhesiveness, motility and chemo- tactic factors. In summary, the data presented define the parameters for performing the isolation and analysis of shape change in bovine neutrOphils. These data can be used as a basis to investigate other areas of bovine neutrOphil function. Chapter 4 ISOLATED BOVINE HEPATOCYTES: A MODEL FOR XENOBIOTIC METABOLISM 156 157 INTRODUCTION Isolation of viable rat hepatocytes with collagenase was first described by Berry and Friend in 1969. In 1972 and 1973, Seglen published detailed descriptions of the factors required for optimizing the isolation of rat hepa- tocytes. Seglen was the first to perform the two-step method which has formed the basis for all isolation procedures. The isolation of rat hepatocytes has Since become a routine procedure in many laboratories. The thrust of isolated hepatocyte research is in two major areas: in expanding the number of applications for iso- lated rat cellsand in establishing long-lived cultures of hepato- cytes which do not dedifferentiate. Recently, many researchers in toxicology have changed from systems based on liver sub- cellular fractions, organ perfusions, or slices,to isolated hepatocytes. This change occurred because isolated hepatocytes seem to mirror the £2.23X2 metabolism of xenobiotics more closely than other tg ytttg systems through the accountability of structure and kinetics (Fry and Bridges, 1977; Menzer, 1979). No reports describe the isolation of hepatocytes from cattle, although hepatocytes have been isolated from non-labor- atory animal Species such as Sheep (Clark gt gt., 1976 ; Ash and Pogson, 1977) and humans (Liddiard gt gt., 1980 ; Reese and Byard, 1981; Strom gt gt., 1982). Since cattle are often too large and expensive for use in toxicokinetic studies, iso- lated bovine hepatocytes would be an ideal alternative. 158 This paper describes an approach that can be used to prepare isolated hepatocytes from bovine liver. The iso- lation procedure results in large numbers of viable hepa- tocytes which are metabolically active, and can be used for metabolizing xenobiotics. 159 MATERIALS AND METHODS Reagents perfusion (CaZI/Mg2+-free) buffer: 8.3 g sodium chloride, 0.59 potassium chloride, 2.4 g HEPES buffer and 0.1mM ethylene- bis (oxyethylene-nitrilo) tetraacetic acid (EGTA) were dis- solved in 1 liter of double-distilled water, and adjusted to a final pH of 7.4 with 1M NaOH. Just prior to use, the solution was saturated with 95% 02/5% C02. Collagenase Solution: prepared by the addition of SmM calcium chloride and 50 mg/dl collagenase1 to the perfusion buffer. The collagenase was added just before use. Wash buffer: same composition as the perfusion buffer, but without EGTA. Trypan blue solution: a filtered solution of 0.4% trypan blue in normal saline. Gluconeogenesis medium: Earle's balanced salt solution free of glucose and phosphate and fortified with 20mM lactic acid, 4mM lysine, 2mM pyruvate, 20mM sodium acetate, 0.2mM dibutyryl cAMP. The pH was adjusted to 7.45 with sodium hydroxide. Con- trol gluconeogenesis medium was the same as the gluconeo- genesis medium but without the addition of 20mM lactic acid, 4mM lysine and 2mM pyruvate. Palmitic acid oxidation media: Calcium-free Krebs-Ringer 1Collagenase II was obtained from Worthington Biochemical Corp., Freehold, NJ and Type I Collagenase from Sigma Chemical Co., St. Louis, MO. 160 bicarbonate containing 1mM-l4c-palmitate2 and 2mM DL-carnitine. The palmitate was complexed with 1.5% bovine serum albumin at a 4:1 fatty acid to bovine serum albumin ratio. The final pH was adjusted to 7.4 with C02. Homogenization buffer for cytochrome P-450: 20mM Tris-hydro- chloric acid, 1.15% potassium chloride, pH 7.4; 2.49 Trizma base and 159 potassium chloride were dissolved in approximately 800 m1 of double distilled water, the pH adjusted to 7.4 using 100mM hydrochloric acid, and brought to a volume of one liter with double—distilled H20. Isolation of Bovine Hepatocytes Livers were generally from mature Holstein-Friesian dairy cattle but were occasionally from calves and steers of other breeds. Preliminary experiments revealed that the caudate process of the bovine liVer was best for preparation of viable hepato- cytes. Viability was not affected if the caudate process was removed within 15 min following euthanasia. The caudate process was removed by cutting across its base of attachment. The cross sectional area of the cut surface was kept to a minimum. Immediately after removal, the caudate process was perfused with approximately 300 ml of cold perfusion buffer. This was accomplished by inserting a plastic syringe tip of a 50 ml syringe into a number of the larger exposed arteries and veins on the cut face and repeatedly perfusing 2l-MC-Palmitate was obtained from Amersham, Arlington Heights, IL. 161 until the tissue was uniformly blanched. Immediate perfusion of the tissue accomplished three objectives required in order to obtain viable hepatocytes: l) removal of blood from the tissue before it clotted; 2) removal of calcium from the tissue which expedites dissocia- tion; and 3) rapid cooling of the tissue to extend viability. The perfused tissue was transported quickly to the laboratory in ice-cold perfusion buffer. Any unblanched tissue located around the cut surface of the lobe was trimmed away. The lobe was perfused as before with an additional 400 ml of perfusion buffer at room temperature. A 20 g portion of the liver was excised from the lobe with as much intact capsule as possible. Usually, greater success was achieved with the apical region of the caudate process because of a higher ratio of intact capsule to cut surface. The 20 g portion of liver then was placed in the perfusion apparatus. The perfusion apparatus was composed of a 600 ml beaker that served as a reservoir and a wide stem glass funnel 100 mm in diameter containing a 150 m1 sintered glass bottomed cru- cible placed on tap of the beaker (reservoir). The crucible, which functioned as a support surface for the lobe, had several 1 to 2 mm diameter holes bored in it to facilitate drainage. The perfusion apparatus was placed in a 37°C water bath. A two-channel perfusion pump (Masterflex, Barrington, IL) cir- culated the collagenase solution through the tissue via cathe- ters sutured into exposed arteries or veins. An alternate but less desirable procedure was to attach 162 20 Ga x 3.8 cm needles to the ends of the perfusion tubing and insert them into the liver tissue. This procedure periodically was used as a back up method to the aforementioned suture technique. When the needles were used, they had to be moved to different locations within the tissue at frequent intervals in order to achieve an even perfusion. The perfusion flow-rate was approximately 15 ml/min from each outlet. The collagenase solution was kept at 38 to 39°C to compensate for the temperature drop in the perfusion apparatus. Optimal cell disaggregation was achieved when the collagenase solution was not recirculated through the tissue and the tissue capsule remained intact. The collagenase per- fusion was terminated after the tissue became swollen, took on a mushy appearance and the capsule became wrinkled with collagenase solution exuding from all intact surfaces. This generally took 30 to 45 minutes. The perfused tissue then was placed in a 150 mm plastic petri dish containing a small amount of collagenase solution. and cut into 1 to 1.5 g pieces. A Stainless steel comb was used to dislodge the cellular material from the capsule and connective tissue. Next, 2 to 3 volumes of ice-cold wash buffer were added to the dispersed tissue, the mixture of Single cells and debris were filtered through 150 micron mesh nylon, and the remaining residue was discarded. Alternatively, a two- step filtration through a 250 micron followed by 150 micron mesh nylon was occasionally necessary to prevent clogging of the finer mesh nylon. The filtered suspension was then poured into 163 cold 15 m1 plastic centrifuge tubes and centrifuged for 3 min at 50 x g with the supernatant being discarded. The cells were washed a total of three times with cold wash buffer and were resuspended by using gentle action with a wide bore Pasteur pipette. After the final wash, cells were reconstituted to a thick suSpension of greater that 1.0 x 107 cells/ml in the wash buffer and either used immediately or stored for short periods in an ice bath. The entire isolation procedure required 2 to 3 hours. Cell Counting_and Trypan Blue Dye Exclusiog Viability Determination A volume of 0.1 m1 of the cell suspension was added to 0.9 ml of trypan solution and left standing for 5 min before the cells were counted in a hemocytometer. Both stained (non- viable) and unstained (viable) cells were counted and the via- bility percentage and number of viable cells per milliliter were calculated. The cell suspension was diluted to a working con- centration of 5 to 10 x 106 viable cells/ml. Two ml of cells at this concentration and at 75 plus percent viability were equivalent to 10 to 20 mg/ml dry weight. Gluconeogenesis Two m1 of lactate and control media (Krebs gt gt., 1976, 1979) were added to respective 30 ml culture flasks along with 2 ml of isolated hepatocytes. Incubation of media and cells was at 37°C in a Shaking water bath. Flasks were gassed with 95% 02/5% CO at the start of the incubations 2 and every 30 min thereafter. Incubations were terminated after 164 various times by addition of 120 ul of concentrated (70%) perchloric acid followed by centrifugation for 15 min at 900 x g. The supernatant was used for the glucose assay, and the pellet was discarded. The glucose concentration was determined as described by Raabo and Terkildsen, (1960). Endogenous glu- cose production was the difference between controls incubated for the various times and a zero time. The zero time value was obtained by immediately stOpping the reaction between media and isolated hepatocytes. In experiments that compared isolated hepatocytes with liver Slices, tissue was obtained from the caudate lobe of the same animals. The isolated hepatocytes were prepared as des- cribed. Slices were prepared by Slicing on a Stadie-Riggs microtome (Thomas Scientific) which was calibrated to deliver slices 0.8mm thick. The Sections were kept in ice-cold wash buffer until used. Dry weights were obtained from the difference between 2 m1 of a working concentration of isolated cells and 2 ml of wash buffer dried for 2 h at 1000C. Viable dry weight is a term that describes the percentage dry weight resulting from viable hepa- tocytes as determined by trypan blue. Palmitic Acid Oxidatigg Liver slices (5 to 30 mg dry weight) and isolated hepa- tocytes (2 ml, 10 to 20 mg dry weight) were placed in 25 m1 Erlenmeyer flasks containing 3 ml of oxidation media for the incubation of Slices or 2 ml for incubation of isolated hepato- cytes. Flasks were equipped with suSpended centerlwells, gassed 165 for 15 s with 95% 02/5% CO2 and incubated for 60 min at 37°C. Reactions were terminated by addition of 3 ml of 1M perchloric acid. Methylbenzethonium hydroxide (0.3 ml) was added to the center wells and incubations were continued for an additional 60 min to collect liberated 14CO . Radioactivity 2 in 14C02, and acid-soluble metabolites was determined by liquid scintillation counting (Nuclear Chicago, Model Mark 1) in ASC scintillant (Amersham). Quench corrections were calculated from a Standard quench curve. Acid soluble metabolites were identified by high per- formance liquid chromatography on Waters Associates equipment (Milford, MA) and consisted of a M45 solvent delivery system, a model 441 detector set at 214 nm and a C reverse phase Radial- l8 Pak cartridge. Oxygen Utilization Oxygen utilization was determined by monitoring the dis- appearance of oxygen at 25°C from a suSpension of isolated hepatocytes in oxygen containing media. An oxygen specific electrode (Zazar Research Laboratories DO-166 dissolved oxygen probe, Los Angeles, CA) was used to monitor oxygen. The oxygen probe was fitted through an aperture in the cap of a 30 ml plastic vial. The vial contained 30 ml of Hanks balanced salt solution oxygenated at 20 ppm, and approximately 2.0 x 107 viable cells. When the oxygen probe was in place, no air pockets were contained within the vial. The vial itself was sealed with layers of parafilm so that exchange of gases with the atmOSphere was minimal. The oxygen probe and its interface unit were attached 166 to a pH meter (Fisher Accumet Model 525 Digital pH/ion) capable of reading millivolts. The mixture of isolated hepatocytes and oxygenated media was mixed gently with a micromagnetic stir bar and a Corning PC-353 magnetic stirrer. The Speed of the micro- magnetic bar was controlled by a variable autotransformer in line with the Corning magnetic stirrer. Once an oxygen baseline was established, the recorder was turned on, and the slope of the oxygen disappearance was monitored. Controls consisted of cyanide-treated honviablé iso- lated hepatocytes analysed in the same manner. The oxygen probe was calibrated to atmospheric oxygen and to a 0 ppm oxygen standard prepared by dissolving 1.6 g of sodium bisulfite in double-distilled water. Cytochromes P-450 and b 5 Cytochromes P-450 and b were quantitated in microsomes 5 prepared from intact liver and microsomes prepared from iso- lated hepatocytes. A portion of the caudate process was used for microsomal preparation. The remainder of the caudate lobe was processed as described to obtain isolated hepatocytes. The method used for the preparation of microsomes was described by Shull gt gt. (1982). Microsomes were resuspended in 150 mM KCL to yield approximately 30 mg of protein/ml. Protein was determined colori— metrically (Gilford Stasar II Spectrophotometer model 136745) by the biuret method. BSA was used as the Standard (Gornall gt gt. , 1949). Microsomal concentrations of cytochromes P-450 and b5 167 were measured spectrophotometrically (Beckman Acta III dual- beam spectrophotometer) by the carbon monoxide difference spectral procedure. The microsomes were suspended in 100 mM tris buffer, pH 7.4, at a protein level of 1.0 mg/ml (Omura and Sato, 1964). Epoxidation of Aldrin Aldrin was dissolved in either DMSO or ethyl alcohol at 2 mg/ml and added to isolated hepatocytes in suspension cul— ture. Either 10 ug, 20 ug or 50 ug of aldrin were added to cul- ture flasks containing 2 ml of phosphate buffered saline and viable hepatocytes equivalent to 8 to 10 mg dry weight. The flasks were gassed with 95% 02/5% CO2 and incubated at 37°C in a shaking water bath for either 15 or 30 min. Reactions were stopped by adding 10 ml of a 3:2 mixture of n-hexane and iSOprOpyl alcohol. This solvent was also used to extract the aldrin and dieldrin (3X). The solvent was evaporated and analy— sed for aldrin and dieldrin with a Varian 2100 gas chromato- graph (Palo Alto, CA) equipped with an electron capture de- tector. The column was glass, 1.83 m long, 2 mm IDrpacked with 4% SE-30 on 100/200 Cromosorb W . Statistical Analysis Data were analyzed by regression analysis and analyses of variance on the Genstat V system at Michigan State University, East Lansing, Michigan. A model describing the relationships among the variables affecting either cytochrome P-450 or cytochrome b5 concentration 168 was used. It was assumed that the same variables affect both cytochrome concentrations. The model describing this relation- ship was: Yijkm = M + T1 + Mj + T.M (ij)k+ eijkm where Yijkm is the P-450 or b5 cytochrome concentration, M is the mean of all responses, Ti is the effect of the i-th tissue (i = 1 isolated cells or 2 intact tissue), Mj is the effect of the j-th time (j = l, ..., 6; representing six different times), T'M(ij)k is the effect of the k-th interaction of tissue with time (k = 1,..., 6: representing six tissue time interactions), is the residual error. eijkm T'M(ij)k is the error term for Ti and Mj. Factors Ti’ Mj’ and T'M(ij)k are fixed, whereas and e. are random (Gill, 1978). Yijkm ijkm 169 RESULTS Viability and yield of hepatocytes isolated from each of 20 caudate processes are presented in Table 4.1 with an example of the ultrastructure of freshly isolated bovine hepa- tocytes shown in Figure 4.1. Figure 4.2a shows total glucose production by isolated bovine hepatocytes over 2 h. This figure shows a Slightly greater rate of increase of total glucose formed in hepatocytes incubated in the lactate substrate than in the control media. This can be attributed to the depletion of endogenous pre- cursors for gluconeogenesis and glycolysnsin hepatocytes in- cubated in the control media. Most of the glucose formed can be attributed to glucose production from endogenous precursors. The difference between the two traces (total glucose production in lactate-fortified cells and glucose production in control cells) represents glucose production attributed to gluconeo- genesis from the nonendogenous Substrate, primarily lactate. The rate of glucose production due to exogenous substrate is presented in Figure 4.2b. The rate of glucose production from exogenous sources decreased during the 2 hr incubation. However, the total amount of glucose produced over the same time increased. The potential advantage of isolated hepatocytes over Slice techniques is shown in Table 4.2. This table shows com- parative data on gluconeogenesis for both isolated hepatocytes and liver slices. The liver for both methods was obtained from the same animal and analysed simultaneously. Isolated 170 Table 4.1. Yield and Viability After Isolation Of Bovine Hepatocytesa Trial # % Viabilityb Total Number of Viable Cells Obtained x 108 1 91.2 .12 2 68.0 2.09 3 81.0 2.64 4 88.2 5.56 5 86.6 .89 6 91.6 12.00 7 88.8 5.73 8 87.7 6.80 9 76.2 1.80 10 63.0 2.85 11 80.2 1.95 12 87.3 4.82 13 80.3 1.21 14 80.2 4.98 15 82.2 2.93 16 70.2 2.95 17 70.3 .77 18 73.2 2.24 19 87.0 1.29 20 92.0 .34 3.20 x 108C mean total viable cells 81.3% mean viability aStarting withliver tissue wet weights of 12 to 20 grams. Viability as determined by trypan blue dye exclusion. C CorreSponds to 3.94 x 108 total cells isolated (viable plus non- viable as determined by trypan blue) and 21.6 x 106 viable hepatocytes per gram of tissue (wet weight). 171 Figure 4.1. Isolated Bovine Hepatocyte Figure 4.2a. Total Glucose Production I72 1 l 90 120 Time (minutes) 60 l 15 30 L l I l I In V (‘0 N 1- IuBIeM Ma (aneIA) male/elnqu/paonpmd esoon|9 salow If Figure 4.2b. Rate of Glucose Production from Exogenous Substrate in Bovine Isolated Hepatocytes. I73 0.00:. .9500 :. 003000000... .3 000300... 00003.0 .000... sIdIld 0.00.... 000000.. n.00c0mo0c003.m e. 000600000: .3 000300.... 00003.0 .000... I 60035:: 08.... Our 0m 00 on m.. _ _ H _ _ H _ H — (00.. I § 1 o O 0) iufiiaM no (main) male/peonpmd asaanla lo sa|ow n' |ela_|_ I GO? IL 00m 174 TABLE 4.2. Comparison of Gluconeogenesis Between Bovine Isolated Hepatocytes. and Liver Tissue Slicesa Animal Substrate Isolated Cells Tissue Slices Lactateb 1.47 i .12e None detectable (1.68): .14f l Pr0pionateC 1.01 + .03 None detectable (1.16); .04 Noned 3.819 1.43 Lactate .88 i .07 .12 i .93 (1.15): .09 2 PrOpionate .13 i .07 None detectable ( .18): .10 None 5.26 2.90 aIsolated hepatocytes and liver slices were from the same animal and were analyzed at the same time. bEarle's balanced salt solution without glucose and phoSphate: with a final concentration of 10 mM lactic acid, lmM lysine, lmM pyruvate, lOmM sodium acetate, 0.2mM dibutyryl cAMP, pH 7.45 saturated with 95% 02/5% CO2 and run in triplicate. CSame as b except pr0pionate (lOmM) is substituted for lactate. dEarle's balanced salt solution without glucose and phosphate: with a final concentration 0f lOmM sodium acetate, 0.1mM dibutyryl cAMP, pH 7.45 and saturated with 95% 02/5% C02. eUmoles of glucose produced/minute/gram of liver (dry weight) at 37°C at 60 minutes :SE. umoles of glucose produced/minute/gram of liver ("viable" dry weight) at 37°C at 60 minutes :SE. gGlucose produced from endogenous sources in umoles produced/ minute/gram of liver (dry weight) at 37°C at 60 minutes. f 175 hepatocytes showed higher gluconeogenesis rates when calculated on a dry weight basis than did tissue slices. It is also possible to calculate results as per gram "viable" dry weight when isolated hepatocyes are used. This calculation is justi- fied because hepatocyte preparations showing virtually 100% nonviable cells by trypan blue dye exclusion will not exhibit gluconeogenesis. Further comparisons between isolated hepatocytes and liver slices were made on their ability to oxidize palmitate (Table 4.3). The rate of oxidation of 1--14 14CO2 and 14C— acid soluble metabolites by isolated hepatocytes was four to five times higher than in liver slices. This C palmitate to 5 comparison is on a pmoles of 1-14C palmitate oxidized to pro- duct/minute per mg tissue (dry weight) basis. The majority (1>70%) of radioactivity in the acid-soluble fraction was B- hydroxybutyrate and acetoacetate as determined by high per- formance liquid chromatography. A comparison of isolated cells and tissue slices on a dry weight basis is not entirely accurate because of the connective tissue in liver slices which contri- butes to the dry weight of the slice. However, it is believed that the difference caused by this discrepancy is small in comparison to the differences between rates of metabolism. This rate of oxygen uptake by viable isolated hepatocytes is presented in Table 4.4 and indicates that the viable hepatocytes consumed oxygen almost three times as fast as nonviable hepato- cytes. Oxygen utilization in the nonviable hepatocytes was probably due to non-biologic utilization of oxygen. The addition 176 TABLE 4.3 Comparison of Palmitic Acid Oxidation by Bovine Liver Slices and Isolated Bovine Hepatocytes 14 l- C-Palmitate Slices Cells oxidation to Wet Weight Drngeight Dry Weight 14 a C02 0.36 i .04 3.16 i .50 14.90 i 2.34 14C-Acid Soluble Metabolites 1.16 + .16 10.77 12.03 47.99 + 8.18 aMean :SE in pmoles 1-14C palmitate oxidized to product/min/mg tissue of quadruplicate incubations from six different tissue preparations. b Significantly different from slices, (P.05). eNot significantly different from tissue (P >.10). 180 TABLE 4.6 Epoxidation of Aldrin By Bovine Isolated Hepatocytesa Incubation Aldrin Delivery Dieldrin Aldrin to Conversion Time added solvent found dieldrinC rate Min. ug -- ug % conver. ug/min/g (viable) dry weight 15 50 ETOH 0.84 2.47 6.4 30 50 ETOH 1.2 3.24 4.6 30 50 DMSO 0.68 1.67 2.6 15 20 ETOH 0.68 5.01 5.2 30 20 ETOH 0.65 5.10 2.5 15 10 ETOH 0.57 6.35 4.4 30 10 ETOH 0.40 4.88 1.5 30 10 DMSO 0.60 5.77 2.3 a5.6 to 10.4 x 106 viable hepatocytes per culture. bEthyl alcohol (ETOH) or dimethyl sulfoxide (DMSO). CNo conversion occurred in cultures containing an equivalent weight of intact nonviable isolated hepatocytes. 181 DISCUSSION AND CONCLUSIONS As shown in Figure 4.1,the isolated hepatocytes were in a morphologically intact state having normal membranes and organelles. However, swelling of the end0p1asmic reticulum is evident. Whether these swollen end0p1asmic reticulum contain lipid or whether they would return to normal with time is not known. High yields of cells are desirable, however, yield should not be used solely to judge the quality_of hepatocyte prepara- tions. Biochemical and physiological function are much more useful to ascertain hepatocyte quality since cell yield depends on factors which have no major influence on the viability of isolated cells. Some factors which affect yield include the number of washing (purification) steps and the centrifugation speed. Seglen (1973b) reported an increase from 30 to 90% in liver weight recovered as suspended cells by decreasing the centrifugation force and number of washing steps. Accompanying this decrease in cell yield was an increase in the purity of parenchymal cells from 80% to greater than 98%. Trypan blue exclusion is not a reliable indicator of cell function (Krebs 23 al., 1979). Vital stains indicate only irreversible damage to cells. Also, staining can be affected by environmental factors such as pH (Baur et_§l., 1975). It has been the author's experience that trypan blue accurately identifies cells that are nonviable, but it sometimes gives false negative (nonstaining)results as compared with other viability measurements such as gluconeogenesis or oxygen 182 utilization. Because of this, it is suggested that several cellular function tests be used to adequately judge hepa- tocyte viability. Perhaps the most sensitive indicator of cellular via- bility is gluconeogenesis from precursors such as lactate (Krebs gt gt., 1979). To synthesize glucose from lactate, a complex anabolic process, cells must possess intact cellular components and metabolic processes such as mitochondrial and plasma membranes, numerous enzyme functions, adequate energy supply and regulatory mechanisms (Hemes gt gt., 1966; Krebs gt gt., 1979). The rates of gluconeogenesis achieved (Figure 4.2 and Table 4.2) are consistent with those for ovine isolated hepa- tocytes fortified with lOmM lactate, (Ash and Pogson, 1977) a gluconeogenesis rate of 1.41 umoles of glucose formed/minute per g of liver dry weight. Also in ovine isolated hepato- cytes,and fortified with lOmM lactate, Clark gt gt. (1976) reported gluconeogenesis rates ranging from 1.6 to 2.2 umoles/ minute per g liver (dry weight). Clark gt gt.,(l976) also evaluated a whole liver per- fusion technique to measure glucose formation from lOmM lactate. With this procedure, Clark obtained gluconeogenesis rates ranging from 1.1 to 2.5 umoles/minute per g liver (dry weight). Cook (1966) reported that the total body glucose turnover rate in a cow was 0.873 grams/min. This figure can be used to calculate umoles of glucose formed/minute per g of liver (dry weight) assuming a cows liver weight at 0.75% to 1% of body 183 weight (Cook, 1966; Kinzell gt gt., 1981) and a wet weight to dry weight conversion factor of 3.7 (determined from the experiments). Thus, a range of gluconeogenesis rates of be— tween 4.3 and 5.7 umoles glucose formed/minute per g of liver (dry weight) is derived. This range of calculated gluconeo- genesis rates is consistent with the data, especially when glucose production from both exogenous and endogenous sources are added together. Taken together, trypan blue dye exclusion, gluconeogenesis, ketogenesis and oxygen uptake results provide excellent evidence that the isolated hepatocytes are viable. The main purpose for establishing a method for isolation of viable and functional hepatocytes from bovine liver was to provide a system for studying xenobiotic metabolism 12.21EEQ' Because of significant differences among Species in the meta- bolism of xenobiotics (Kulkarni and Hodgson, 1980), it is best to employ the species of research interest in order to obtain relevant data. Although sometimes preferred, xenobiotic studies carried out in a live animal such as the cow are impractical. Isolated rat hepatocytes have been used in xenobiotic metabolism studies (Dougherty gt gt., 1980; Salocks gt gt,1981) and should become even more practical for studies in nonlaboratory Species such as cattle. Since statistically significant changes in cytochrome levels were not detected, neither cytochrome, P-450 or b5, should be rate limiting in short studies of xenobiotic metabol- ism. There were, however, numerical reduction of P-450 in 184 some cell preparations. This phenomenon was noted in rats (MichalOpoulos gt gt., 1979) in which only 10% of the ori- ginal cytochrome P-450 remained after four days of culture. In humans, 50% remained after four days (Strom gt gt., 1982). These findings illustrate the importance of short xenobiotic metabolism studies until P—450 either can be induced in culture or maintained longer. Research indicates that maintenance of P-450 may soon be possible. Nutritional requirements (Paine and Hockin, 1980) are being defined so P-450 can be main- tained 12 XiEEQ- Additives such as ascorbic acid (Bissel and Guzelian, 1979), 5-aminolevulinic acid, and various hormones (Micha10poulos gt gt., 1976; Decad gt gt., 1977) also seem to be beneficial. Losses of cytochrome P-450 during the postisolation period is the major problem in using isolated hepatocytes for metabolism studies. Most of the variance in cytochrome levels accounted for by statistical analysis was attributed to the particular tissue used for analysis (28.2%) and was not due to the amount of time elapsed after removal of the liver (14.5%). This indicates that the majority of the cytochrome lost was due to something associated with the tissue. It is suspected that cytochrome P-450 may be lost from nonviable and damaged cells and is being removed during the washing procedure. The aldrin epoxidation model illustrates how intact isolated hepatocytes can be used to activate chemicals 12 gittg. This activation system may more accurately model chemi- cal activation in mammals than do microsomal systems. 185 Studies using isolated hepatocytes are rapidly emerg- ing as an effective approach for evaluating biochemical path- ways. This chapter provides a description of the means in which viable isolated hepatocytes can be easily prepared from large animals. Bovine isolated hepatocytes should be useful to the bovine nutritionist who is studying metabolism kinetics, as well as to the toxicologist. SUMMARY This research supports the following conclusions: First, exposure to environmentally relevant levels of penta- chlorophenol (less than 1 mg/kg body wt/day) does not adversely affect the immune system of either lactating adults or bull calves. This statement does not extend to all situations of pentachlorophenol exposure. Exposure to pentachlorophenol may cause altered host resistance when acting concomitantly with another stress factor such as inadequate nutrition, poor ventilation, or simultaneous eXposure to other toxic substances. Second, at high levels of pentachlorophenol ingestion (greater than 1 to 2 mg/kg body weight per day in calves) the immune system is adversely affected. Prolonged exposure to pentachlorOphenol at these levels causes thymic atrOphy, and a decreased lymphocyte blastogenesis in thymocytes. Clinico- pathological changes include elevation of serum gamma-glutamyl transferase, a result of microsomal induction by the dioxins and other contaminants in pentachlorophenol, and reductions in serum total protein and albumin. The reduced total protein and albumin is probably a secondary effect associated with protein deficiency caused by reduced feed intake and gastointestinal irritation. These are both attributable to pentachlorophenol ingestion. High levels of pentachlorophenol also inhibit shape 186 187 change in bovine neutrOphils £2.XEE£91 However, no effect was seen in any neutrOphil parameter studied 12 yizg_and neutrOphil chemotaxis was not studied. Third, it is apparent that adverse effects of penta- chlorOphenol exposure were caused more by the toxic impurities than the pentachlorophenol itself. 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