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DATE DUE DATE DUE DATE DUE , MAG 1:; 2 [7'15 AFDA THE S 6 [add MSU Is An Affirmative Action/Equal Opportunity Institution c:\circ\dutoduo.pm3-o.‘ WITHDRAWAL OF POLYCHLORINATED BIPHENYL (PCB) AND POLYBROMINATED BIPHENYL (PBB) RESIDUES FROM RATS USING FEED RESTRICTION AND/OR MINERAL OIL IN THE DIET BY Patricia A. Wiggers A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1990 ABSTRACT WITHDRAWAL OF POLYCHLORINATED BIPHENYL (PCB) AND POLYBROMINATED BIPHENYL (PBB) RESIDUES FROM RATS USING FEED RESTRICTION AND/OR MINERAL OIL IN THE DIET BY Patricia A. Wiggers Rats were fed diets containing 10 mg Aroclor 1254/kg diet or 10 mg fireMaster‘Eful/kg diet for 14 days followed by 21 days of withdrawal treatment involving 50% feed restriction, 5% mineral oil, or a combination of the two treatments. PCB and. PBB residues in ground whole-body rat samples were determined using gas chromatography. Body burdens of PCBs and PBBs on day 0 withdrawal were 1505 and 181 ug/rat, respectively. Feed restriction or mineral oil alone significantly (p< 0.5) reduced PCB body burdens by approximately 27%. The combination of feed restriction and mineral oil enhanced withdrawal of PCB and PBB body burdens significantly' (p< 0.5), with. losses of 49.8% and. 45.4%, respectively. In memory of my grandfather, Alfred R. Stanley, PhD, whose search for knowledge I share. ii ACKNOWLEDGEMENT S I would like to thank the members of’ my guidance committee, Dr. Donald Polin, Dr. Steven Bursian, and Dr. Patricia O’Handley, for their assistance during the preparation of this manuscript. Special thanks goes to my major professor, Dr. Polin, for his encouragement, patience, and faith in my ability to complete this degree. I would also like to thank Dr. Richard Leavitt, Bob Kon, Bob Schuetz, and Leister Geissel of the Pesticide Research Center for ‘their- valuable assistance in. teaching' me 'the preparation and.method of analysis of samples for PBB and PCB residues. My fellow graduate students, Ellen Lehning, Barb Olson, Natalie Biondo, and Mike Underwood, deserve thanks for their input and help along the way. Special thanks go to my parents, sisters, the Lintzenich family, Carol Gamlen, and Rich Hammer for their support, encouragement, and confidence in me. iii ACKNOWLEDGEMENTS I would like to thank the members of my guidance committee, Dr. Donald Polin, Dr. Steven Bursian, and Dr. Patricia O’Handley, for their assistance during the preparation of this manuscript. Special thanks goes to my major professor, Dr. Polin, for his encouragement, patience, and faith in my ability to complete this degree. I would also like to thank Dr. Richard Leavitt, Bob Kon, Bob Schuetz, and Leister Geissel of the Pesticide Research Center for 'their 'valuable assistance in “teaching' me 'the preparation and method of analysis of samples for PBB and PCB residues. My fellow graduate students, Ellen Lehning, Barb Olson, Natalie Biondo, and Mike Underwood, deserve thanks for their input and help along the way. Special thanks go to my parents, sisters, the Lintzenich family, Carol Gamlen, and Rich Hammer for their support, encouragement, and confidence in me. iii TABLE OF CONTENTS Page LIST OF TABLES .......................................... vi LIST OF FIGURES ......................................... Vii INTRODUCTION ............................................ 1 LITERATURE REVIEW ....................................... 4 Chemical Properties of PCBs ............................ 4 Production and Uses of PCBs ............................ 6 Environmental Distribution and Metabolism of PCBs ..... 8 Distribution, Metabolism, and Excretion in Animals ..... 9 Toxicity of PCBs ...................................... 12 Chemical Properties of PBBs ........................... 18 Production and Uses of PBBs ........................... 20 Environmental Distribution and Metabolism of PBBs ..... 21 Distribution, Metabolism, and Excretion in Animals .... 22 Toxicity of PBBs ...................................... 23 Enhanced Withdrawal of Xenobiotics .................... 32 MATERIALS AND METHODS ................................... 38 Experimental Methods .................................. 38 Lipid and Water Determination on Tissue Samples ....... 42 Gas Chromatographic Analysis of PCB and PBB Residues in Rat Whole-Body Samples ................................ 44 Statistical Analysis .................................. 51 RESULTS ................................................. 52 Residues and Body Burdens of PCBs and PBBs ............ 52 Gas Chromatographic Peaks for PCBs and PBBs ........... 58 Feed Intake, Body Weights, and % Lipid ................ 60 DISCUSSION .............................................. 68 APPENDICES .............................................. 72 A. Data for feed consumption and body weight gains of rats fed PBBs and PCBs for 14 days, and then after withdrawal of these xenobiotics for 21 days ...... 72 iv B. Coding of rats by cage, treatment, day killed, and for residue analysis ............................. C. Raw data for percent water and percent lipid ...... BIBLIOGRAPHY ............................................ Table 1. 2. LIST OF TABLES Page Experimental design ............................... 40 Residues and body burdens of PCBs and PBBs in rats fed non-contaminated diet or diets containing 10 ppm PCBs or PBBs for 14 days (day 0 withdrawal) .... 53 Residues and body burdens of PCBs and PBBs in rats fed non-contaminated diet or diets containing 10 ppm PCBs or PBBs for 14 days followed by a 21 day withdrawal period involving no treatment, 5% mineral oil (MO), 50% feed restriction (FR), or 10% MO and 50% FR (day 21 withdrawal) .................. 54 PBB residues and body burdens in rats fed 10 mg/kg PBB in the diet for 14 days followed (on day 0) by a 21 day withdrawal involving no treatment, 5% mineral oil (MO), 50% feed restriction, or a combination of 10% MO and FR (FR + MO)-based on multiple peaks excluding peak # 4 ................................. 57 Total PBB residues and body burdens (based on all peaks) in rats fed PBBs in the diet at 10 mg/kg for 14 days followed (on day 0) by a 21 day withdrawal involving no treatment, 5% mineral oil (MO), 50% feed restriction (FR), or a combination of 10% MO and 50% FR (FR + MO) ............................... 58 Feed intake, body weights, and % lipid of rats fed non-contaminated diet or diets containing 10 ppm PCBs or PBBs for 14 days (day 0 withdrawal) ........ 63 Feed intake, body weights, and % lipid of rats fed non-contaminated diet or diets containing 10 ppm PCBs or PBBs for 14 days followed by a 21 day withdrawal period involving no treatment, 5% mineral oil (MO), 50% feed restriction (FR), or a combination of 10% MO and 50% FR (MO + FR) ..... 65 vi Fi 1. LIST OF FIGURES re Page Gas chromatograms to measure peak # 4 of a 0.1 ug PBB/ml standard and a rat whole-body extract (at 1/3 dilution), day 21-no withdrawal treatment .. 59 Gas chromatograms to measure multiple peaks (excluding peak # 4) of a 0.5 ug PBB/ml standard and a rat whole-body extract, day 21-no withdrawal treatment .......................................... 61 Gas chromatograms to measure peaks of a 1.2 ug PCB/ml standard and a rat whole—body extract, day 21- 10% mineral oil and 50% feed restriction ........... 62 vii INTRODUCT I ON In July 1973, 10-20 50# bags of fireMaster‘Efndq a fire- retardant containing polybrominated biphenyls (PBBs) , was accidentally mixed into Michigan Farm Bureau Service’ 5 (Battle Creek, MI) dairy pellets instead of Nutrimasterfi a magnesium oxide supplement. Both fireMaster‘ETHl and.Nutrimaster'were made by Michigan Chemical Company (St. Louis, MI) and packaging was identical, except for the name. The mix-up and the nine month delay in determining the cause of the toxicity observed resulted in widespread PBB contamination in Michigan. The result was the destruction of approximately 30,000 cattle, 5,900 swine, 1,470 sheep, and 1,500,000 chickens (FDA, 1975; Carter, 1976). The quarantine, destruction of animals, disposal of milk, eggs, and feed, and the cleanup resulted in losses of $75-100 million or greater (Carter, 1976). Several thousand farm families and their neighbors had consumed.meat, eggs, and milk contaminated with PBBs. The level of exposure of the general public was less due to the mixing of milk (Carter, 1976). At about the same time, the toxic effects of polychlorinated biphenyls (PCBs) were being examined. PCBs l 2 were used in industry worldwide and had. become widely distributed in the environment. PCB residues had been found in sediments, fish, wildlife, domestic animals and humans (Hutzinger g; l., 1979; Kimbrough, 1980; Safe, 1984). Both.PCBs and.PBBs are very stable, resist breakdown, and therefore persist in the environment. They are both lipophilic, bioaccumulate in the adipose tissue, and remain in the body indefinitely. As PCBs and PBBs produce toxic effects and have been identified as possible carcinogens (Kimbrough, 1980; Safe, 1984), it would be advantageous to be able to remove them from the body. In addition, many high quality' breeding animals and. animals 'with. 10W’ level iPBB contamination had to be destroyed following the Michigan PBB accident. They may have been salvaged if some method of removal was available. Several studies have shown that the use of mineral oil in conjunction with feed restriction enhanced PBB and PCB elimination (70-80% eliminated in 21 days) in. chickens (Polin and Leavitt, 1984; Polin _e__t;_ $4 1985; Polin t al., 1989). The objectives of this study were: 1) To quantify the PCB and PBB residues in ground whole-body rat samples obtained from rats fed diets containing 10 mg PCBs or PBBs/kg diet. 2) To determine if addition of 5% mineral oil to the diet during the 21 day withdrawal period would enhance elimination of the PCB or PBB residues from rats. 3) 4) 3 To ascertain if 50% feed restriction would enhance the elimination of PCB or PBB residues from rats over a 21 day period. To determine if 50% feed restriction in combination with 10% mineral oil in the diet enhanced elimination of PCB or PBB residues from rats during a 21 day withdrawal period. L I TERA‘I'URE REVIEW I. Polychlorinated Biphenyls A. Chemical Properties of PCBs Polychlorinated biphenyls (PCBs) are chemical compounds with the empirical formula anuHrum, where n=l-10. Theoretically, there are 209 possible PCB congeners, although at least 20 have not been found in any technical PCB mixture analyzed. Monsanto Chemical Company produced PCBs in the United States under the tradename, Aroclor. Aroclors contain a mixture of different PCBs, which are specific to a particular Aroclor. Production of these mixtures of PCBs is by chlorination of biphenyl with subsequent separation and purification of the desired chlorinated biphenyl fractions. Contaminants which include polychlorinated dibenzofurans and polychlorinated naphthalenes are occasionally present and vary from batch to batch. The first two numbers of the Aroclors, with the exception of 1016, indicate the number of carbons in the biphenyl molecule, and the last two numbers indicate the percent chlorination by weight. Individual chlorinated biphenyls are colorless crystals in their pure form, and commercial mixtures (i.e. Aroclors) are liquids due to 4 5 depression of the melting point occurring with.mixing of'PCBs. These commercial mixtures of' PCBs have ‘properties which include thermal stability, resistance to chemical and biological degradation, low water solubility, high dielectric constants, high electrical resistivity, stability' to conditions of oxidation and hydrolysis encountered in industrial use, and low vapor pressures. Water solubility, vapor pressure and the ability to be degraded decrease as the chlorination of the compound increases. Aroclor 1254, which was used in the present study, is a light-yellow viscous liquid. It is soluble in ethyl acetate and very soluble in toluene. Aroclor 1254 has the following properties: Chlorine % = 54% Specific gravity = l.495-l.505 (65°/15.5°C) Density = 12.82 lb/gal at 25%: Distillation range = 365-390%: Viscosity = 1400-2500 sec at 37.8%: Vapor pressure = 1.8 x 10“ mmHg at 20%: Vaporization rate 0.053 mg/cmz/hr Pour point = 10°C Principal components = CL.--Cl6 # of components reported = 27-116 Flash and Fire points none to boiling Dielectric constant = 5.0 at 20%3, 4.3 at 100%: Aroclor 1254 is primarily pentachlorobiphenyl and contains 54% chlorine. At least 85 components have been detected in Aroclor 1254 using high resolution capillary columns, although with gas chromatographic analysis using packed columns there are 12 to 15 peaks present on the 6 chromatogrann Webb and McCall (1973) described 13 peaks where the number of chlorines on the biphenyl for each peak was determined. Peaks 1-3 contained tetrachlorobiphenyls, peaks 4-5 contained 25% tetra- and 75% pentachlorobiphenyls, peaks 6-8 contained.pentachlorobiphenyls, peaks 9-10 70% penta- and 30% hexachlorobiphenyls, peaks 11-12 contained hexachloro- biphenyls, and peak 13 contained heptachlorobiphenyls. The preceding information was taken from Webb and McCall (1973), Mieure et a1. (1976), Hutzinger gt _1. (1979), Kimbrough (1980), Richardson and Waid (1982), Safe (1984), Erickson (1986) and Alford-Stevens (1986). B. Production and Uses of PCBs PCBs were produced under the tradename Aroclor in the U.S. by Monsanto Chemical Company from 1929 through 1977. From 1971 to 1973'approximately 1 million pounds of PCB-based heat-transfer oil were manufactured by Geneva Industries (Houston, TX). In the period 1930 to 1975, total production of PCBs in the U.S. was 1400 million pounds. Imports equalled 3 million pounds, domestic sales were at 1253 million pounds, and exports equalled 150 million pounds. In April 1971, Monsanto voluntarily ceased PCB production that was for use in open-ended or nominally closed systems. Production and sales of PCBs were at the maximum in 1970 and by 1974 had declined to one-half that level. Monsanto ceased production of PCBs in mid-1977 and had shipped its last inventory by October 1977. Non-U.S. production of PCBs was 80-85 million pounds annually prior to 1971. In 1971, production was 100 million pounds. Production fell after 1971 to 43 million pounds in 1973 and to 30 million pounds in 1976. Japan was a major producer of PCBs sold under the tradename of Kanechlor, from 1954 to 1972. Other producers of PCBs were the German Federal Republic, France, Italy, and the USSR” Total world production through 1980 was 2.4 billion pounds. PCBs were used as coolants and dielectric fluids in transformers and. capacitors, heat transfer fluids, flame retardant coatings for'wood products, components of carbonless paper, paints, inks, dust control agents, pesticides, hydraulic fluids, and lubricants, plasticizers in rubbers and resins, adhesives, and as wax extenders. By far the greatest single use of PCBs was in capacitors for fire protection and increased service life, although in 1968 to 1971, use as plasticizers was the largest. In 1976, the manufacture, processing, distribution and use of PCBs, except in totally enclosed systems (transformers, capacitors, and electromagnets) was banned by Congress. This was in response to evidence that PCBs were possible promoters of cancer. The information on production and uses of PCBs was compiled from Hutzinger e; 1. (1979), Kimbrough (1980), 8 Richardson and Waid (1982), Safe (1984), Erickson (1986), and Alford-Stevens (1986). C. Environmental Distribution and Metabolism of PCBs Due to the worldwide use and production of PCBs, they can be detected in nearly all niches of the global ecosystem. Residues have been found in highly industrial areas to remote areas like the Arctic. PCB residues have been detected in sediments, fish, wildlife, domestic animals, and humans. The highly lipophilic nature of PCBs is evident by high residue levels due to bioaccumulation in fat of carnivores. The resistance of PCBs to breakdown by acids, bases, heat, light, oxidizing and reducing agents contributes to their stability and environmental persistence. Uptake by plants does not readily occur. The most important method for the destruction of waste PCBs seems to be thermal degradation at temperatures of greater than 800%3, which results in the formation of organic compounds like C0, C02, HCl, and C12 (Hutzinger et, al., 1979; Kimbrough, 1980; Safe, 1984). Photolysis occurs under laboratory conditions with.the primary reaction being reductive dechlorination. Chlorines in the 921:; positions are lost preferentially and dechlorination occurs more rapidly in polar solvents (Hutzinger-e§_al., 1972; Ruzo and Zabik, 1975; Kimbrough, 1980). 9 D. Distribution, Metabolism, and Excretion in Animals The distribution and metabolism of PCBs in animals has been studied by many researchers. When i.v. doses of 2,4,5,2',4’,5’-hexachlorobiphenyl (6-HCB) were given to 1., 1984), 1., 1982; Ryerson e; beagles, monkeys (Sipes e; and rats (Birnbaum, 1986), it was found that 70-82% of 6-HCB in the blood was redistributed to the liver and muscle within 30 minutes to 2 hours. By 24 hours, 6-HCB was redistributed from the liver and muscle to fat, omentum and skin. The fat continued to accumulate 6-HCB over the course of the study (up to 90 days in the monkeys). In all three species, the major route of excretion was found to be through the feces, with a small amount being excreted in the urine. The dog excreted 66% of the total dose of 6-HCB in 15 days, the monkey 18% in 90 days, and the rat 2% in 21 days. The greater excretion of 6-HCB by the dog appeared to be due to a higher rate of biotransformation, as the quantity of metabolites found in the blood of dogs was 4-8 times that found in monkeys. In the dog, there was no significant enterohepatic circulation, whereas the monkey had more parent compound in the bile than was excreted in the feces, indicating that reabsorption occurred. In the rats, the fecal excretion was primarily of metabolites. Other PCBs were also studied for distribution and metabolism in animals. Birnbaum (1986) injected rats i.v. 10 with 2,3,6,2’,3',6’-hexachlorobiphenyl. Greater than 50% of radiolabeled 2,3,6,2’,3’,6’-HCB was metabolized and excreted via the bile into the feces within 2 days (Birnbaum, 1986). Yoshimura and‘Yamamoto (1975) injected rats which had the bile duct ligated with 2,4,3’,4’-tetrachlorobiphenyl (2,4,3’,4'- TCB) i.v. They found an average of 0.6% of the dose was excreted unchanged from the small intestine daily. No other parts of the gastrointestinal tract were found.to be secreting the parent compound or metabolites, indicating that the small intestinal wall serves as a major site of secretion of unchanged 2,4,3’,4’-TCB. Several studies were done to determine the role of lipoproteins in the mobilization and distribution of 6-HCB in the rat and human. It was found (Vomachka g al., 1983; 1., 1984; Rau and Vodicnik, 1986) that Spindler-Vomachka g; hyperlipidemic conditions in humans and rats, like those occurring during pregnancy, cause an increase in the release of 6-HCB from the liver in association with very low density lipoproteins (VLDL). The distribution of 6-HCB within the different fractions of plasma (VLDL, low density lipoproteins or LDL, high density lipoproteins or HDL, and the bottom fraction consisting of albumin and corticosterone- binding globulin) was found. to be dependent on the content of triglyceride (TG) and protein in the plasma. Therefore, it appeared that 6-HCB was released from hepatocytes in 11 association with newly synthesized TG, and then distributed in the circulation based on the TGzprotein ratio. Kimbrough (1980) and Erickson (1986) reviewed the in vivo metabolism of individual PCB congeners and indicated that phenolic products were the major metabolites with lesser amounts of sulfur metabolites (methylsolfones), trans- dihydrodiols, polyhydroxylated PCBs and their methyl ether derivatives, and ring-degraded microbial oxidation products. Several rules appear to describe the metabolism of PCBs: 1. Hydroxylation is favored at the Bag position of the less chlorinated phenyl ring unless this site is hindered. sterically (ie. 3,5-dichloro substituted. congener). 2. The para position of both biphenyl rings and.the carbon atoms, which are para to the chloro substituent in the lower chlorinated biphenyls are all readily hydroxylated. 3. Oxidative metabolism of the PCB substrate is enhanced by the availability of 2 neighboring unsubstituted carbon atoms (especially Cg 67-90% of control levels. Colestipol and petroleum jelly were the least effective. From the data provided. in ‘the combination. experiments above, it appears that the use of feed restriction in combination with bile-binding resins or lipotropic agents cause the greatest reductions in the body burdens of lipophilic xenobiotics that accumulate and persist in the body. MATERIAL AND METHODS I. Experimental Methods A. Preparation of Experimental Diets The residue build-up phase of this study used a PCB- contaminated diet, a PBB—contaminated diet and a non- contaminated diet. The non—contaminated diet used for all three diets was Purina Certified Rodent Chow #5002. The rodent chow, in large pellet form, was ground to a mash using a Hammermill feed grinder to facilitate mixing of PCBs or PBBs evenly throughout the diet. Non-contaminated diet consisted of ground rodent chow only. A PCB-contaminated diet was formulated using Arochlor 1254 (Monsanto Chemical Corp., St. Louis, MO), a commercial mixture of polychlorinated biphenyls. Incorporation of Aroclor 1254 into the ground rodent chow was accomplished by diluting a weighed amount of Arochlor 1254 into a measured.volume of hexane. The volume needed to make 10 ppm in the diet was blended into a premix composed of finely ground rodent chow. The premix was then mixed into 4 kg ground rodent chow by tumbling for 5 minutes in a modified paint tumbling machine. fireMaster‘ FF-l (Michigan Chemical Co., St. Louis, MI), a commercial mixture of polybrominated biphenyls, was the source of PBBs. The PBB material used was obtained from the original batch of PBBs 38 39 accidentally introduced into Michigan agriculture in the fall of 1973. The same mixing method was used to prepare the PBB- contaminated diet as was used to prepare the PCB-contaminated diet. The final concentration of PBBs in the diet was 10 ppm. The withdrawal phase of this study required the preparation of non-contaminated diet with mineral oil at 0, 5 or 10% by weight. The mineral oil was obtained from the Veterinary Clinical Center at Michigan State University. It was mixed thoroughly into the diet by hand-stirring the mixture in a 20 gallon plastic container. B. Husbandry Sprague-Dawley male rats, 12 weeks of age and weighing 300-350 grams, were received through University Laboratory Animal Resources (ULAR) at Michigan State University from a commercial breeder. All the animals fed PCBs or PBBs were isolated in a room at a ULAR facility located in the MSU’s Clinical Center. Rats were housed three per polypropylene cage, with wire tops, measuring 33 x 38 x 18 cm. Ground corn cobs were used as bedding which was changed two times per week. Artificial lighting was supplied on a schedule of 16 hours light : 8 hours dark. The temperature was maintained.at 22 +/- ZTL Water was provided ad libitum. C. Schedule Rats were received on November 12, 1985 and were allowed a week to adapt to their new environment and.experimental conditions. The experimental design is presented in Table 1. 40 Table 1. Experimental design. Cage1 Contamination Withdrawal Numbers Diet Treatment (14 day period) (21 day period) Control rats: 7,11,15 Non-contaminated None2 1,2,3 Non-contaminated None3 4,5,6 Non-contaminated 50% feed restriction 8,9,10 Non-contaminated 5% Mineral oil 12,13,14 Non—contaminated 10% Mineral on @ 50% FR4 PBB rats: 22,26,30 10 ppm PBBs None2 16,17,18 10 ppm PBBs None3 19,20,21 10 ppm PBBs 50% feed restriction 23,24,25 10 ppm PBBs 5% Mineral oil 27,28,29 10 ppm PBBs 10% Mineral on @ 50% FR4 PCB rats: 34,38,42 10 ppm PCBs None2 31,32,33 10 ppm PCBs None3 35,36,37 10 ppm PCBs 50% feed restriction 39, 40,41 10 ppm PCBs 5% Mineral oil 43,44,45 10 ppm PCBs 10% Mineral on @ 50% FR4 1 Rats were housed three per cage. 2 The three rats in each of these cages were euthanized at the end of the 14 day contamination phase and analyzed for PCB and PBB residues. Rats in these cages were fed ad libitum. 4 FR = feed restriction. b.) 41 The contamination phase of the experiment lasted 14 days, during which time PCB— or PBB-contaminated diets were fed. The rats received feed ad libitum. Rats in fifteen cages (45 rats) were on each diet including the control diet at this time. Feed intake was determined two times per week and body weights were obtained weekly (see appendix A for data on feed consumption and body weights). On day 14 of the contamination phase, after an overnight fast, three rats from three different cages from each of the three treatment groups were randomly selected and euthanized.bloodlessly with excess C02. The three euthanized rats from each cage were sealed in a plastic bag and frozen at -20%:. They were later prepared for analysis of PCBs, PBBs, lipid and water content as described in the section on tissue preparation. After completion of this phase of the experiment, the room was thoroughly cleaned to remove all PCB and PBB contamination. During the 21 day withdrawal phase, the rats were fed non-contaminated diet containing 0, 5 or 10% (by weight) mineral oil. Three rats from three different cages previously fed each of the contamination diets and from three cages fed the non- contaminated diet were fed non-contaminated diet containing 0% mineral oil, ad libitum (Table 1). Nine rats from another three cages from each of the original three diet groups were fed non- contaminated diet at a 50% feed restriction. As indicated in Table 1, nine rats from three more cages from each group were fed 5% mineral oil diet ad libitum, while the nine rats in the final three cages from each group were fed a diet containing 10% 42 mineral oil at a 50% feed restriction. The 50% feed restriction was based on feed intake of ad libitum rats which were measured every other day. On day 21 of the withdrawal phase all remaining rats were euthanized with excess C02. The three rats from each cage were put in a plastic bag and frozen at -20°C. Later, preparation of the rats for analyses was conducted as described in the section on tissue preparation. Appendix B outlines the coding and treatment associated with each cage of rats. D. Safety Methods and Contaminated Waste Disposal Protective clothing was mandated for all personnel who entered the animal room at the ULAR facility. All cages and other equipment used for the experiments were rinsed with hexane prior to their removal from the room. Bedding, feces, and all inorganic and organic waste were sealed in 50 gallon plastic or steel barrels. Hexane used to rinse equipment and cages was collected and sealed in plastic jugs. The barrels and plastic jugs were disposed of by Michigan State University’s Office of Radiation, Chemical and Biological Safety in accordance with state and federal regulations. II. Lipid and Water Determination on Tissue Samples A. Tissue Preparation Plastic bags containing the three rats from each cage were removed from the freezer and thawed overnight at 4°C. Carcasses were then cut into several small pieces with a Hobart 5212 F electric saw, and ground to a hamburger-like consistency 43 in a Hobart 4732 SS electric meat grinder. Samples were put through the grinder five times to produce a homogeneous blend of the three rats from each cage. Grab samples were removed and two Nasco whirl-pakiibags were filled and labelled for each set of rats. Samples in whirl-paksa'were refrozen and stored at -20%:. B. Water Determination The percent water in whole-body samples was determined by weighing out approximately 60 grams of the ground sample, to the nearest 0.1 gram, into a tared aluminum dish. The sample in the dish was covered with cheesecloth, to prevent sample loss. The labelled aluminum dishes containing samples were placed into a Virtis ZSSRC freeze drier for 48 hours, to reach a constant weight. The weights of the dish and sample were taken immediately upon removal from the freeze drier to determine the weight lost as water. The percent water in the sample was then determined by dividing the lost weight by the initial carcass sample weight, and multiplying by 100%. See Appendix C for raw data on percent water. C. Lipid Determination Lipid was determined gravimetrically by soxhlet extraction of the sample using petroleum ether in a biosafety cabinet. Extraction thimbles were dried prior to use for 12 hours in a Precision Scientific Oven (either Model 19 or 26) at 80%:. Attare for each thimble was obtained.by weighing them dry. Approximately five grams of freeze-dried sample were added to each weighed thimble. Each of the freeze-dried samples was 44 analyzed in duplicate. The thimbles containing samples were then dried in the oven for 12 hours at 8GTL Samples in thimbles were removed from the oven, cooled in a desiccator and then immediately weighed to obtain a pre-extraction dry weight. Thimbles were placed in the soxhlet apparatus where samples were repeatedly extracted with petroleum ether for 18 hours. Then, thimbles containing extracted samples were set in racks under the fumehood to allow evaporation of petroleum ether. Once the ether had evaporated, samples were oven-dried at 8UTZfor 12 hours, and a post-extraction dry weight obtained. The formula for percent crude lipid is as follows: Percent crude lipid = Pre-extraction drv weight - Post-extraction dry weight X 100% Pre-extraction dry weight See Appendix C for raw data on percent lipid from whole- body ground rat samples. III. Gas Chromatographic Analysis of PCB and PBB Residues in Rat Whole-Body Samples A. Gas Chromatographic (GC) Conditions Residues of PCBs and PBBs in whole body samples were determined with a Varian Aerograph 3700 Gas Chromatograph with a 63Ni electron capture detector. Chromatograms were printed by a Varian 9176 Chromatogram.Recorder. For analysis of both PCBs and PBBs, the gas Chromatograph was equipped with a 6 meter x 2 millimeter i.d. glass column containing 3% OV-l liquid phase on 45 100/120 mesh Chromosorb W-HP solid support. conditions were as follows: Injector temperature Column temperature Detector temperature Carrier gas Carrier flow rate Chart speed Attenuation = For PBB analysis, conditions were Injector temperature Column temperature Detector temperature Carrier gas = Carrier flow rate = For PCB analysis, 220°C 200°C 270°C 99.99% pure Nitrogen gas 40 ml/min. 1.0 cm/min. 32 as follows: 270°C 250°C 27 0°C 99.99% pure Nitrogen gas 40 ml/min. Chart speed(single peak) = 1.0 cm/min. To prevent leakage of air through Chart speed(mu1tip1e peaks) = 0.5 in/min. Attenuation(single peak) 128 Attenuation(multiple peaks) = 16 septums multiple injections, they were changed weekly. B. General Procedures for Preparing Samples damaged. by for Gas Chromatographic Analysis PCBs and PBBs were analyzed in duplicate ground.whole- body samples chromatography. according to the following procedure: (coded for identification - see Appendix B) by gas They were extracted and the extracts clarified 1. Take a portion of thawed sample out of the whirl-pakR and chop it with a razor blade to a fine consistency. 2. From the finely chopped sample, a 50 ml Erlenmeyer flask. weigh out 2 grams into 3. Homogenize for one minute with 25 ml of toluene/ethyl acetate (1:3) solvent using a Tekmar tissuemizer. Db U1 46 Decant fluid through a 5 cm Buchner funnel containing solvent wetted 5 cm Whatman glass fiber filter, under vacuum, into a 250 ml filter flask, leaving any solid portion of the sample in the Erlenmeyer flask. Repeat steps 3 and 4 two more times. Combine all extracts in the filter flask. Rinse the Erlenmeyer flask and tissuemizer blades with toluene/ethyl acetate and collect that solvent as well. Pour combined filtered solvent through a glass funnel containing a small plug of glass wool and 5 grams of granular anhydrous sodium sulfate (NagKL, Mallinckrodt , Analytical grade) into a 250 m1 flat bottom flask with a 24/40 top. Rinse filter flask and funnel with three 5 m1 portions of solvent. Rinse joint of flat bottom flask and attach flask with a clip to a rotoevaporator unit. Rotoevaporate in a 350W3'waterbath to about 5 ml and pour through a small funnel into a 10 ml volumetric flask. Rinse the 250 ml flask and funnel with several small portions of solvent and add to the volumetric flask. Fill the volumetric flask to the 10 m1 line with solvent, cap, and seal with parafilm (American Can Company) and store refrigerated until further preparation. Five ml of the 10 ml from each sample were processed through a ABC Lab Autoprep 1001 gel permeation Chromatograph (GPC) using S-X3 Biobeads, 200/400 mesh packing, to remove high molecular weight lipids, etc. from the sample. The solvent used was toluene/ethyl acetate (1:3). GPC collection was made into a 250 ml flat bottom flask with a 24/40 top. GPC parameters were as follows: Dump 21 minutes Collect 15 minutes Wash = 5 minutes Flow rate = 5 ml/min. These parameters were determined with standards prior to doing samples to ensure that all the PCBs and PBBs residues were recovered. 9. The GPC output was rotoevaporated to approximately 5 ml, and transferred quantitatively with rinsing through a small funnel into a 10 m1 volumetric flask. Then, the solution in the volumetric flask was brought to 10 ml with toluene/ethyl acetate, and the contents transferred into a screw top tube that was stored in a refrigerator until analysis. 47 C. Preparation and Storage of PCB and PBB Standard Solutions and Spiked Samples Stock solutions of PCBs were made using Arochlor 1254 diluted with toluene/ethyl acetate (1:3). PCB stock solutions contained 12, 20 and 598 ug PCBs/ml. Standard solutions were made from these stock solutions by diluting the stock solutions with toluene/ethyl acetate (1:3) to pmoduce PCB standards of 0.24, 0.48, 0.84 and 1.2 ug/ml. Spiked samples were prepared from 1 ml of 12 ug PCB/ml stock, 7 ml of 1.2 ug PCB/ml solution, 4 ml of 1.2 ug PCB/ml solution and 2 ml of a 1.2 ug PCB/ml solution by adding these to 2 gram samples of ground non- contaminated whole body rat samples. After allowing the solutions to soak into the carcass sample for 10 minutes, they were processed as the regular samples to produce 1.2 ug/ml, 0.84 ug/ml, 0.48 ug/ml and 0.24 ug/ml, respectively, in the final 10 ml extract if 100% recovery occurred. PBB stock solutions were prepared from fireMaster‘ diluted in toluene/ethyl acetate (1:3). Stock solutions contained 10, 100 and 1000 ug PBB/ml. Standard solutions of 0.2, 0.5, 0.8 and 1.0 ug/ml were made by diluting the stock solutions with toluene/ethyl acetate to produce the proper concentration of PBBs. Spiked samples were prepared similarly to the PCBs to produce spikes of 0.2, 0.5, 0.8 and 1.0 ug/ml in the final extraction volume of 10 ml. All spiked sample extracts, stock and standard solutions were stored under refrigeration. 48 D. General Techniques for GC Injections All injections into the GC were made using Hamilton microliter syringes. Syringes were rinsed 20 times with toluene/ethyl acetate (1:3) before filling with a sample or standard for injection. The amount injected was determined by reading a total volume of solution prior to injection as compared to the volume remaining after injection into the GC. All samples and standards were warmed.to room.temperature prior to injection. The time needed for a sample to completely pass through the column was determined as follows. A.PCB-spiked sample and.a PBB- spiked sample, prepared earlier, were injected separately to determine the point where no more peaks appeared on the chromatogram” The point in time where no more peaks appeared was chosen to be the minimum analysis time for each PCB and PBB residue. E. Analysis of PCB Residues in Tissues Using the GC conditions as described for PCBs, three injections of approximately 7 ul of 1.2 ug/ml standard solution were injected in rapid succession to load the column, decreasing daily variation 1J1 detector response t1) standards. Once the column had been loaded and peaks ceased to form on the chromatogram, standards were injected, followed by samples and then another set of standards. This procedure was followed each day, with no:more than 10 samples being injected before standards were injected again. Standards for PCBs were 0.24, 0.48, 0.84 and 1.2 ug/ml. Quantification of PCB residues was performed by 49 measuring the peak heights of six major peaks (peaks #4-6,and 8,9 and 11) and peak areas for four smaller broad.peaks (#12-15) that appeared on chromatograms for both samples and standards with retention times of two to ten minutes. The heights or areas for each peak of each standard injected.on a particular day were used to determine a dose-response line for each peak. The equations for these dose-response lines were then used to quantitate the PCB residues in the final sample extracts by peak, after which the ug PCBs/ml for all peaks in the sample were totaled. The ug PCBs/ml calculated for final sample extracts was corrected for recovery based on the recovery of 84.8% determined from the spiked samples, which yielded the ug PCB/g tissue residues in the rat carcasses. Body burdens of PCBs were then determined as ug/rat by multiplying the ug PCBs/g tissue by the average weight of a single rat in each cage. F. Analysis of PBB Residues in Tissues Using the GC conditions as described for PBBs-single peak, three injections of approximately 7 ul of 1.0 ug/ml standard solution were injected in rapid succession to load the column. Once the column had been loaded and peaks had ceased to form on the chromatogram, standards were injected, followed by samples, and then another set of standards. This procedure was followed each day, with no more than 10 samples being injected before standards were injected again. Standards for PBBs were 0.2, 0.5, 0.8, and 1.0 ug/ml. Quantification of PBB residues was performed by measuring the peak area of one large peak (peak #4) 50 representing 2,4,5,2’,4’,5’-hexabromobiphenyl which is the major congener of fireMaster‘ETH1. To be able to measure this peak, the samples had to be diluted, 1:2 with toluene/ethyl acetate (1:3). Peak areas for the standards run each day were used to form a daily dose-response curve, allowing quantitation of PBB levels in the diluted final sample extracts. The ug PBB/ml calculated for these sample extracts had to be corrected for the one-third dilution and for 88.4% recovery determined from spiked samples. Once the corrections had been made, PBB residues in ug/g was the end result. Body burdens, as ug/rat, were determined by nmltiplying the ug PBB residue/g tissue by the average weight of a single rat in the cage the sample represented. The areas of 5 smaller peaks (#1-3,5, and 6) were measured without diluting the samples using the GC conditions described for PBB-multiple peaks. The same procedures for loading the column and order of injections were used as with the large peak analysis. Total peak areas for each standard were used to form a daily dose-response curve, with an equation allowing quantitation of PBB levels in final sample extracts. These values were corrected for recovery of 88.4%, to give ug/ml PBBs or residue of PBB in ug/g. Body burdens were calculated as above. 51 IV. Statistical Analysis All statistical analyses were performed using the computer program Statview 512+, marketed by Brainpower Inc., 24009 Ventura Blvd. Suite 250, Calabasos, CA 91302. RESULTS I. Residues and Body Burdens of PCBs and PBBs PBB residues and body burdens in this section are based on single peak measurement. Rats euthanized on day 0 of withdrawal, after 14 days feeding of diet containing 10 ppm PCBs (Aroclor 1254), or 10 txxn PBBs (fireMaster‘ FF-l), contained. average residue concentrations of 4.123 ug/g tissue or 0.500 ug/g tissue, respectively (Table 2). Those rats receiving gg libitum diet during the 21 day withdrawal period showed no significant (p> 0.05) loss of residues from day 0 levels, with residues of PCBs and PBBs of 3.967 ug/g and 0.499 ug/g, respectively (Table 3). Residues in rats restricted in feed intake during the withdrawal period were 3.675 ug PCBs/g tissue and 0.471 ug PBBs/g tissue (Table 3), representing insignificant (p> 0.05) losses of 7.4% and 5.6%, respectively. Mineral oil added to the diet, resulted in residues of 2.943 ug PCBs/g tissue (significant at pg 0.05) and 0.424 ug/g (non-significant at p> 0.05) PBBs (Table 3), equivalent to losses of 25.8% and 15%, respectively, over levels in rats receiving no treatment. The combination of feed restriction and mineral oil resulted in residue reductions of 35.9% and 32% (both significant at pg 0.05), to 2.543 ug/g and 0.339 ug/g (Table 3), over nontreated rats for PCBs and PBBs, 52 53 Table 2. Residues and body burdens of PCBs and PBBs in rats fed non-contaminated diet or diets containing 10 ppm PCBs or PBBs for 14 days (day 0 withdrawal). Residue of PBBs Body Burden Pre-treatment Diet1 Cage #2 or PCBs (ug/g b.w.)3 (ug/rat)4 Non-contaminated: 7 ND5 ND 11 ND ND 15 ND ND Mean i SE ND ND 10 ppm PBBs: 22 0.626 225 26 0.444 165 30 0.430 153 Mean i SE 0.5 i 0.063 181 -_+-_ 22 10 ppm PCBs: 34 4.253 1517 38 3.891 1463 42 4.225 1535 MeaniSE 4.123 ~_I-_ 0.116 1505 i 22 1 Pre-treatment diets were fed for 14 days prior to withdrawal phase. 2 Rats were housed three per cage and were analyzed as a composite. 3 Values represent the average concentration of PBBs/PCBs in whole body rat samples, as detected by gas chromatography. 4 Body burden was calculated by multiplying body weight (g) by residue of PBBs/PCBs in the sample (ug/g body wt.). 5 ND = not detectable; no peaks on the chromatogram. Residues manna: Source: di MS 1 df MS f PBBs: Between groups 1 0.375 62.73“ 1 49357.2 66.4“ Within groups 4 5.978E-3 4 743.1 PCBs: Between groups 1 25.499 1257.21 " 1 3397541.5 4837.4" Within groups 4 0.02 4 702.3 ' p s 0.05 n p s 0.01 54- mwdm mez 20 QmDZHBZOU .Emuooumeowco on» co mxmmm oc "oabmuoouoplcoc .c0fiuowwumou poow u mm «Hfio Howe: E." oz.m oz m .Efiuflnfld Um 09m v .A.3.b m\msv oHQEmm ecu cw mmOm no mmmm wo ospflmow an Am. ucmfloz moon ocaaamfluane an poumasoamo we: copusn zoom m .asmnumoumeowso and an pouoouop mm .mpadfipm you moon odor: ca mmom no mmmm mo coaumuucoocoo omnwo>m on» acomoumou mozam> N .ouamOQeoo m on poumamcm ouoz ppm mono Hod owns» condo: one: mumm H mm H can NON.o H mwm.N 44 H mofl Hmo.o H mmm.o oz oz mm.” amps who mm4.N m4 ~04 omm.o ow oz oz 44 was mom + 02 wow Hum p4m.~ 44 so oo~.o om oz oz ma woo woo + oz wow moo omo.m M4 mmH a04.o pm oz oz N4 was woo + or «OH 44.“ mmHH 4mo.o n.m4m.m NH n.oofln omo.o a 4~4.o oz oz om n.cpoz. 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In summary, combination of 50% feed restriction and 10% mineral oil in the diet produced significant (p5 0.05) reduction in both PCB and PBB residues, while mineral oil at 5% in the diet produced significant (pg 0.05) reduction only in PCB residues. Body burdens of rats on day 0 of withdrawal for PCBs were 1505 ug/rat and for PBBs 181 ug/rat (Table 2). Day 21 body burdens for rats receiving no withdrawal treatment were 1589 ug/rat and 198 ug/rat (Table 3) for PCBs and PBBs, respectively. These values did not represent a significant (p> 0.05) difference in PBB or PCB body burdens. Rats on 50% feed restriction during the withdrawal period had body burdens of 1149 ug PCBs/rat and 150 ug PBBs/rat (Table 3), which represent reductions of body burden greater than nontreated rats of 27.7% (significant at p5 0.05) and 24.2% (not significant at p> 0.05), respectively. Use of mineral oil alone resulted in body burdens of PCBs of 1153 ug/rat (reduction of 27.4% when compared to nontreated rats, significant at pg 0.05) and a reduction of PBBs to 169 ug/rat (less by 14.6% when compared to nontreated rats, not significant at p> 0.05) (Table 3). The combination of feed restriction and mineral oil resulted in significant (pg 0.05) reductions in body burdens of 49.8% and 45.4% or concentrations of 797 ug PCBs/rat and 108 ug PBBs/rat (Table 3), respectively. In summary, both PCB and PBB body burdens were significantly (pg 0.05) reduced by 50% feed restriction in conjunction with 10% mineral oil in the diet. Both feed restriction and mineral oil, alone, resulted in 57 comparable but significant (PS 0.05) reductions in PCB body burdens as compared to rats not receiving withdrawal treatment. When multiple peaks were measured for PBBs, excluding the largest peak that had been measured previously, the values for PBB residues and body burdens were as follows (Table 4). Table 4. PBB residues and body burdens in rats fed 10 mg/kg PBB in the diet for 14 days followed (on Day 0) by a 21 day withdrawal involving no treatment, 5% mineral oil (MO), 50% feed restriction (PR), or a combination of 10% MO and FR (FR + MO) -based on multiple peaks excluding peak #4. Day killed & Treatment Residue (nu/Q) Body Burden (uq/rat) killed Day 0 0.36 i 0.074 (100%) 132.4 1 29.4 (100%) killed Day 21 None 0.32 i 0.031 (89%) 125.8 1 11.6 (95%) MO 0.30 i 0.036 (83%) 120.1 i 14.7 (91%) FR 0.30 i 0.012 (83%) 94.9 i 2.9 (72%) MO + FR 0.29 i 0.043 (81%) 91.4 i 18.0 (69%) PBBs were not lost from rats receiving no treatment during the withdrawal period, as residues and body burdens on day 0 and day 21 were not significantly (p> 0.05) different. Residues of PBBs were not significantly (p> 0.05) reduced by mineral oil or feed restriction treatments. IFeed restriction alone, and ill combination with mineral oil resulted in comparable significant (p< 0.05) reductions in body burdens of PBBs over nontreated and mineral oil treated rats. When considering all the peaks in PBB samples, the total reduction occurring in body burdens with a combination of feed restriction and mineral oil was 46% of the day 0 burdens (Table 5). The combination of feed restriction and mineral oil decreased body residue concentrations significantly (pg 0.05) to 73% of day 0 values (a reduction of 27%) or 0.63 ug PBBs/g tissue 58 (Table 5). Body burdens of PBBs were significantly (pg 0.05) reduced by feed restriction alone or in combination with mineral oil (Table 5). Reductions in PBB body burdens were 22% for feed restriction alone and 36% for the combination of feed restriction and mineral oil. Table 5. Total PBB residues and body burdens (based on all peaks) in rats fed P338 in the diet at 10 mg/kg for 14 days followed (on day 0) by a 21 day withdrawal involving no treatment, 5% mineral oil (MO), 50% feed restriction (FR), or a combination of 10% MO and 50% FR (FR + MO). Day killed Treatment Residue (ug/g) Body Burden (ug/rat) day 0 None 0.86 (100%) 313.4 (100%) day 21 None 0.82 (95%) 323.8 (103%) day 21 MO 0.72 (84%) 289.1 (92%) day 21 FR 0.77 (90%) 244.9 (78%) day 21 FR + MO 0.63 (73%) 199.4 (64%) II. Gas Chromatographic Peaks for PCBs and PBBs In analysis of PBBs, the one major peak present, representing 2,4,5,2’,4’,5’-hexabromobiphenyl, which is peak #4 (Figure 1) was measured. Figure 1 compares chromatograms for detecting peak #4 for a PBB standard solution made from fireMaster" FF-l, and the PBB extracted from whole body rat sample. The extract from the whole body rat was diluted to one- third to allow measurement of peak #4. The chromatogram is for a 0.1 ug PBB/ml standard and the sample extract is from rats killed on day 21 of withdrawal after receiving no withdrawal treatment. In comparing the two chromatograms there is a loss of the earlier eluting peaks (peaks 1 and 3). After injection of the sample into the gas Chromatograph it takes longer for the whole-body extract to come back down to the baseline. At the .vnoavaonv Hmzmnonpas onism awe .Acoapzawo m\4 any pomwpwo zoonimaons pow m one unmannem Ha\mmm M: 4.0 m we 4* room musmmoe op msmnmopmeOngo wow .4 onswam o I! 9 4.5.5 445.4333 oz 5 4m smoupopnpwm ppm A Sex»: 4.8 hoomuoaonz n4 mmm ohmocmpm mmm 60 tail end of the chromatogram, the two last peaks also disappear (peaks 6 and 7) which may be due to their small size. Figure 2 has chromatograms for a PBB standard solution, and an extract of a whole-body sample. The later represented an undiluted sample for measuring the smaller peaks seen previously in Figure l. The standard is 0.1 ug/ml and the extract is from rats killed on day 21 withdrawal after receiving no withdrawal treatment. Again the earlier eluting peaks (peaks 1 and 3) are lost from the chromatogram. The chromatogram for a PCB standard (1.2 ug/ml) made from Aroclor 1254 has 15 peaks (Figure 3). A.chromatogram from an extracted whole-body PCB sample has only 9 peaks (Figure 3). The extracted sample was from a group of rats killed on day 21 withdrawal that had been treated with combined feed restriction and mineral oil. The peaks present in the standard and not in the sample are peaks # 1-3, 6, 7, and 10. As with the PBB samples, it takes longer for the extract sample to approach the baseline directly after injection into the gas Chromatograph. Also present was a negative peak that was not seen in the standard chromatograms. III. Feed Intake, Body Weights, and % Lipid Feed intakes during the 14 day contamination phase were 19.3, 19.1, and 19 g/rat/d (Table 6) for rats being fed non- contaminated, PBB-contaminated, or PCB-contaminated diets, respectively. There were no significant (p> 0.05) differences ..P:oapoonpam3mwonpaz ooiwm hoe .pompvwo zoopaoaosz pop a can onmonmpm Ha\mmm mo m.o a mo A4» room mnaosaoxov mxmom manapase museums op maonmopm80nno new .N onsmam Jo Ill 61 poppy Hospnaapaz 02 4m smaupopnpxm ppm Aaa\ma m.ov soomuoaooz so mom pupaappm mom 62 .coavoanpmon comm XOm can pop a one anpooppm Ha\mom mo N.4 me or D 2 m4 m4 coapownpmon doom XOm USN HHO HmeGHE XOP so smoupopnpxm ppm 44 saomuoaozz :4 moo m HHo Hanonae Xoriwm boo .pownvwo hoopnoaoss o no mxmoo onsmmos op memumomeowno new A m4 4? he NF Aae\mo N.PV a oumazppm mom UP .m «woman e 0“; 63 Table 6. Feed intake. body weights, and % lipid of rats fed nonocontaminated diet or diets containing 10 ppm P083 or PBBs for 14 days (day 0 withdrawal). Pro-treatment Diet1 Cage2 Feed Intake (g/rat/day) Body Wt (g/rat) % lipid Non-contaminated: 7 378 4.64 1 1 377 5.86 1 5 363 5.02 Mean¢SE 19.253 372 i 5 5.17 a». 0.36 10 ppm P883: 22 360 5.62 26 373 6.14 30 357 4.45 MeanisE 19.063 363 _t 5 5.40 i 0.50 10 ppm PCBs: 34 357 4.36 38 376 4.85 42 363 4.67 Mean¢SE 18.953 365 _t 6 4.63 1. 0.14 1 Contamination diets were fed for 14 days prior to withdrawal phase. 2 Rats were housed three per cage and were analyzed as a composite. 3 Average for the three cages. Summary of ANOVA Body Wt % lipid Source: df MS 1 df MS 1 P885: Between groups 1 133.8 0.2463NS 1 0.079 0.728”S Within groups 4 72.6 4 0.569 PCBs: Between groups 1 78.2 0.3923NS 1 0.448 0.231 NS Within groups 4 85.2 4 0.226 NS Not significant p> 0.05. 64 in feed intakes based on the diets fed during the contamination phase. During the 21 day withdrawal phase the feed intakes for the non-restricted rats were 20.6, 22.0, and 21.6 g/rat/d (Table 7) for rats receiving no treatment that had previously been fed non-, PBB-, and PCB-contaminated.diet. There were no significant (p> 0.05) differences among feed intakes of rats run: on feed restriction. Rats on 50% feed restriction received 11.1 g/rat/d whereas rats on the combination of mineral oil and feed restriction were fed 11.9 g/rat/d (Table 7). Body weight of rats on day 0 withdrawal were 372, 363, and 365 g/rat (Table 6) for non-, PBB-, and PCB-contaminated diets, respectively. No significant (p> 0.05) differences among body weights due to content of the diet were detected on day 0 of withdrawal. On day 21 of withdrawal, the rats receiving no withdrawal treatment had average body weights of 384-401 g/rat, with no significant (p> 0.05) difference among them based on prior diet fed (Table '7). Body ‘weights were found. to Ibe significantly (pg 0.05) decreased by feed restriction alone, or in combination with mineral oil as compared to nontreated and mineral oil treated rats. Average body weights for rats on feed restriction alone were 312-328 g/rat, compared to 313-318 g/rat when feed restriction and mineral oil were combined (Table 7). Body weights were not significantly different (p> 0.05) between feed restriction alone and in combination with mineral oil. 65 w0oz< .0 00.00.00 .0008. 4 0.00.. 67 The percent lipid in whole-body samples on day 0 of the withdrawal phase from rats fed noncontaminated diet was 5.17% (Table 6). The lipid content of rats on PBB and PCB contaminated diets were 5.40 and 4.63 %, respectively (Table 6). There was no significant (p> 0.05) difference in the % lipid on day 0 of withdrawal among the rats fed different diets. (I) day 21 of withdrawal, the % lipid values were 4.9 to 5.4 % for the rats receiving no withdrawal treatment (Table 7). A significant (pg 0.05) decrease in % lipid was caused by feed restriction either alone or in combination with mineral oil. With feed restriction alone, the values for % lipid were 2.4-2.7%, in comparison to 1.9-2.3% for feed restriction with mineral oil (Table 7). Only the rats that were initially fed PCBs had a significantly (pg 0.05) greater loss in % lipid with the combined treatment (1.9%) than with feed restriction alone (2.7%). In rats previously treated with P888, there was a significant (pg 0.05) increase in % lipid with mineral oil treatment as compared to nontreated rats. DISCUSSION The combination of 50% feed restriction and 10% mineral oil in the diet produced a marked reduction in body burdens of both PCBs and P833 in rats during a 21 day withdrawal period. Feed restriction at 50% of _a_d_ libitum intake with addition of 10% mineral oil to the diet reduced rat body burdens of PCBs and PBBs by 49.8% and 45.4%, respectively. These results are in accordance with studies performed.earlier with chickens (Polin.gt al., 1985; Polin e; l., 1989), in which a combination of feed restriction and mineral oil resulted in approximately 70% reduction in body burdens of PCBs and P883. When considering residue concentrations in the carcass, the combined feed restriction and.mineral oil treatment again proved to be the most effective in reducing PCBs and PBBs. The effectiveness of the combination of the two is presumably due to the nonbiliary intestinal secretion, as described by Yoshimura and Yamamoto (1975), which is increased as lipid stores of PCBs and PBBs are mobilized due to feed restriction. 1., 1985), As with previous studies in chickens (Polin g; feed restriction or mineral oil used alone were not proven effective in reducing body burdens of PBBs in rats. Rat body burdens of PCBs were reduced with both feed restriction and 68 69 mineral oil independently, but to a lesser degree than when used in combination. Polin gt al. (1989) reported that PCB body burdens were reduced with 5% mineral oil in the diet, but that 50% feed restriction alone did not significantly reduce burdens in chickens. Conversely, feed restriction at 25% of ad libitum intake had been found to enhance the elimination of 2,4,5,2',4’,5’-HCB, a major congener in Aroclor 1254, by 50% from rats (Matthews and Anderson, 1975; Wyss is; al., 1982). Feed restriction or mineral oil alone, either were not effective or were less effective than the combination of the tan) in their ability to remove PCBs and PBBs from the body. Each alone serves a function in increasing the loss of xenobiotic from the body, but together there is a additive effect on PCB elimination and a synergistic effect on PBB elimination. Feed restriction’ mobilized adipose tissue, as demonstrated by the % lipid reduction by nearly 50% in rats restricted to 50% of ad libitum intake. This mobilization would increase the levels of PCBs and PBBs in the circulation resulting in higher concentrations being presented to and eliminated through the intestinal wall. In this case, it would be expected that feed restriction itself should cause a greater reduction than has been demonstrated in the literature. The use of mineral oil presumably would not allow reabsorption of the xenobiotics and speed passage out of the body via the feces. Runeral oil also stimulates the excretion of xenobiotics directly through.the intestinal.wallq as demonstrated with hexachlorobenzene (Rozman, K., et al., 1983). The mechanisn1 70 involved in the enhanced elimination seen with the use of feed restriction and mineral oil needs to be further studied to determine if the level of feed restriction and mineral oil used is Optimal. It would be advantageous to be able to increase the elimination of xenobiotics without such a high restriction of feed intake with its associated reduction of body weight gains. Use of the combined feed restriction and mineral oil withdrawal treatment has many possible applications. Some livestock destroyed during the PBB incident in bfihmigan could have been salvaged, especially those animals in which a short-term reduction in body weight gains would not be a problem (i.e. valuable breeding stock). Use in humans to reduce levels of xenobiotics which accumulate in the adipose tissue may be a future application after more research has been done. There was a 5-fold difference between total PBB residues (total of all peaks equalling 0.86 ug/g b.w.) and PCB residue (4.123 ug/g b.w.) in whole body rat samples at day 0 withdrawal. Other studies conducted in our laboratory using 10 mg/kg PCBs or PBBs in the diet have produced day 0 residue values in rats equivalent to those for PCB residues in this experiment. In double checking calculations for adding 10 mg/kg of Aroclor 1254 or fireMaster‘FTHl to the ground rodent chow, there was no error evident. Unfortunately, the diet saved for analysis was inadvertently thrown out, and therefore the actual concentrations present in the diets were not available. The cause of the low concentration of PBB residue in the rat tissues is unknown, but 71 the ability to measure the reduction in residues due to the different withdrawal treatments was not affected. Rats treated with 5% mineral oil in their diets and previously fed diet containing 10 ug/kg PBBs, a significant (pg 0.05) increase in the % lipid was seen. No other references to this occurring were found in the literature. The significance of this increase would require further research to determine if it is repeatable or not. APPEND ICES 72 mo<0 mez 20 9.322.200 4.0 000 000 EN 040. 400 «.40 now 2n men 00.0. 00. mm... moo. mod. 0:4 0.00 4.00. 00. .3 00.0. mmm 04.. mom com 34.“ no. NNNN 24.7w ©...N mod. mmua E00 0. <2 000 :40 m. .4. 400 00 00:... non 0mm own 04.. own oom mvm on. in 0mm 5mm 0N. NNN. .0... .m... 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Km 5.3 00.0 0 00... 0 00.0.0. 0... 00 .0 A000. 0. 00.0...00. 0.03 0.0. 026.05.; 05.00 0 .0: 00 00... 0 .400 00.....0000 00 02.0 .00 u <2 0.0 040 000 00.0 0.0 400 000 000 4.0 040 .00 000 00.0. .4... .0... <2 000 000 00 v. 400 00 00...... 000 000 000 0.... 000 000 000 000 000 400 000 000 00.00 00.0 00.00 <2 040 000 00 v. 400 00 00.... :0 000 000 040 0.0 000 400 000 0.0 000 000 000 00.... 00.0. 00.0. <2 400 000 00 v. 400 00 00.... .00 000 000 000 00... 000 000 000 000 040 400 0.0 .000 40.0 00.0 00.0. 0000 0.000. <2 400 0.00 00 v. 400 00 00:0. 000 040 .000 000 .00 000 000 000 000 000 000 040 00.0. .4... .0... <2 040 .00 00 v. 40.. 00 00.00. 000 .40 000 000 000 040 000 000 000 000 SH 000 00.00 00.0 00.00 <2 000 400 00 v. 400 00 00.00. 8030.558. < x.QZmaa< 74 Appendix B. Coding of rats by cage, treatment, day killed and for residue analysis. A. Control rats Cage #1 Withdrawal Treatment Day killed2 Codes 1 None3 21 30,87 2 None3 21 10,50 3 None3 21 17,59 4 50% feed restriction 21 4,75 5 50% feed restriction 21 38,91 6 50% feed restriction 21 20,82 7 None 0 2,74 8 5% mineral oil 21 27,64 9 5% mineral oil 21 36,90 10 5% mineral oil 21 43.83 11 None 0 26.85 12 10% MO@50%FR4 21 11,56 13 10% MO@50% FR4 21 41,71 14 10% MO@50% FR4 21 9,55 15 None 0 1.51 1 Each cage housed three rats, which were analyzed as a composite sample. 2 Represents the day of withdrawal. These rats were fed ad libitum. 4 MO - mineral oil; FR - feed restriction. Do 75 Appendix B (con't) B. PBBs rats Cage #1 Withdrawal Treatment Day killed2 Codes 16 None3 21 15.58 17 None3 21 31,66 18 None3 21 21,61 19 50% feed restriction 21 32,88 20 50% feed restriction 21 13.57 21 50% feed restriction 21 3.52 22 None 0 7,54 23 5% mineral oil 21 14,79 24 5% mineral oil 21 33,67 25 5% mineral oil 21 12,78 26 None 0 24,84 27 10% MO@50%FR4 21 16,80 28 10% MO @ 50% PR4 21 22,72 29 10% MO @ 50% FR4 21 29,65 30 None 0 35,68 1 Each cage housed three rats, which were analyzed as a composite sample. 2 Represents the day of withdrawal. 3 These rats were fed ad libitum. 4 MO .. mineral oil; FR - feed restriction. 76 Appendix B (con't) C. PCBs rats Cage #1 Withdrawal Treatment Day killed2 Codes 31 None3 21 8,77 32 None3 21 6,76 33 None3 21 23.62 34 None 0 42,93 35 50% feed restriction 21 39.70 36 50% feed restriction 21 34.89 37 50% feed restriction 21 25,63 38 None 0 28,86 39 5% mineral oil 21 37.69 40 5% mineral oil 21 40,92 41 5% mineral oil 21 5.53 42 None 0 19,60 43 10% MO©50%FR4 21 18.81 44 10% MO @ 50% FR4 21 45.73 45 10% MO @ 5091 FR4 21 44,94 1 Each cage housed three rats. which were analyzed as a composite sample. 2 Represents the day of withdrawal. 3 These rats were fed ad libitum. 4 MO - mineral oil; FR .. feed restriction. '77 Appendix C. Raw data for percent water and percent lipid. I. Body water content - % A. Control rats Withdrawal Treatment Cage 4' % Water Cage * % Water Cage 3 7. Water None1 1 65.83 16 65.26 31 66.68 None 1 2 62.97 17 67.00 32 66.65 None1 3 65.24 18 66.09 33 65.73 507. feed restriction 4 67.16 19 68.62 35 68.03 50% feed restriction 5 68.59 20 69.63 36 67.39 50% feed restriction 6 68.12 21 68.81 37 68.06 None2 7 68.67 22 67.90 38 68.49 5% mineral oil 8 64.29 23 65.70 39 65.69 5% mineral on 9 66.98 24 64.37 40 65.30 5% mineral oil 10 65.03 25 64.95 41 66.42 None2 1 1 65.41 26 66.53 42 68. 19 10% M0 0 50% FR3 12 68.90 27 70.77 43 68.74 10% r10 6 50% FR3 13 68.93 28 68.78 44 69.34 10% r10 @ 50% FR3 14 68.61 29 68.78 45 69.23 None2 15 69.21 30 69.64 34 69.82 l Rats were fed ad libitum ground rodent chow. 2 Rats were euthanized on day 0 0f the withdrawal phase and analyzed for PCBs and PBBs residue. 3 M0 = mineral oil; FR = feed restriction. 78 Appendix C (con't) 11. Body lipid content - %. A. Control rats % lipid % lipid Actual Cage # Withdrawal Treatment (DM)l (as is)2 lipid (g/rat)3 1 None4 13.80 4.71 17.52 2 None4 16.00 5.92 23.03 3 None4 16.38 5.69 22.25 4 50% feed restriction 7.21 2.37 7.65 5 50% feed restriction 8.11 2.55 8.39 6 50% feed restriction 7.57 2.41 8.02 7 None5 14.80 4.64 17.54 8 5% mineral oil 18.71 6.68 26.92 9 5% mineral oil 14.87 4.91 19.00 10 5% mineral oil 16.75 5.86 22.56 11 None5 16.93 5.86 22.09 12 10% MO 6 50% FR6 8.02 2.49 7.89 13 10% MO @ 50% FR6 6.82 2.12 6.78 14 10% MO 6 50% FR6 7.25 2.28 7.00 15 None5 16.32 5.02 18.22 1 Percent lipid is based on dry matter (DM) - this value is the average of duplicate samples. Percent lipid is based on an £1.15. basis - this value was calculated using the formula (100 - percent water) 1 (DM percent lipid/100) - percent lipid 2.8.1:. 1 body weight in grams - grams of lipid. Rats were fed ad libitum. Rats were euthanized on day 0 of the withdrawal phase and analyzed for PCBs and PBBs residue. 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