RETURNING MATERIALS: IV1£3I.] PIace in book drop to LIBRARJES remove this checkout from —_. your record. FINES will be charged if book is returned after the date stamped below. ORGAN METABOLIC CLEARANCE: ROLE OF FLOW, ENZYMIC CAPACITY, AND BINDING IN THE METABOLIC CLEARANCE OF BENZO(A)PYRENE BY RAT LIVER AND LUNG By David Arthur Wiersma A DISSERTATION Submitted to Michigan State University in partial fulfilIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacoiogy and Toxicology 1982 ABSTRACT Organ Metabolic Clearance: Role of Flow, Enzymic Capacity, and Binding in the Metabolic Clearance of Benzo(a)pyrene by Rat Liver and Lung By David Arthur Wiersma The physiological model of clearance shows that the organ metabolic clearance of circulating xenobiotic compounds can be affected by altered blood flow, organ metabolic capacity, and binding to blood components. In this study, the role of these factors in the metabolic clearance of a model lipophilic xenobiotic compound, benzo(a)pyrene (B(a)P), by rat liver and lung was examined. In microsomal preparations, the metabolic capacity (intrinsic free clearance) of liver toward B(a)P was 80 times greater than that of lung in control rats and 5000-fold greater in rats pretreated with 3-methyl- cholanthrene (3MC), an inducer of microsomal arylhydrocarbon hydroxylase activity. In isolated organs of control rats, B(a)P clearance by liver was high and increased with flow, while clearance in lungs was low and unaffected by flow. In contrast, in organs from 3MC pretreated rats lung B(a)P clearance was high and flow dependent, while liver clearance was similar to that of controls. Both flow and 3MC pretreatment en— hanced the production of B(a)P metabolites by isolated lungs. Alter- ing perfusion medium composition demonstrated that addition of serum David Arthur Niersma albumin or lipoproteins to red blood cell containing medium enhanced B(a)P clearance. B(a)P was rapidly eliminated from the blood of conscious rats. Pharmacokinetic analysis showed that 3MC pretreatment enhanced total body clearance. Results from isolated organs suggested that this en- hancement arises through increased clearance by extrahepatic organs. In addition in conscious 3MC pretreated rats, first-pass extraction by lung and liver was nearly equal. These results suggest that the organ metabolic clearance of xeno- biotic compounds, such as B(a)P, may be predicted from enzyme kinetic data using the physiological model of clearance. Altered flow, meta- bolic capacity, and binding to blood components may under certain circumstances each affect organ clearance of B(a)P. Finally, the re- sults suggest that at normal organ flows in control rats, liver B(a)P clearance (5.6:D.2 ml/min) will be about five-fold greater than lung clearance, while in 3MC pretreated rats clearance by these two organs will be about equal (lung, 8.8:0.5 ml/min; liver, 7.4:0.6 ml/min), despite large differences in their enzymic capacity. For Brenda Yahweh, my heart has no lofty ambitions, my eyes do not look too high. I am not concerned with great affairs or marvels beyond my scope. Enough for me to keep my soul tranquil and quiet like a child in its mother's arms, as content as a child that has been weaned. Israel, rely on Yahweh, now and for always. Psalm I31 ii ACKNOWLEDGEMENTS I thank Dr. Robert A. Roth, my guidance committee chairman, for his advice, patience and prodding throughout this project. I also thank him for instilling in me the desire and capacity to do research. I also am thankful for his friendship. I am also indebted to the other members of my guidance committee, Dr. Theodore M. Brody, Dr. N. Emmett Braselton, and Dr. Jay I. Goodman of the Department of Pharmacology and Toxicology and Dr. Steven Aust of the Department of Biochemistry for their contributions to the direction of this project. I also thank Dr. Gregory D. Fink of the Department of Pharmacology for his aid in the development of surgical procedures and statistical analysis of the data. I acknowledge the support of the Pharmaceutical Manufacturers Association Foundation in the form of an advanced predoctoral fellowship during l980 and 198l. I also thank Mr. Martin Miner, Mrs. I. Mao, Ms. Jessica DeForest- Towns, Miss Cynthia Amon, Mr. Mark Nuzzolilo and Miss Michelle M. Imlay for their technical assistance and Miss Diane Hummel for her help in typing this dissertation and other manuscripts. TABLE OF CONTENTS Page DEDICATION-----------—-------------e ----------------------------- ii ACKNOWLEDGEMENTS ------------------------------------------------- iii LIST OF TABLES --------------------------------------------------- viii LIST OF FIGURES -------------------------------------------------- x INTRODUCTION ----------------------------------------------------- l A. Metabolism of Xenobiotic Compounds --------------------- 2 B. Relationship Between Elimination and Metabolism -------- 3 l. Physiological Model of Metabolic Clearance -------- 3 2. Factors Affecting Hepatic Metabolic Clearance ----- 8 a. Altered flow --------------------------------- 9 b. Altered metabolic capacity ------------------- 10 c. Altered binding to blood components ---------- l4 3. Effect of Altered Blood Flow, Metabolic Capacity, and Binding to Blood Components on Extrahepatic Metabolic Clearance ------------------------------- l8 4. Conditions Which May Alter the Relative Metabolic Clearance of Organs ------------------------------- 22 C. Approaches to the Study of Organ Metabolic Clearance--- 25 l. Broken Cell Studies ------------------------------- 26 2. Isolated Organ Studies ---------------------------- 26 3. Studies In_Vivo ----------------------------------- 27 D. Benzo(a)pyrene as a Model Compound in the Study of Metabolic Clearance ------------------------------------ 28 E. Purpose ------------------------------------------------ 32 F. Experimental Approach ------------ ; --------------------- 33 MATERIALS AND METHODS -------------------------------------------- 35 A. Animals ------------------------------------------------ 35 B. Pretreatment of Animals -------------------------------- 35 C. Preparation of H-Benzo(a)pyrene ----------------------- 35 iv TABLE OF CONTENTS (continued) Page MATERIALS AND METHODS (continued) D. Studies Using Broken Cell Preparations ----------------- 36 1. Preparation of Microsomes ------------------------- 36 2. Determination of AHH Activity --------------------- 37 3. Calculation of Metabolic Activity ----------------- 38 4. Calculation of Apparent Enzyme Kinetic Parameters, Vmax and Km --------------------------------------- 38 5. Estimation of fB ---------------------------------- 38 E. Studies Using Isolated Perfused Organs ----------------- 39 l. Surgical Procedures ------------------------------- 39 2. Perfusion Apparatus ------------------------------- 4O 3. Perfusion Media ----------------------------------- 4l a. Preparation of red blood cells --------------- 4l b. Isolation of serum lipoproteins -------------- 42 4. General Protocol For Perfused Organs -------------- 42 5. Calculation of B(a)P Clearance -------------------- 43 6. Details of Specific Experiments Utilizing Isolated Organs -------------------------------------------- 44 a. Flow dependence of B(a)P elimination --------- 44 b. Flow dependence of B(a)P metabolism ---------- 44 c. Efflux of B(a)P from isolated lungs ---------- 44 d. Concentration dependence of B(a)P elimination 44 e. Distribution of H within perfused livers---- 45 f. Determination of hepatic portal-hepatic venous shunts -------------------------------- 45 9. Dependence of B(a)P elimination on medium composition ---------------------------------- 46 F. Association of B(a)P with Blood Fractions -------------- 46 1. Injection with 3H-B(a)P --------------------------- 46 2. Separation of Blood Componentse ------------------- 47 G. Studies of B(a)P Disposition In_Vivo ------------------- 47 1. Preparation of Cannulas --------------------------- 47 2. Cannula Implantation ------------------------------ 48 3. Administration of B(a)P and Collection of Blood Samples ------------------------------------------- 48 4. Pharmacokinetic Analysis -------------------------- 5l 5. Calculation of Organ Extraction ------------------- 54 TABLE OF CONTENTS (continued) Page MATERIALS AND METHODS (continued) H. Analytical Methods ------------------------------------- 55 1. Protein Determination ----------------------------- 55 2. Lipid Determination ------------------------------- 55 3. Separation and Quantification of B(a)P and B(a)P Metabolites --------------------------------------- 56 a. Extraction of B(a)P from samples with hexane- 56 b. Separation and quantification of 3H-B(a)) and individual metabolites in methanol extracts-- 57 1. Chemicals ---------------------------------------------- 58 J. Statistical Analysis ----------------------------------- 59 RESULTS ---------------------------------------------------------- 6D A. Broken Cell Studies ------------------------------------ 60 l. Determination of Apparent Enzyme Kinetic Parameters and Intrinsic Free Clearance ---------------------- 6O 2. Effect of Added Bovine Serum Albumin on Microsomal B(a)P Metabolism ---------------------------------- 76 3. Organ B(a)P Clearances Predicted from Results of Broken Cell Studies ------------------------------- 80 B. Isolated Organ Studies --------------------------------- 80 l. Effect of Altered Flow on B(a)P Clearance --------- 82 2. Effect of Altered Metabolic Capacity on B(a)P Clearance ----------------------------------------- 89 3. Effect of Altered Flow or Metabolic Capacity on B(a)P Metabolite Production ----------------------- 96 4. Effect of Altered Perfusion Medium Composition on B(a)P Clearance and Metabolism -------------------- 112 5. Other causes for Altered B(a)P Clearance ---------- lZl a. Reversible clearance of B(a)P by isolated lungs ---------------------------------------- l2] b. Concentration dependent B(a)P clearance ------ l24 c. Non- uniform distribution of B(a)P ------------ l24 d. Intra- -hepatic shunts ------------------------- l28 C. Association of B(a)P and B(a)P Metabolites with Blood Fractions—— -------------------------------------------- 128 D. B(a)P Elimination From the Blood of Conscious Rats ----- lBl l. Assessment of Animals ----------------------------- l3l 2. Disappearance of B(a)P from Blood ----------------- l3l 3. Effects of 3MC Pretreatment ----------------------- 131 4 Effects of Varied Route of Administration --------- l35 vi TABLE OF CONTENTS (continued) Page RESULTS (continued) E. Comparison of Organ Extractions Based on Results From Broken Cell, Isolated Organ, and Conscious Rat Studies- I46 DISCUSSION ------------------------------------------------------- 148 A. Broken Cell Studies ------------------------------------ 148 1, Determination of Apparent Enzyme Kinetic Parame- ters and CI'int"" ------------------------------- I48 2. Determination of f ------------------------------- 153 3. Prediction of B(a) Clearance --------------------- l54 B. Isolated Organ Studies --------------------------------- l55 l. Organ Flow and B(a)P Clearance -------------------- l56 2. Metabolic Capacity and B(a)P Clearance ------------ l57 3. Binding to Blood Components and B(a)P Clearance--- l59 4. Flow and Metabolic Capacity Influence B(a)P Meta- bolism in Perfused Lungs -------------------------- 166 5. Comparison of Isolated Organ Results with Predic- tions from Enzyme Kinetic Data -------------------- l7O C. Studies in Conscious Rats ------------------------------ l73 l. Metabolic Capacity and B(a)P Clearance ------------ 174 2. Varied Route of Administration and B(a)P Clearance 176 3. Role of Metabolic Capacity and Route of Admini- stration in the Metabolism and Tissue Distribution of B(a)P ------------------------------------------ I76 4. Comparison of Results In_Vivo with Those in Iso- lated Organs -------------------------------------- l78 D. Significance ------------------------------------------- l8l SUMMARY AND CONCLUSIONS ------------------------------------------ l84 BIBLIOGRAPHY ----------------------------------------------------- l88 vii Table 10 II 12 13 LIST OF TABLES Page Assay conditions for determination of apparent enzyme kinetic parameters of hepatic and pulmonary microsomal AHH activity ------------------------------------------- 69 Apparent enzyme kinetic parameters and intrinsic free clearance for microsomal B(a)P metabolism -------------- 75 Effect of added BSA on the microsomal metabolism of B(a)P ...................... 79 Hepatic and pulmonary clearance of B(a)P at normal organ flow as predicted from microsomal metabolic activity ----------------------------------------------- Bl Effect of flow on isolated rat liver parameters -------- 83 Effect of flow on isolated rat lung parameters --------- 84 Effect of flow on pharmacokinetic parameters of B(a)P elimination by isolated livers ------------------------- 90 Effect of flow on pharmacokinetic parameters of B(a)P elimination by isolated lungs -------------------------- 9l B(a)P clearance by isolated rat livers and lungs per- fused of normal organ flow ----------------------------- 97 The concentration of B(a)P and methanol-extractable metabolites in isolated, perfused lungs ---------------- lO9 The ratio of B(a)P and metabolite concentrations in lung tissue to that in the perfusion medium ------------ lll Effect of perfusion medium composition on isolated rat liver parameters --------------------------------------- ll3 Effect of perfusion medium composition on pharmacoki- netic parameters of isolated rat livers --------------- ll7 viii LIST OF TABLES (continued) Table 14 15 16 17 18 19 20 21 22 Page Effect of perstion medium composition on the amount of B(a)P and metabolites appearing in the bile of isolated livers ------------------------------------------------- 120 Concentration dependence of B(a)P clearance in isolated livers and lungs of 3MC pretreated rats ---------------- l27 Fraction of microspheres appearing in the effluent of isolated, perfused livers of 3MC pretreated rats ------- l29 Association of B(a)P and metabolites with blood fractions ---------------------------------------------- l3O Effect of cannula implantation and pretreatment on rat body weights ------------------------------------------- l32 Pharmacokinetic parameters of B(a)P disposition in con- scious rats -------------------------------------------- l36 Area under the arterial blood B(a)P metabolite concen- tration vs time curves --------------------------------- l4l Fraction of the dose available and the fraction ex- tracted by liver and lung of 3MC pretreated rats in_ vivo --------------------------------------------------- l45 Extraction of B(a)P by rat livers and lungs at normal organ flow as predicted from enzyme kinetic data, observed in isolated organs, and determined ig_vivo---- l47 ix Figure Chm-{>00 10 11 12 LIST OF FIGURES Page The relationship between organ clearance and flow ------ 7 Clearance of 5-hydroxytryptamine (5-HT) by isolated rat livers and lungs perfused at several flows ------------- 2l Pathways of B(a)P metabolism --------------------------- 30 Sites of cannula implantation -------------------------- 50 Two-compartment open model ----------------------------- 53 Incubation time and microsomal protein concentration dependence of microsomal liver AHH activity of control rats --------------------------------------------------- 62 Incubation time and microsomal protein concentration dependence of microsomal liver AHH activity of 3MC pre- treated rats ------------------------------------------- 64 Incubation time and microsomal protein concentration dependence of microsomal lung AHH activity of control rats --------------------------------------------------- 66 Incubation time and microsomal protein concentration dependence of microsomal lung AHH activity of 3MC pre- treated rats ------------------------------------------- 68 Lineweaver-Burk plots of the substrate concentration dependence of liver and lung microsomal AHH activity of control rats ---------------------------------------- 72 Lineweaver-Burk plots of the substrate concentration dependence of liver and lung microsomal AHH activity of 3MC pretreated rats --------------------------------- 74 The influence of added BSA on liver microsomal AHH activity of control rats ------------------------------- 78 LIST OF FIGURES (continued) Figure 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Page Effect of altered flow on the disappearance of B(a)P from the reservoir of isolated, perfused rat livers---- 85 Effect of altered flow on the disappearance of B(a)P from the reservoir of isolated, perfused rat lungs ----- 88 B(a)P clearance by isolated livers and lungs of con- trol rats perfused at several flows -------------------- 93 B(a)P clearance by isolated livers and lungs of 3MC pretreated rats perfused at several flows -------------- 95 Elution pattern of B(a)P and its metabolites separated by high pressure liquid chromatography using gradient elution ------------------------------------------------ 99 Concentration of B(a)P and total B(a)P metabolites in the reservoir of isolated rat lungs perfused at low and high flow ------------------------------------------ 102 Appearance of individual B(a)P metabolites in the re- servoir of isolated lungs perfused at low and high flow 104 Disappearance of B(a)P from the reservoir of isolated livers perfused with media of varied composition ------- 116 Appearance of B(a)P metabolites in the reservoir of isolated livers perfused with media of varied composi- tion --------------------------------------------------- 119 The efflux of B(a)P and sucrose from isolated 1ungs---- 123 The distribution of 3H within isolated perfused livers- 126 Elimination of B(a)P from the blood of conscious rats-- 134 B(a)P concentration in rat organs ---------------------- 138 'Concentration of B(a)P metabolites in the blood of con- scious rats -------------------------------------------- 140 B(a)P metabolite concentration in rat organs ----------- 143 Delivery of B(a)P to hepatocytes by red blood cells and serum albumin -------------------------------------- 163 xi INTRODUCTION Removal from the body of endogenous waste products occurs through three major mechanisms: renal excretion, biliary excretion, and exha- lation. Water soluble wastes pass out of the body with the urine. Some of the endogenous wastes formed in the liver are excreted into the bile and eliminated with the feces, while others are transferred to the blood and excreted renally. Volatile wastes are exhaled from the lung. Wastes which are not water soluble, volatile, or capable of being transported by the hepatocytes into the bile are metabolized to com- pounds which can participate in these elimination mechanisms. Xeno- biotic agents are also eliminated from the body by these same mecha- nisms. For example, water soluble antibiotics, such as penicillin, are eliminated by urinary excretion (Weinstein, 1975), inhalation anesthe- tics by exhalation, and the diagnostic agent indocyanine green by biliary excretion (Cherrick gt_gl,, 1960). However, many xenobiotic compounds do not readily participate in these processes due to their low volatility and extremely lipid solubility. These compounds tend to accumulate and remain in the fatty tissues of the body (Bickel and Meuhlebach, 1980). R. T. Williams deduced that only hydrophilic xenobiotic compounds are excreted from the body in unchanged form (Williams, 1959). Non- volatile, lipophilic compounds are either eliminated very slowly or 2 transformed into more polar compounds which can be excreted. In fact, if not metabolized many lipophilic xenobiotic compounds would remain in the body for the lifetime of the individual (Brodie, 1956). There- fore, xenobiotic compounds undergo hydrolysis, oxidation, reduction and conjugation reactions which increase polarity and, thus, renal and biliary excretion. A. Metabolism of Xenobiotic Compounds Many of the metabolic transformations of xenobiotic compounds are oxidative reactions performed by mixed function oxidases. This family of cytochrome P-450 containing enzymes is located in the endoplasmic reticulum of cells and requires oxygen, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and a lipid environment for activity (Brodie et_al,, 1955). Exposure jn_!jyg_to certain environmental pollu- tants, such as cigarette smoke, or to polycyclic aromatic hydrocarbons, such as 3-methy1cholanthrene (3MC), can greatly enhance the activity of this enzyme system (Conney, 1967). All organs of the body (with the possible exception of bone) possess the capacity to metabolize xenobiotic compounds. Litterst 35 31: (1975) examined the ability of liver, lung, and kidney of several labo- ratory species to metabolize drug substrates. They found in all the species examined that drug metabolizing activities in extrahepatic tissues were generally less than those of liver. Similar results were obtained by Lake et_al, (1973), Weibel et_al, (1973), Zampaglione and Mannering (1973), and Heitanen and Vainio (1973) using a variety of substrates. 3 The activity of a metabolic process determined in broken cell preparations is not, however, the only factor limiting the ability of an organ to metabolize circulating compounds in_ijg, Cofactor concentra- tion and availability, total amount of enzyme in the tissue, the affi- nity of the enzyme for its substrate and substrate availability can each influence metabolic activity. Substrate availability (i.e., substrate concentration at the site of the enzyme) is affected by a number of physiological as well as physical factors. Physical factors include dissociability of the substrate from carriers in the blood, the amount of capillary surface area, distance from the blood to the enzyme site, the number and types of barriers to diffusion, and nature of entry of the compound into cells (passive diffusion or carrier mediated uptake). A major physiological limit of organ drug metabolism is the rate of delivery (blood flow) of the substrate to the organ. B. Relationship between Elimination and Metabolism 1. Physiological Model of Metabolic Clearance The rate of enzyme-mediated reactions may be described by the Michaelis-Menten equation: V [S] v = max (1) Isn+|S| in which v is reaction velocity, [S] is the concentration of free (un- bound) substrate, Vmax is the maximal velocity and Km is the Michaelis- Menten constant (Segel, 1975). This equation describes a parabolic relationship between reaction velocity and substrate concentration where Km is the concentration value at which v equals 1/2 Vmax“ 4 Under first-order conditions, that is, when substrate concentration is much less than the Km value (generally 20% or less), the denominator of equation 1 is essentially equal to the Km. Thus, reaction velocity max/Km and is characteristic for each substrate-enzyme interaction. This factor has approaches the substrate concentration multiplied by V been called the intrinsic free clearance (Cl'int) by Gillette (1971) and others (Rowland gt_al,, 1973; Wilkinson and Shand, 1975). V - "‘3‘" (2) tI'int ' TKEF' Vmax is expressed in terms of the whole organ. Intrinsic free clearance is that volume of organ water cleared completely of a substrate per unit time by metabolism (Wilkinson and Shand, 1975; Rane £5 21,, 1979). It is implicit in this definition that the substrate is free in solution in the organ water. The intrinsic free clearance is a term which describes the metabolic capacity of the organ (i.e., the organ's ability to metabolize the compound when factors other than enzyme are not limiting). It is a composite term reflecting both the metabolic activity of the organ (Vmax) and the affinity of the enzyme for its substrate (Km). Thus, this term is probably a better estimation of organ metabolic capacity under first-order conditions than enzyme activity (i.e., Vmax) alone. As implied in the definition of intrinsic metabolic clearance, organs may not be capable of performing the maximum clearance suggested by the Cl'"It value. Two limiting factors which may reduce this maximal clearance are organ blood flow and the relative proportions of free and bound substrate in the blood. The physiological model of organ drug 5 clearance is a model which describes the relationship of clearance, metabolic capacity, flow, and the free fraction of substrate (Rowland gt 31,, 1973; Wilkinson and Shand, 1975; Keiding and Andreasen, 1979). In this model (also known as the perfusion-limited model) elimination from the blood is described as a clearance term, that volume of blood from which the substrate is completely and irreversibly removed per unit of time. Under steady-state first-order conditions, this metabolic clear- ance (Cl) depends on flow (0), the free fraction of substrate in the blood (f3), and CI'int: Q ' (fB ° CPint) C1 = Q + (£3 . c11 3 int) ( ) The product, fB - Cl'int, describes the intrinsic clearance of total drug (Clint) rather than that of the free fraction alone. Equation 3 describes a parabolic relationship between clearance and flow bounded by two limiting conditions as shown in Figure 1. When intrinsic clear- ance is much greater than flow to the organ, clearance approaches flow (condition A in Figure 1). This occurs with compounds for which the organ has a very high metabolic capacity. Under such conditions clear- ance is limited by delivery of substrate to the organ. The value of organ clearance, of course, can be no greater than the organ flow. Thus, clearance is flow-limited. In contrast, when flow to the organ is much greater than intrinsic clearance, clearance is limited by the value of the intrinsic clearance (as in condition 8 of Figure 1). This occurs in organs with low metabolic capacity and relatively high flow. Thus, clearance is enzyme-limited, or flow-independent. Between these two extremes clearance depends on both flow and intrinsic clearance. be, .a=_m> “=2_u ago mmsomocaam mucmcmmFU wcommcwcu . ._u cusp prwmgm m? 30”; .mmv mcowuwucoo umpwew_-wechw cmucs .mzpw> 20F; ms“ mmcumocaam mucmgmmFQ mcommcmgp .Au wpov mucmcmmFU umeVchw mg“ on m>wmemL sop my 30F; .Amg 30—4 use mocmcmmpo .mucmgmmpu cameo eo Ponce umpwswfi scowmzmgma mg» op mcwucouu< .zopm use wucmgmmpu cameo :mmmea a_;m:owpm_mc «sh .F mcamvd _ mgzmwu A375 03.0.9; 30.”— ov on m V m N — 11 md _______;________ (sigun aAynlaJ) aaumoap 8 Brauer and coworkers were among the first investigators to report that physiological variables could affect the ability of the liver to remove circulating compounds from the blood. In a series of experiments examining the uptake of chromic phosphate colloid (Brauer et_ 31,, 1957; Brauer, 1958) and bromosulfophthalein (Brauer, 1963a,b) in the isolated liver, they discovered that removal was a function of hepatic blood flow. However, it was not until later (Gillette, 1971) that the implications for drug metabolism by organs jn_vjy9_were real- ized. With the theoretical treatment by Rowland gt_al, (1973) and subsequent studies by others (Wilkinson and Shand, 1975; Nies gt_al,, 1976; Wilkinson, 1976; Branch and Shand, 1976; Shand gt_al,, 1976; Colburn and Gibaldi, 1977; Pang and Rowland, 1977a,b,c,; Keiding and Andreasen, 1979), the importance of hepatic blood flow, metabolic capa- city, and binding to blood components in determining the ability of liver to remove circulating drugs from the blood has been established. Furthermore, experiments in which the value of Cl'int was predicted from enzyme kinetic parameters determined in broken cell preparations have verified that under first-order conditions this perfusion-limited model accurately described the hepatic elimination of many circulating com- pounds (Rane §t_al,, 1977; Wiersma and Roth, 1980; Hilliker and Roth, 1980). B2. Factors Affecting Hepatic Metabolic Clearance Alterations in blood flow, binding to blood components, and metabolic capacity may affect the participation of an organ in total body metabolic elimination. The importance of these variables is 9 demonstrated in the effects which physiological, pathophysiological, and pharmacological interventions have on the hepatic clearance of drugs (Nies 9321., 1976; Branch and Shand, 1976; Wilkinson, 1976; Jusko, T 976; Blaschke, 1977; Williams and Mamelok, 1980; Piafsky, 1980). a. Altered flow Alterations in hepatic blood flow can occur from numerous causes. Some are the result of altered cardiac output, while others are independent of an effect on cardiac output. Changes in hepatic flow W1 1 'l have the greatest effect on the removal of those compounds with f1 ow-limited hepatic clearance. Both Nies e_tal. (1976) and Wilkinson ( 1 976) have reviewed causes of altered hepatic flow and their known effects on drug disposition. Experimentally, the effect of flow on hepatic metabolic ‘31 earance has been investigated in isolated liver preparations perfused at constant flow. Extractions of propranolol (Branch e_t_ §_1_., 1973), 1 ‘3 docaine (Shand gt__a__l_., 1975; Pang and Rowland, 1977b), hexobarbital (-ROth and Rubin, 1976a), serotonin (Wiersma and Roth, 1980), and tauro- ChO‘late (Pries 31391., 1981) were high and flow dependent, while the e>Fn|:10unds in humans, were not affected by the transition from bed rest to exercise (Swartz e_t_a_1_., 1974; Klotz and Lucke, 1977; Theilade _et 11: . 1979). b. Altered metabolic capacity Altered metabolic capacity will be most effective in changing the enzyme-limited metabolic clearance of low extraction com— Dounds, such as antipyrine. This effect has not as yet been demon- strated in isolated perfused livers. However, other methods have been uSEd to investigate the influence of this factor. 11 An increase in hepatic intrinsic clearance will not markedly affect the metabolic clearance of highly extracted compounds following systemic administration. However, when given orally or d irectly into the hepatic portal vein, highly extracted compounds are subject to a considerable hepatic first-pass effect (Routledge and Shand, 1979). reducing the fraction of the dose available to the sys- temic circulation. Thus. availability, as well as clearance, reflects hepatic intrinsic clearance. Accordingly, changes in intrinsic clear- ance will alter availability. For example, if extraction rises from 0 - 85 to 0.90 (a 6% increase) with increased intrinsic clearance, avail- a bi 1ity would decrease from 15% to 10% of the dose, a reduction of 33%. This concept was used in an experimental paradigm to determine the effect of pentobarbital, an inducer of hepatic microsomal drug metabolizing enzymes. on the disposition of alprenolol in humans (A1 van 31],, 1977). Alprenolol, like propranolol, is avidly removed 1’iflom the circulation by the liver. The area under the concentration vs. 1:1 me curve following intravenous administration was not significantly 31"? acted by the pentobarbital treatment. However, the area under the COUcentration vs. time curve (AUCm) following oral administration was r‘ecluced by 80%. Liver blood flow and plasma binding were unaffected. Thus, capacity to metabolize alprenolol was significantly increased. S‘Nnilar treatment of rats resulted in increased capacity of liver cells to metabolize alprenolol (Grundin _e_t_ 31.. 1974). Changes in the availability of propranolol occurred c“Wing treatment of patients with chlorpromazine (Vestal e_t_ a_l_., 1979). chlorpromazine exposure resulted in elevated systemic blood concentra- T-Ions of orally administered propranolol. Since neither liver blood I'D 12 f1 ow nor plasma binding was altered, these results suggested that chlorpromazine reduced the intrinsic clearance of propranolol by inhi- bition of liver metabolism. Thus, availability was increased. Similar experiments in rats have demonstrated the ability of phenothiazines to i nhibit hepatic metabolism of propranolol (Shand and Oates, 1971). In contrast to the lack of an effect of elevated Cl'int on the clearance of highly extracted compounds, increasing this factor 5 i gnificantly enhanced the hepatic clearance of poorly extracted com- pounds. Nies e_t_al. (1976) noted that phenobarbital treatment of rats increased their antipyrine clearance. This increase was greater than that due to the increased hepatic blood flow produced by the pheno- barbital treatment alone. Therefore, a shift to a different clearance VS - flow curve with a greater limit (Cl'int) likely occurred accounting for the additional increased clearance. However, no direct experiments 7 h isolated livers were performed to test this hypothesis. Hepatic Cl'int can also be altered by mechanisms other than an increase or decrease in the amount of enzyme in the liver cells. FOY‘ example, in chronic liver disease in humans, hepatic clearance of arrtipyrine is reduced. This reduced clearance suggests that the disease has reduced the amount of enzyme within the liver. However, specimens of liver tissue examined for metabolic activity showed no reduction in metabolic capacity (Schoene 3311;, 1972). Since liver blood flow is not reduced, the hypothesis has been suggested (Branch and Shand, 1976) that the reduced C1 'int is due to the development of intrahepatic shunts. These shunts divert flow from the hepatic portal vein and the hepatic sinusoids directly into the hepatic veins, thus bypassing a 13 significant portion of the hepatocytes. Such anatomical shunts have been described in livers of patients with cirrhotic disease (Popper gt 2.1.: , 1954; Groszmann e_tal” 1977). They arise as areas of regenerating ti ssues replace those damaged by disease. Such shunts also appear to reduce the hepatic clearance of bile acids in cirrhotic patients (Poupon _e_t_:_ _a_l_., 1981). Organ metabolic capacity, Cllint’ describes the ability of an organ to metabolize circulating compounds. The measurement of C1 ' i nt in intact organs, however, may be influenced by factors other than metabolism. For example, Stegmann and Bickel (1977) examined the uptake of imipramine by the isolated perfused rat liver. They found that clearance of imipramine from the perfusion medium was better corre- 1 ated with uptake into intrahepatic binding sites than with the rate of 1mi pramine metabolite formation. Colburn (1981) examined changes in the Pharmacokinetics of lidocaine in epileptic patients receiving anticon- V11 1 sant therapy. He found that the rate constant of lidocaine elimina- t‘i on in these patients was not increased, but that total body clearance 01" 1idocaine was. This increased clearance was reflected in an elevated aDparent volume of distribution (Vd) which was attributed to an in- creased effective liver volume. In an investigation of ampicillin phalr‘macokinetics in cirrhotic patients Lewis and Jusko (1975) observed an increase in Vd. This was due to an increase in tissue retention and resulted in a three-fold increase in the metabolic (biliary) clearance of this antibiotic. Thus, for some drugs tissue uptake was not neces- sarily followed by immediate metabolism. As a result this empirically determined Clint may have reflected two components of uptake, metabolism l4 azrid filling of binding sites. Presumably, as the blood concentration decreased bound drug was released from these sites and metabolized. c. Altered binding to blood components In addition to altered organ blood flow and metabolic capacity, the metabolic clearance of xenobiotic compounds can be affected by an altered distribution between the free compound in solu- ti on and that associated with blood components. In general, only the free fraction is available for removal, however, if rapid dissociation from the bound forms occurs, the fraction of the compound removed may exceed the free fraction (Wilkinson and Shand, 1975; Gillette and Pang, 1 97 7 ). Lipophilic xenobiotic compounds are carried in the blood bound to various blood components. Due to their hydrophobic nature, the fraction of these compounds free in solution is very small. Xeno- b‘i otic compounds are associated with serum albumin, globulins, glyco- F’Y‘Oteins, lipoproteins and red blood cells (Meyer and Guttman, 1968; V31 1ner, 1977; Piafsky, 1980). Although altered binding to any compo- "eht may alter organ metabolic clearance, only changes in binding to a‘l bumin have received much attention. Investigations using isolated organs or IBM in patho- 1 °9ical conditions which altered serum albumin concentration have esta- b1 ished the influence of binding of drugs to albumin on pharmacoki- netics. In accord with the perfusion-limited model (Equation 3), Shand g; 91. (1976) found that the hepatic clearance of phenytoin in isolated Y‘at livers perfused with media containing varying amounts of albumin Was proportional to the free fraction. Furthermore, using data from 15 various species, Evans _e__t__a]_. (1973) showed that propranolol clearance and fB were also related as the model predicted; those species showing a high degree of plasma binding had lower clearances. Colburn and Gibaldi ( 1 977) investigated the metabolic clearance of phenytoin in intact rats. When the free fraction was increased by coadministration of oleate to d i splace the phenytoin from its albumin binding sites, clearance in- creased. However, removal of circulating compounds does not always correlate with the free fraction. Using isolated livers perfused with media composed of varying albumin concentration, Forker and Luxon (1981) and Weisiger gt__a_l_. (1981) found that extraction of taurocholate and 01 eate corresponded with the bound fraction rather than that which was free. They also found specific, high affinity sites on the hepatocyte Surface for albumin. These results suggested that albumin may enhance the removal of some compounds by transporting them to efficient uptake 3 ‘i tes. Another possibility, suggested by Forker and Luxon, is that a'l bumin facilitates diffusion across the space of Disse. This space may act as a barrier to diffusion of the free compound. Changes in hepatic function, inflammation, and extensive bUr‘ns alter the free fraction of compounds in the blood (Jusko, 1976; K1 Otz, 1976; Piafsky, 1980). In addition, nephrosis and uremia, asso- ciated with various renal diseases, may also affect the metabolic c1 earance of xenobiotic compounds. Nephrosis alters normal glomerular function allowing maSS'lve amounts of protein, including albumin, to be excreted in the uY‘ine. This results in a severe hypoalbuminemia which can have one or 16 more effects on metabolic clearance. (1) Metabolic clearance could be reduced as renal clearance of bound drug increased. (2) f8 could also be increased, resulting in an increased filtered load. If renal reab- sorptive mechanisms were unable to compensate, renal excretion could be increased. (3) Furthermore, an increased fB increases Vd, and thus, the concentration at enzymic sites could be enhanced. Therefore, metabolic clearance could be enhanced. In uremia normal renal function is reduced. Since excre- to ry function is reduced, endogenous waste products (such as urea) begin to build up in the blood. Little or no hypoalbuminemia occurs in uremia. However, binding to plasma proteins can be severely reduced. Thi s is due to displacement of xenobiotic compounds from albumin binding 5 1‘ tes by the endogenous waste products (Reidenberg e_t_ _a_l_., 1971; McNamara Si‘ _a_1__., 1981). The net result is an increase in Vd which for reasons 31 ready described may result in an increased metabolic clearance. Whether such altered binding results in changes in meta- bo'l ic clearance is as yet unclear. Two recent reports are the only Studies to date in which the consequences of altered protein binding haVe been examined. Murray _etal. (1981) examined the effect of renal i I“Pairment on oxazepam kinetics. They found that metabolic clearance of this benzodiazepine was increased in those patients suffering from uremic disease. This was associated with an increased Vd as the unbound fraction was twice as large in these patients as that of the healthy COHtrols. Piroli e_t_al. (1981) investigated the disposition of anti- pyrine (little protein binding) and warfarin (highly protein bound) in a 17 gazitient with idopathic hypoalbuminemia. Except for the reduced serum a1 bumin, this patient was healthy. Antipyrine clearance by this person was not different from control subjects. Warfarin clearance, however, was double that of the control subjects. The patient exhibited a normal Vd , despite a doubling of f3. However, warfarin half-time was signi- f'l' cantly shorter, suggesting a faster elimination rate constant and, hence, a greater clearance. These observations suggest that, indeed, aa'l‘i:eared binding does influence metabolic clearance of xenobiotic com- FD¢DDtJl1dS. Other pathological conditions increase the binding of xenobiotic compounds to blood components. In inflammatory conditions such as Crohn's disease, certain forms of arthritis, and chronic renal d ‘i sease, elevations in the plasma concentration of 0:1 acid glycoprotein Occur. A number of drugs, including dipyridamole, quinidine, imipra- m'i ne and alprenolol, bind to this protein in the plasma (Piafsky, 1980). P1 afsky 2391. (1978) examined the plasma binding of propranolol and Ch1 orpromazine in patients with these diseases. They found that, ‘3 hdeed, changes in binding correlated with alterations in a] acid glyco- protein concentrations. However, the relationship of this binding to a 'l terations in hepatic clearance was not examined. Lipoproteins also bind xenobiotic compounds. Diphenyl- h.ydantoin, bishydroxycoumarin, pentobarbital (Rudman gt 31;, 1972), tesztosterone and other steroids (Hobbelen e_t_a_1_., 1975), quinidine (Nilsen, 1976), chlorpromazine (Bickel, 1975), imipramine (Bickel, 1975; Damon and Chen, 1979), certain insecticides (Skalsky and Guthrie, 1978), beIizo(a)pyrene and other polycyclic aromatic hydrocarbons (Avignon, 18 1959; Shu and Nichols, 1979; Remsen and Shireman, 1981) have all been shown to associate with serum lipoproteins. Increased serum lipoprotein concentrations have altered binding within the serum, increasing the portion of these compounds associated with the lipoprotein fraction (Rudman gt_al,, 1972). Unfortunately, no studies have appeared in which the effect of lipemia or the lack of serum lipids on the uptake, dis- tribution and elimination of xenobiotic compounds were examined. Lipophilic drugs and xenobiotic compounds also bind to and are transported by red blood cells. Binding to this blood component has received little attention, and no experimental or clinical evidence is available to define its role in influencing metabolic elimination. Investgations of red blood cell binding have shown that lipophilic drugs such as chlorpromazine, imipramine (Bickel, 1975) and diphenylhydantoin (Kurata and Wilkinson, 1974) were associated with red blood cells. In addition, Kurata and Wilkinson noted that when albumin binding was decreased, thus increasing the unbound fraction of the drug in the plasma, binding to red blood cells was increased. B3. Effect of Altered Blood Flow, Metabolic Capacity, and Binding to BloodFComponents on Extrahepatic Metabolic Clearance From the foregoing discussion it is clear that significant evidence exists supporting the perfusion-limited model of hepatic meta- bolic clearance. In recent years, however, it has become evident that extrahepatic organs are also capable of metabolizing xenobiotic com- pounds (for example, see Gram, 1980a). Differences in flow and metabo- lic capacity between liver and other organs might influence the relative roles these organs play in the total body disposition of circulating foreign compounds. 19 There exist few reports of the roles that blood flow, metabo- lic capacity and binding to blood components have in determinining the disposition of xenobiotic compounds by extrahepatic organs. One such describes the influence of altered flow on metabolic clearance of sero- tonin (5-hydroxytryptamine; 5-HT) in isolated rat lungs (Wiersma and Roth, 1980). This biogenic amine is removed from the pulmonary circu- lation by a facilitated transport process localized to the vascular endothelium (Strum and Junod, 1972; Cross et_al,, 1974; Iwasawa et_gl,, 1973). Within the endothelial cell 5-HT is quickly metabolized by monoamine oxidase and an aldehyde dehydrogenase to an acid metabolite which is excreted from the body (Alabaster and Bakhle, 1970; Junod, 1972). As shown in Figure 2, alteration of flow markedly affected pulmonary 5-HT clearance. Clearance increased from 12 ml/min at a flow of 13 ml/min to 19 ml/min at a normal pulmonary flow (OP) of 45 ml/min. In order to determine the relative roles of liver and lung in the total body disposition of 5-HT, we also examined 5-HT clearance in rat livers perfused at several flows (see Figure 2). Hepatic 5-HT clearance was also dependent on flow and had a value of 7 m1/min when perfused at normal organ flow (OH). From this study we concluded that jg_vj!9_lung probably has a greater role than liver in the total meta- bolic clearance of 5-HT in the rat. Unlike 5-HT, the pulmonary clearance of norepinephrine is low and independent of flow in rat isolated lungs (Roth, 1982). Similarly, metabolic clearance of mescaline by rabbit lungs is also likely inde- pendent of flow (Hilliker and Roth, 1979). 20 Figure 2. Clearance of 5-hydroxytryptamine (5-HT) by isolated rat livers and lungs perfused at several flows. Isolated organs were perfused in a recirculating manner. Four lungs and five livers were perfused at each flow. Each organ was perfused at only one flow. 5-HT clearance was calculated as the product of the slope of the reservoir 5-HT disappearance curve and the apparent volume of distribution. Circles represent clearance values in lungs; squares represent clearance values in livers. The dashed lines represent normal organ flows; OH, hepatic; OP, pulmonary. 21 T nu Kg 3 2 2 EE \ 15 plan .0 320.505 O 15" 20 3O 4O 50 60 10 Flow (ml / min) Figure 2 22 B4. Conditions which May Alter the Relative Metabolic Clearance of Organs Each organ has its own clearance vs. flow relationship for each substrate. However, many physiological, pathological, pharmaco- logical, or toxicological conditions may change these clearance and flow relationships. Such changes may alter the relative contribution of an organ to total metabolic clearance. If clearance of a compound was enzyme-limited in both liver and extrahepatic organs, flow alterations may not affect their relative roles. However, metabolic capacity changes could. Similarly, if clearance in both was flow-limited, altered metabolic capacity might not substantially affect relative contributions to total metabolic clearance. Flow changes, however, could alter them. Since its Cl' for many compounds is usually much greater int than flow, metabolic clearance by liver is often flow-limited. Extra- hepatic organs commonly have low metabolic capacity and consequently clearance is often enzyme-limited. Thus, major shifts in relative con- tributions of organs to total body metabolic clearance most likely will follow altered relative perfusion of liver and extrahepatic organs. For example, during exercise hepatic flow decreases while cardiac output (pulmonary flow) increases markedly (Chapman and Fraser, 1954). Thus, for a compound metabolically cleared by liver and lung the relative contribution of lung may increase as hepatic flow decreases. Such relative decreases in hepatic flow also occur during fasting, in hypoxic individuals (Roth and Rubin, 1976b), during the transition from supine to upright posture and in patients with liver disease (Nies et_al,, 23 1976). However, if metabolic clearance by lung is flow-limited, its contribution to total body clearance could also increase in these conditions as pulmonary flow increases. Therefore, exposure to atmos- pheres of reduced oxygen content (Gorkin and Lewis, 1954; Cross gt_al,, 1959), and diseases such as beriberi, hyperthyroidism and anemia (Guyton gt 31,, 1973) which increase cardiac output might also increase relative pulmonary clearance. Decreased pulmonary clearance of flow-limited compounds could be expected with valvular disease, circulatory shock or in cardiac failure (Guyton §t_al,, 1973; Benowitz et_al,, 1974) as cardiac output decreases. Changes in organ metabolic capacity might also alter the roles of liver and extrahepatic organs in the clearance of compounds with enzyme-limited metabolic clearance. For example, Lake §t_al, (1973) found that some metabolic activities of extrahepatic tissues were in- creased more than corresponding hepatic activities on exposure of rats to 3-methylcholanthrene. In the absence of compensating alterations in affinity (Km) this would produce an increase in the total metabolic capacity (Cl'1nt) of extrahepatic organs that is greater than the in- crease in liver (since Cl'int = Vmax/Km)° Therefore, whether hepatic clearance is flow- or enzyme-limited, the relative contribution of extrahepatic organs would increase in the absence of hepatic flow changes. A possible example of this effect is the action of cigarette smoking on drug metabolism. In humans, smoking reduces the action of a number of drugs apparently by enhancement of metabolic elimination (Cohn, 1978; Jusko, 1978; McGovern gt_a1,, 1976; Parsons and Neims, 24 1978). Short-term studies in rats exposed to cigarette smoke have shown large increases in pulmonary and renal drug metabolizing activity with- out increases in hepatic metabolism (Van Cantfort and Gielen, 1975; Welch gt_al,, 1971). If humans respond in this manner, the increased elimination of some drugs in smokers may be the result of enhanced extrahepatic metabolic clearance. Exposure to toxic compounds could also affect the relative ability of organs to clear circulating compounds. Carbon tetrachloride, for example, is toxic to cells containing microsomal mixed function oxidases. In both liver and lung, systemic administration of carbon tetrachloride results in the destruction of cytochrome P-450 containing cells (Boyd gt_al,, 1980; Recknagel and Glende, 1973). Thus, Cl'int in each organ is likely to be reduced. Therefore, exposure to carbon tetrachloride may cause a severe reduction in the contribution of lung to total body clearance of metabolically cleared compounds. Exposure of rabbits to certain aldehydes (such as phthaldehyde and p-tolualdehyde) reduces both pulmonary and hepatic microsomal cyto- chrome P-450 in_yjtrg_(Patel et_al,, 1978; Patel, 1979). However, in the presence of the hepatic cytosolic fraction of cells this toxicity is prevented. Hepatic cytosol contains an aldehyde dehydrogenase which rapidly converts the toxic aldehydes to non-toxic carboxylic acids. Cytosol from lung cells, however, does not contain this enzyme and is not capable of detoxifying these aldehydes. Therefore, on exposure lg yjyg_liver cells might escape injury while lung cells would be damaged by these toxic agents. Thus, Cl'int for mixed function oxidase sub- strates in liver would be unaffected, but that of lung reduced. This 25 possibility suggests that the importance of lung could be reduced in total body metabolic drug clearance. Similar results might be expected in rabbits in which a selective destruction of.one form of cytochrome P- 450 occurs following exposure to polychlorinated biphenyls (Ueng and Alvares, 1981). In contrast, exposure of rats to allyl alcohol may increase the relative role of lung in the metabolic clearance of drug metabo- lizing enzyme substrates” For the same reason that phthaldehyde and p- tolualdehyde may reduce pulmonary C].int’ the lung is protected from the toxic actions of allyl alcohol (Patel gt 21,, 1980). Allyl alcohol is converted by liver cytosol to acrolein which causes periportal necrosis in liver. However, rat lung does not contain the requisite alcohol dehydrogenase to transform allyl alcohol to this toxic metabolite. Therefore, liver may be so damaged by this exposure that its Cl'int is reduced, thus raising the possibility that extrahepatic organs may participate to-a greater extent in the metabolic clearance of certain circulating xenobiotic compounds. C. Approaches to theJStudy of Organ Metabolic Clearance To determine the roles which organs play in the total body dispo- sition of xenobiotic compounds, it is necessary to examine those factors which determine metabolic clearance. For the studies described in this thesis, the influence of a number of factors which modify the ability of rat liver and lung to remove circulating compounds was examined. Liver was chosen because of its high specific activity as well as total metabolic capacity. Lung was examined because it has the highest rate of substrate delivery (flow) of any organ in the body, since it receives 26 all of the cardiac output. In addition, recent reports have established that lung has significant metabolic capability (Heineman and Fishman, 1969; Bakhle and Vane, 1974, 1977; Hook and Bend, 1976; Gram, 1980b). The relative ability of these two organs to eliminate xenobiotic com- pounds was examined at three levels: (1) biochemically using subcellu- 1ar fractions of organ homogenates, (2) in isolated organs and (3);yl 3119, Each of these methods has limitations, but each has distinct advantages as well. Cl. Broken Cell Studies Utilizing broken cell preparations the metabolic activity of the enzyme, the influence of substrate concentration changes and the role of free vs. bound forms of the substrate can be assessed without interference by organ structural barriers. Cofactor concentrations can be controlled so that resulting activities can be related to enzyme- substrate interactions. The major limitation of broken cell studies is that extrapolation to the situation jg_vjvg_is often not possible because the influences of cell structure, enzyme environment and dilu- tion of endogenous cofactors on enzyme-substrate interactions are un- known. C2. Isolated Organ Studies In recent years isolated organs have been used to investigate the ability of organs to metabolize xenobiotic compounds (see reviews by Thurman and Reinke, 1979; Roth, 1979; Roth and Wiersma, 1979; Mehendale §t_§1,, 1981). Although viable for only short periods of time, such preparations are useful and have certain advantages over broken cell preparations and studies jfl_V1VO. 27 Compared to broken cell studies, isolated organs offer the opportunity to investigate the role of organ structure on metabolic processes. Structure may influence cofactor availability and the trans- port of substrate into the organ or the cells containing degradative enzyme. Results in isolated organs may differ from those in broken cell preparations. For example, Law gt_al, (1974) found rabbit pulmonary subcellular fractions capable of metabolizing imipramine and chlor- cyclizine. In isolated, perfused lungs extensive uptake and sequestra- tion of these compounds occurred, but no metabolites were detected. Thus, these substrates were not available to cells containing enzymes capable of the metabolism. Similar differences between broken cell studies and isolated lungs occur with histamine, dopamine, tyramine, and epinephrine (Fishman and Pietra, 1974; Alabaster, 1977). Isolated organs are often a good approximation of the organ jg_ vjvg_and enable the functions of the organ to be examined without inter- ference by other organs. A major advantage over jg_vjyg_studies is control over physiological factors which influence organ function, such as flow, perfusion pressure, metabolic capacity and composition of perfusion medium. C3. Studies In M Despite the advantages of isolated organs, they only approxi- mate organ function jg_vivo. At best, an isolated organ is a deteriorat- ing preparation and can be used for only short times. Removal of the organ from the body may alter structural features, tissue viability, and thus function so that results may not reflect the situation jg_vivo. An advantage of studies j__vivo is that the influence of other organs on 28 the function of an organ can be examined. For example, the effect of toxic metabolites produced in the liver on extrahepatic organs is best examined jp_ijg, D. Benzo(a)pyrene as a Model Compound in the Study_of Metabolic Clearance Benzo(a)pyrene (B(a)P) was chosen as a model xenobiotic compound for these studies of organ metabolic clearance. B(a)P is a ubiquitous environmental contaminant (IARC, 1973) formed during the incomplete combustion of organic matter. Principal sources of exposure to B(a)P are through inhalation of polluted air or cigarette smoke and by in- gestion of many cooked foods. Metabolism of B(a)P by mammalian liver produces metabolites which are mutagenic, cytotoxic and carcinogenic to laboratory animals (DePierre and Ernster, 1978; IARC, 1973; Malaveille §t_al,, 1975; Wood §t_al,, 1975). The disposition of B(a)P is governed by its lipophilic nature. B(a)P distributes to fatty tissues of the body and is not excreted to any great extent in unchanged form (Kotin gt_al,, 1959; Levine, 1970). The transformation of B(a)P into water soluble excretable compounds is initiated by the enzymic action of a family of microsomal mixed function oxidases, arylhydrocarbon hydroxylase (AHH), which convert B(a)P into one of a number of epoxide intermediates (see Figure 3 for the pathways of B(a)P metabolism). Subsequent spontaneous degeneration or enzymic action convert these reactive epoxides to yet more polar and less reactive metabolites (Yang §t_al,, 1981). For example, one of the initial epoxides produced by the action of AHH upon B(a)P is the 7,8— epoxide. This epoxide may (1) spontaneously rearrange to 7-hydroxy 29 Figure 3. Pathways of B(a)P metabolism. Conjugates are formed by the action of glutathione-S-, uridine diphosphoglucuronic acid - , and sulfotransferases. Boxed metabolites elute in the peak of the HPLC chromatogram labelled "polar metabolites" (Figure 17); those in parentheses are usually unstable. AHH, arylhydrocarbon hydroxy- lase. EH, epoxide hydrase. SR, spontaneous rearranagement. 30 (emxuoss) 5y w QUINONES <—-— PHENOL\S DIHYDRODIOLS \ \\1\AHH [CONJUGATEJ F:—- 4”, 1» TRIOLS EH PHENOL AND AND <-—— DIHYDRODIOL TETROLS EPOXIDES Figure 3 31 B(a)P (7-phenol), (2) become conjugated with reduced glutathione, or (3) be converted to the 7,8-dihydrodiol by the action of the enzyme, epoxide hydrase. The phenol could then be (1) converted by non-enzymic re- arrangement and oxidation to a quinone or (2) conjugated enzymically with sulfate or glucuronic acid. The 7,8-dihydrodiol could (l) undergo conjugation with sulfate or glucuronic acid or (2) be further metabo- lized by AHH to a diol-epoxide (such as the 7,8-dihydrodiol-9,10-epoxide) which could subsequently become a trial, a tetraol, or a conjugate of glutathione, sulfate, or glucuronic acid. It is obvious that the pro- ducts of B(a)P metabolism are myriad. (A recent review by Gelboin (1980) summarized the current state of knowledge concerning the pathways of B(a)P metabolism.) Despite this vast body of knowledge concerning the mechanism of B(a)P metabolism, the formation and consqeuences of reactive metabolites, and the carcinogenic nature of B(a)P, the relative roles that various organs might play in the total body disposition of B(a)P is not known. A number of investigators have compared the metabolic capacity of rat livers and lungs using broken cell preparations at saturating con- centrations of B(a)P (Lake et_gl,, 1973; Weibel §t_gl,, 1971; Matsubara §t_gl,, 1974; Vahakangas gt_al,, 1977; Prough gt_al,, 1979; Lee et_al,, 1980). The ratio of rat liver/rat lung AHH activity in these studies ranged from 25-370; thus, it is clear that liver has greater specific activity. In one study (Prough gt_gl,, 1979) in which the individual metabolites were separated and identified, the only major difference was that a slightly greater fraction of dihydrodiols and a slightly lower fraction of phenols were produced by lung microsomal incubations. 32 The disposition of B(a)P has also been investigated in isolated rat livers (Levine, 1970; Vahakangas, 1979) and lungs (Vainio gt_al,, 1976; Cohen gt_al,, 1977; Vahakangas et_al,, 1977, 1979; Vahakangas, 1979). However, in none of these investigations were livers and lungs compared with regard to their ability to eliminate B(a)P from the system. In addition, physiological factors, such as flow and perfusion medium composition, which may influence the disposition of B(a)P vary between these preparations and are often inappropriate to relate these results to normal organ function jg_yiyg, The disposition of B(a)P in rats has also been examined jp_gj!9_for a variety of purposes (Kotin gt 21,, 1959; Iqbal gt_gl,, 1979; Vauhkonen .gt_gl,, 1980; Reeve and Gallagher, 1981; Chipman gt_§1,, 1981a,b). Following intravascular administration, B(a)P is rapidly removed from the blood and only a small amount is excreted into the bile unchanged. Thus, elimination of B(a)P does require metabolism. Metabolites also appear rapidly in both blood and bile. However, only a small portion of the B(a)P or metabolites is excreted with the urine. In most of these studies B(a)P metabolism is attributed to liver, although there is no data to support this claim. None of these investigations established the role of individual organs in the total body clearance of B(a)P. E. Purpose Organ metabolic clearance is influenced by metabolic capacity, flow and binding to blood components. B(a)P is eliminated from the body through metabolism; in part, by liver and lung. The overall purpose of the experiments reported in this thesis was to examine how metabolic 33 capacity, flow and binding to blood components affect the ability of rat liver and lung to clear B(a)P from the circulation. These studies should provide the information needed to assess the effect that such factors may have on organ metabolic clearance of lipophilic xenobiotic compounds. They should also provide information to assess the role that liver and lung may play in the total body metabolic clearance of certain xenobiotic compounds under normal conditions, as well as similar condi- tions in which relative flow, metabolic capacity, or blood composition may change. F. Experimental Approach Studies in broken cell preparations were used to assess the metabo- lic capacity of these two organs in both control and 3MC pretreated rats. Apparent enzyme kinetic parameters of AHH determined in micro- somal preparations were used to calculate Cl'int. The influence of binding was assessed by determining the effect of altered albumin con- centration on microsomal AHH activity. Isolated, perfused organs were used to assess the role of flow in determining metabolic clearance of livers and lungs with normal and enhanced metabolic capacity (control and 3MC pretreated rats, respec- tively). The influence of flow and metabolic capacity on B(a)P metabo- lite production was also examined. The role of albumin, lipoproteins and red blood cells on metabolic clearance was assessed in isolated livers. Causes for differences in B(a)P clearance between isolated organs and that predicted by the perfusion limited model were also examined. 34 The disposition of B(a)P in vivo was studied in conscious rats. The ability of liver and lung to extract B(a)P from the circulation as well as the influence of increased metabolic capacity following 3MC pretreatment on total body clearance were examined. MATERIALS AND METHODS A. Animals Male, Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, MI) weighing approximately 200-300 g were used in these studies. The animals were housed in plastic cages on corn cob bedding in 12-hour light cycled, temperature controlled quarters. Food (WayneR Lab Blox, Allied Mills, Chicago, IL) and tap water were allowed ag_libitum. Animals were acclimated to their quarters for a minimum of one week prior to use. B. Pretreatment of Animals 3-Methylcholanthrene was suspended in corn oil to a final concen- tration of 10 mg/ml. Each rat pretreated with 3MC received an injection (20 mg/kg, i.p.) 24 and 48 hr prior to use. This procedure produces near maximal induction of B(a)P metabolism in both rat liver and lung (Warren and Bellward, 1978). Control animals received an equal volume of corn oil. C. Preparation of 3 H-Benzo(a)pyrene [G-3H]-Benzo(a)pyrene, specific activity 17.4-26 Ci/mmol was puri- fied by the procedure of Van Cantfort gt_al: (1977) and mixed with non- radioactively labelled B(a)P (99+%, Aldrich Chemical Co., Milwaukee, WI) to the required concentration and specific activity in either acetone or 35 36 methanol for the broken cell assays and perfusions, respectively. These solutions were stored under nitrogen at -20°C in the dark until use. Selected solutions were analyzed by high-performance liquid chromato- graphy (Selkirk at al,, 1974) to confirm nominal concentrations. In experiments in which the identity of individual B(a)P metabolites was to be established, the purified 3H-B(a)P and non-radioactively labelled B(a)P were combined and subjected to further purification by high- 3H-B(a)P for injection performance liquid chromatography. Solutions of jp_yjyg_were prepared in serum. 0.6 mCi of 3H-B(a)P and 12 nmol of unlabelled B(a)P in acetone were placed in a glass tube. The acetone was evaporated under nitrogen at room temperature and the residue stored under nitrogen at -20°C. On the day of the experiment the residue was resuspended in 0.6 ml of serum prepared from a donor rat and an aliquot analyzed for radioactivity. 0. Studies Usinngroken Cell Preparations 01. Preparation of Microsomes Rats were sacrificed by decapitation and their livers and lungs were removed and immediately placed in ice-cold 0.05 M Tris-HCl buffer, pH 7.4, containing 1.15% KCl. The organs were then blotted, weighed, and placed in fresh buffer (2 volumes for liver, 4 volumes for lungs). The organs were homogenized using a PolytronR homogenizer (Brinkman Instruments, Westbury, NY) for 30-45 sec at a setting of 5.5- 6.0 and the homogenate centrifuged at 10,000 x g for 10 min at 4°C. Following centrifugation, the lipid was aspirated from the supernatant fraction, which was recentrifuged at 100,000 x g for 60 min at 4°C. The resultant supernatant fraction was decanted and discarded, and the 37 pellet resuspended in 0.05 M Tris-HCl (pH 7.4 for livers and pH 8.0 for lungs) containing 0.25 M sucrose and 10 mM EDTA to a final volume of 1 ml per initial gram of liver and 2 ml per initial gram of lung. Five microliters per 9 initial weight of an absolute ethanol solution con- taining 2% (w/v) butylated hydroxytoluene was added and the resuspended pellet homogenized with six passes of a teflon-glass tissue grinder. Appropriate dilutions of these preparations were prepared in Tris-HCl buffer (pH 7.4 for liver, pH 8.0 for lung) prior to determination of metabolic activity. 02. Determination of AHH Activity Ability of microsomal preparations to metabolize B(a)P was determined in duplicate essentially according to the method of Van Cantfort gt_al, (1977). Incubations were carried out at 37°C in a shaking incubator in 16x100 mm screw cap tubes. The incubation medium (0.5 ml) consisted of 0.05 M Tris-HCl buffer (pH 7.4 for liver pH 8.0 for lung) containing 1 mg/ml bovine serum albumin (Fraction V, Schwartz- Mann), 3 mM M9012, 0.1 mM EDTA, 0.4 mM NADPH, and an appropriate con- centration of microsomal protein. Reaction was initiated following a 2 min preincubation at 37°C by the addition of B(a)P (10 pCi/ml at various B(a)P concentrations) in 20 ul of acetone. Incubations were continued for two to four min as indicated in the figures and tables and stepped by the addition of 1.0 ml ice-cold 0.15 N KOH in 85% DMSO. Extraction of unreacted B(a)P was accomplished by the addition of 2x5 ml of hexane. Following 10 min of shaking the layers were allowed to separate and the hexane aspirated off. Samples (0.5 m1) of the remaining aqueous phase were removed, placed in scintillation vials 38 and neutralized by the addition of 0.5 ml of 0.15 N HCl. Fifteen ml of ACS (aqueous counting scintillant) was added and the radioactivity of each vial quantified by liquid scintillation spectrometry. External standard quench correction was used to correct the raw counts. 03. Calculation of Metabolic Activity The nmol of B(a)P metabolites present in each sample vial were calculated according to the following equation: DS 7 DB x nmol B(a)P added to (4) 0T the assay mixture where 05 is the dpm in a sample vial, 0B is the dpm of a sample blank vial (identical to an activity vial, but incubated in the absence of NADPH), and DT is the dpm in a sample which had not been extracted with hexane. This value was divided by the time of incubation in min and the mg of microsomal protein present in the assay to express the results as the nmol of B(a)P metabolites formed per min per mg of microsomal pro- tein. D4. Calculation of the Apparent Enzyme Kinetic Parameters, Vma and Km x Enzymic reaction rates were determined at 8 to 10 B(a)P con- centrations for each microsomal preparation, and the apparent enzyme kinetic parameters, Km and Vmax’ were calculated using a computer pro- gram based on the weighting procedure of Wilkinson (1961). 05. Estimation of fB Direct determination of the fraction of the total B(a)P free in the incubation mixture or perfusion medium was not possible using conventional equilibrium dialysis or column chromatography methods 39 (Hummel and Drayer, 1962). As an alternative, the B(a)P metabolic activity of microsomal preparations of lung and liver from control and 3MC pretreated rats was determined at two concentrations of added bovine serum albumin (BSA). The concentrations chosen were 1 mg/ml and 32 mg/ml reflecting the added BSA concentration in the microsomal assay mixtures used in the kinetics experiments and that in the perfusion medium of the isolated organs, respectively. The total B(a)P concen- tration in these incubations was 0.2 p”, the initial concentration at the beginning of the organ perfusions. fB was estimated as follows: f- ___ AHH aCtIVIty at 32 mg BSA/m1 (5) B AHH activity at 1 mg BSA/m1 Thus, the fB used in the clearance calculations was not actually the fraction unbound but was a "correction factor" reflecting the influence of the binding of B(a)P to BSA in the microsomal assays of AHH activity and its binding to BSA in the perfusion medium of isolated organs. E. Studies Using Isolated Perfused Organs El. Surgical Procedures Donor rats for isolated organ studies were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and given heparin (500 U, i.v.) to prevent clotting. To remove the livers from the rats, the abdomen was opened, the bile duct cannulated with PE-50 tubing (Clay-Adams, Par- sippany, NJ), the hepatic portal vein isolated, and ties placed around the hepatic portal vein and the inferior vena cava at the entrance of the right renal vein. Following cannulation of the hepatic portal vein the liver was perfused with cold, oxygenated, heparinized (10 U/ml) saline, the chest opened, and the hepatic vein cannulated by way of the 4O vena cava. The entire liver and diaphragm were then separated from the rest of the body and transferred to the perfusion apparatus. For lung removal, rats were anesthetized, the trachea cannu- lated, and the pulmonary artery cannulated through the right ventricle. After excision of the heart, the lungs were carefully removed from the body, inflated and deflated several times to prevent atalectasis, and transferred to the perfusion apparatus. E2. Perfusion Apparatus Isolated rat livers and lungs were perfused at 37°C in a constant flow perfusion system. Each organ was perfused at only one flow. Unless indicated, all perfusions were performed with recirculat- ing perfusion medium. Livers were perfused in an apparatus (MRA Corp., Clearwater, FL) similar to that used by Miller §t_al, (1951) while isolated lungs were perfused using an apparatus like that of Gillis and Iwasawa (1972) adapted for recirculating perfusion medium. In both preparations the perfusion medium was pumped (Cole-Parmer Instrument Co., Chicago, IL) from the reservoir through a silk filter, through a bubble trap, and into the organ. Prior to the bubble trap, the perfu- sion medium for livers was oxygenated with 95% 02-5% CO2 in a silastic tubing oxygenator (Hamilton gt_al,, 1974). The perfusion medium for lungs was oxygenated by ventilation of the lung with the same gas mixture as it passed through the organ. The ventilation was accom- plished by means of a modified Baby Bird respirator (Pearson Medical, Plymouth, MI) connected to the tracheal cannula. The gas was delivered to the lung at a frequency of 90 strokes/min with maximum inspiratory pressure of 15 cm H20 and end expiratory pressure of 2 cm H20. In both preparations, inflow pressure was monitored with a Statham P2310 41 pressure transducer and Grass model 7 polygraph. Following perfusion of the organ, the perfusion medium was returned to the reservoir. The pH of the perfusion medium was maintained between 7.3 and 7.4 in liver experiments by a pH controller (Horizon Ecology Co., Chicago, IL) which added small amounts of sodium bicarbonate to the perfusion medium reservoir. E3. Perfusion Media Three different perfusion media were prepared. All contained washed human red blood cells at a final packed cell volume of 20%. The remaining 80% of the 100 ml of medium for each liver or lung consisted of either (1) Kreb's bicarbonate buffer (pH 7.4) alone, (2) buffer with 4% bovine serum albumin (Fraction V, PentexR), or (3) buffer with the serum lipoproteins prepared from 100 ml of rat blood obtained from untreated rats. The buffer was equilibrated with 95% 02-5% CO2 prior to the addition of the albumin or serum lipoproteins. Perfusion medium (2) is referred to in this thesis as regular perfusion medium. a. Preparation of red blood cells Human red blood cells, obtained as outdated whole blood or packed cells from the American Red Cross (Great Lakes Regional Center, Lansing MI) were alternately centrifuged (7,000 x g, 15 min) and suspended in heparinized (1 U/ml) Kreb's bicarbonate buffer and two changes of non-heparinized Kreb's bicarbonate buffer at 4°C. Following the final centrifugation, the buffer was removed and the remaining cells transferred to glass tubes containing 25 mg of glucose. These tubes were stored at 5°C until time of use. 42 b. Isolation of serum lipoproteins The total lipoprotein fraction was isolated from rat serum according to the method of Nervi and Dietschy (1975). 580 ml of blood were allowed to clot and then centrifuged at 2,000 x g for 20 min to prepare serum. The density of the serum was adjusted to 1.215 g/ml by the addition 0.302 g of KBr per m1 serum. The adjusted serum was centrifuged at 43,000 rpm in a 50.2 Ti rotor for 35.5 hr at 5°C in a preparative ultracentrifuge (Beckman Instruments, Inc., Spinco Division, Palo Alto, CA, Model L8-55). The resultant layer of lipoproteins was removed from the top of the tube and dialyzed (Spectropor 2, Spectrum Medical Industries, Inc., Los Angeles, CA) against 3x4 L of 0.9% NaCl, containing 0.1% EDTA, for 24 hr at 4°C. This preparation was divided into aliquots corresponding to 100 ml of rat blood and stored at 4°C until use. E4. General Protocol for Perfused Organs Following a period of equilibration in which the inflow per- fusion pressure was allowed to stabilize (5-10 min for lungs, 35-40 min for livers) 3H-B(a)P (20 nmol, 40 uCT unless indicated otherwise) was added to the perfusion medium reservoir as a single bolus. Periodi- cally, following the B(a)P addition, 1.0 ml samples of the perfusion medium were removed from the reservoir and analyzed as described under "analytical methods". At the end of the B(a)P perfusion period, bile and, if required, tissue samples were also collected for analysis. In all cases, at the end of the perfusion the organs were removed from the apparatus, blotted, separated from extraneous tissue and weighed. Lungs in which the identity and concentration of specific metabolites were to be 43 determined were chilled with freon spray (CryokwikR, Damon/IEC Division, Needham Heights, MS) immediately on cessation of perfusion. In addition, identical "perfusions" were performed in which no organs were present to determine loss in the system due to uptake by the apparatus and/or "metabolism" by the perfusion medium. These clearance values (ranging from 0.30 to 0.48 ml/min for the liver apparatus and 0.59 to 1.02 ml/min for the lung apparatus) were subtracted from the results obtained when organs were present in the apparatus. E5. Calculation of B(a)P Clearance From semilogarithmic plots of the disappearance of B(a)P from the perfusion medium reservoir with time, the clearance (C1) of B(a)P was calculated using the following equations: C1 vd - ke (6) V dose/Co (7) d where ke’ the first-order elimination rate constant, is the slope of the disappearance curve determined by linear regression analysis and Vd is the apparent volume of distribution, determined as the dose of B(a)P divided by the perfusion medium concentration of B(a)P at zero time, Co. Co was determined by extrapolation of the disappearance curve to the ordinate. Co and k8 are the pharmacokinetic parameters for a one- compartment open model which correspond to B and B. respectively, in a two-compartment open model (see Figure 5). These clearances were cor- rected for uptake by the apparatus by subtracting clearance values ob- tained when an organ was not present in the apparatus and calculated' using equations 6 and 7. 44 E6. Details for Specific Experiments Utilizing Isolated Organs a. Flow dependence of B(a)P elimination Isolated livers from control and 3MC pretreated rats were perfused as described above with regular perfusion medium for 45 min after the addition of the B(a)P at 7, 10, or 20 ml/min. Lungs from similarly treated rats were perfused with 3H-B(a)P for 60 min at 10, 20, or 45 ml/min. b. Flow dependence of B(a)P metabolism Isolated lungs from control and 3MC pretreated rats were perfused with regular perfusion medium at either 10 or 45 ml/min. Samples of perfusion medium and lung tissue were analyzed for B(a)P and individual metabolite content as described below. c. Efflux of B(a)P from isolated lungs In order to determine whether B(a)P removal was irrever- sible isolated lungs from control or 3MC pretreated rats were perfused 14C-sucrose at 10 m1/min in a single pass mode with medium containing and 3H-B(a)P and lacking red blood cells. When sucrose and B(a)P con- centrations in the effluent medium reached steady-state, perfusion was continued with sucrose and B(a)P-free medium. The effluent medium from the lungs was collected in approximately 40 fractions over the following 20 min and the concentration of sucrose, B(a)P, and total B(a)P metabo- lites determined in each fraction. d. Concentration dependence of B(a)P elimination Isolated livers and lungs from 3MC pretreated rats were perfused with regular perfusion medium at 10 ml/min as described above except that either 0.56 or 20 nmol of 3H-B(a)P was added to the 45 perfusion medium reservoir. The amount of radioactivity added was the same at both concentrations. e. Distribution of 3 H within perfused livers Isolated livers from control rats were perfused with regular perfusion medium at 10 m1/min as described above. Twenty nmol of 3H-B(a)P was added to the reservoir at zero time. At 15 min after the addition of the B(a)P to the reservoir the perfusion was ended, the liver removed from the apparatus, and portions sampled from 23 selected sites (see Figure 23). The samples were digested with Soluene-350R, R scintillation decolorized with 30% hydrogen peroxide, 10 ml of 3a20 cocktail (Research Products International Corp., Elk Grove Village, IL) added, and the radioactivity of the samples quantified by liquid scin- tillation spectrometry. Counting efficiency was determined by external standardization. f. Determination of hepatic portal-hepatic venous shunts Isolated livers of 3MC pretreated rats were perfused with regular perfusion medium at 10 m1/min as described. Following recircu- lating perfusion with B(a)P the system was switched to a single pass mode and approximately 4.4 uCi of strontium 85 labelled microspheres (20,000 spheres, 15 micron diameter; 3M Co., Medical Products Division, St. Paul, MN) injected into the perfusion tubing in a retrograde manner just upstream from the inflow cannula. The effluent was collected for 4 min and the perfusion stopped. All tubing and cannulas, the entire liver, and effluent perfusion medium were placed in appropriate vessels and the radioactivity quantified in a gamma scintillation counter. The counts were summed after subtraction of appropriate blank values and the 46 fraction of the radioactivity appearing in the effluent calculated. This value was taken as the fraction of the portal flow which entered the hepatic veins without passing through the hepatic sinusoids. 9. Dependence of B(a)P elimination on medium composition Isolated livers from 3MC pretreated rats were perfused as described above at 10 ml/min with perfusion media of varying composi- tion. In all cases the 100 m1 of perfusion medium for each liver con- tained red blood cells at a packed cell volume of 20%. The remaining 80 ml of medium was composed of Kreb's bicarbonate buffer (1) alone, (2) containing 4 9 BSA, or (3) the serum fraction containing lipoproteins from 100 m1 of rat blood. F. Association of B(a)P with Blood Fractions Fl. Injection with B(a)P Rats to be given B(a)P were placed in a restrainer and a 25 ga needle attached to PE-20 tubing inserted into a tail vein. After insuring that the needle was in the vein by drawing a small amount of blood, 1.0 ml/kg of 3H—B(a)P solution was injected (117 nmol, 2 mCi/ml in donor rat serum). Residual solution was flushed from the tubing with an equal volume of 0.9% NaCl, and the rat was then removed from the restrainer. Approximately 17 min after administration of the B(a)P the rats were anesthetized with ether, the abdomen opened and blood with- drawn from the descending aorta through a 20 ga hypodermic needle. The sample was placed in a glass tube, a portion removed for determination of B(a)P and total metabolite content, and the remainder allowed to coagulate. 47 F2. Separation of Blood Components After coagulation the blood was spun (2,000 x g, 20 min) to separate the serum. A sample was removed for B(a)P and metabolite analysis and the serum lipoproteins were isolated as described above for perfusion medium preparation, except that a 50 Ti rotor was used at 40,000 rpm and the dialysis was omitted. The lipoprotein fractions obtained were diluted to 10 ml and a portion analyzed for B(a)P and metabolites as described below. These lipoprotein fractions, prepared from 4.3:0.2 m1 of serum, contained l.l:0.l mg of protein/ml and 4.3:0.4 mg lipid/ml serum. These results are similar to those reported for rat blood by Mills and Taylaur (1971). G. Studies of B(a)P Disposition In Vivo G1. Preparation of Cannulas Cannulas for the removal of blood from the aorta were prepared R tubing of two sizes (Cole-Farmer, Inc., Chicago, from microbore Tygon IL). Tubing of 0.01" 1.0. x 0.03" 0.0. was soaked in 1:1 petroleum ether-toluene and stretched over 14 gauge stainless steel wire to main- tain internal diameter and reduce wall thickness. Following curing in a steam oven overnight the wire was removed, the tubing trimmed to about 4 cm and inserted into a 9" segment of 0.02" 1.0. x 0.06" 0.0. tubing which had been looped at one end. The junction was sealed with an acrylic glue. Cannulas for the administration of the B(a)P solution were also constructed of 0.02" x 0.06" TygonR tubing into which was inserted a second piece of tubing which was to be placed within the blood vessel. (For the intra-arterial and intravenous cannulas this 48 second piece was a short length of PE20 tubing; for the intra-hepatic portal cannula a piece of stretched 0.01" x 0.03" tubing was used.) As before, the junctions were sealed with acrylic glue. G2. Cannula Implantation Under pentobarbital anesthesia (50 mg/kg, i.p.) cannulas for the administration of benzo(a)pyrene and removal of blood samples were placed in appropriate blood vessels (schematically shown in Figure 4). Cannulas for intra-arterial (i.a.) administration were placed in the left carotid artery. The tip of the cannula just entered the aorta. The cannula for intravenous (i.v.) administration was placed in the left jugular vein with the cannula tip in the vena cava. The intra-hepatic portal vein (h.p.v.) cannulas were placed into the ileocaecocolic vein. Cannulas for the removal of blood samples were placed in the left femoral artery. The tip of the cannula resided in the aorta at about the level of the lumbar artery. All cannulas were filled with isotonic saline (0.9% NaCl containing heparin at 10 U/ml), tunnelled underneath the skin to an incision in the back between the scapulae and exteriorized. Incisions were closed with sutures and painted with antiseptic solution (Acu- dyneR , Acme United Corp., Medical Products 0iv., Bridgeport, CT) to retard infection. The rats were allowed 48 hours of recovery prior to initiation of the pretreatment regimen. G3. Administration of B(a)P and Collection of Blood Samples On the day following the last corn oil or 3MC injection the Pharmacokinetics of B(a)P was determined in conscious rats. All can- "UTas were flushed with a small volume of saline containing heparin (10 49 Figure 4. Sites of cannula implantation. Cannulas for B(a)P admini- stration and blood sampling were placed in vessels at the indicated sites as described in "Methods". Abbreviations: i.a., intra-arterial, i.v., intravenous, i.v.; h.p.v., intra-hepatic portal vein. 50 «U NG A omen L“i TISSUES Figure 4 blood samples in _E.“ a 1.1' J if 51 units/m1) and 300-400 units of heparin administered. Following the collection of a blood sample, 1 ml/kg of the 3H-B(a)P solution was administered (117 nmol, 2 mCi/kg) and the cannula flushed with an equal volume of heparinized saline. Samples of blood (approximately 150 u1) were removed from the aorta 5 min before and 2, 5, 10, 20, 30, 45, 60, 105, 150, 195, 240, and 300 min following the administration of the B(a)P. Residual blood in the cannula after sampling was flushed into the aorta with heparinized saline. 100 pl of blood was analyzed for the presence of B(a)P and B(a)P metabolites as described below. Packed cell volumes were determined at -5, 60, and 300 min. After the final blood sample was removed sodium pentobarbital (300 mg) was administered. Liver, lungs, epididymal fat, kidneys, spleen, and muscle (diaphragm) were removed and the position of the cannulas verified. The organs were immediately placed on ice, weighed, frozen at -70°C, and analyzed for B(a)P and metabolites. G4. Pharmacokinetic Analysis Blood B(a)P concentration vs. time data were subjected to pharmacokinetic analysis using a two-compartment open model (Figure 5) and the program of Nielsen-Kudsk (1980) for mini-calculators. This program fits the data to a line described by the equation: where Ct is the blood B(a)P concentration at time t, A and B are the B(a)P concentrations at zero time and a and B are the values of the COlnposite first-order rate constants for the initial and terminal phases 01’ B(a)P concentration decline. The apparent volume of distribution 52 Figure 5. Two-compartment open model. The pharmacokinetics of B(a)P disposition in vivo were determined using this model. In the upper portion of the figure is a scheme showing the central (1) and peripheral (2) compartments with their associated transfer rate constants k12, kg], and k] . In the lower portion of the figure is an idealized blood con- centra ion vs. time curve described by the equation: ct = A . e'O‘t + B . e'8t The composite first-order_rate constants, a and B, were determined from blood B(a)P concentration vs. time data as the slopes from the terminal phase (B) and residuals (a) using linear regression. A and B are the ordinal intercepts. The parameters are related to the transfer rate constants by the following equations: k12 = a ' 8 ' k10 ' k21 _ A8 k21 ‘ a 1 5" ‘T‘IFK7B k10 = a ' B / k21 Clearance was calculated as: dose of B(a)P B ' k C1 = - B = V 1 10 where dose/B is the apparent volume of distribution and V] is the volume of compartment 1. 54 (Vd), clearance (Cl), and area under the concentration curve from zero to infinity (AUCm) were calucated as follows: dose of B(a)P vd = B (9) c1 = vd - s (10) AUC” = (A/a) + (B/B) (11) GS. Calculation of Organ Extraction 3 The fraction of the H-B(a)P extracted by the lung and liver jp_vivo was calculated from the fraction of the dose available following first-pass through the organ (Cassidy and Houston, 1980). A dose of B(a)P administered i.a. is 100% available to the arterial circulation (see Figure 4). This is reflected in the arterial AUC0° following i.a. administration. Conversely, a dose given i.v. must pass through the pulmonary circulation prior to entering the arterial circulation. Therefore, the arterial AUCco will be reduced in proportion to the B(a)P removed during the first-pass through pulmonary circulation. The frac- tion of the dose of B(a)P available to the arterial circulation follow- ing passage through the lung (fP) is given by the following equation: = AUC i.v. fP AUC i.a. (12) where AUC i.v. and AUC i.a. are the areas under blood B(a)P concentra- tion vs. time curve from zero to infinity for intravenous and intra- arterial administration, respectively. Similarly, the fraction of the 55 dose escaping removal on single passage through the liver (fH) is given by the following equation: _ AUC h.p.v. fH ‘ AUC i.v. (13) where AUC h.p.v. is the area under the arterial blood B(a)P concentra- tion vs. time curve following an intra-hepatic portal vein administra- tion. The fraction of the dose extracted by the organ is the frac- tion which is not available. Thus, the fraction of the dose extracted by the lung (EP) and that extracted by the liver (EH), respectively, are given by the following equations: ['11 II —-I I -h (14) “'1 II ._| I —h H (is) H. Analytical Methods I H1. Protein Determination The protein content of microsomal and lipoprotein preparations was determined by the method of Lowry at_a1, (1951). Human serum pro- tein standard was used as the standard reference. H2. Lipid Determination Lipid content of lipoprotein preparations was determined by gravimetric analysis following extraction of the lipids with chloroform- methanol (Sperry and Brand, 1955). 56 H3. Separation and Quantification of B(a)P and B(a)P Metabolites Two methods of analysis were used depending on whether or not the identity and quantity of individual B(a)P metabolites was to be determined. a. Extraction of B(a)P from samples with hexane B(a)P and total B(a)P metabolites in samples of blood, serum, bile, lipoprotein fractions, perfusion medium of isolated livers, or rat tissues were separated by the method of Van Cantfort at_a1, (1977). Prior to analysis, samples of blood, serum, or bile were diluted to 0.50 ml with 0.9% NaCl. Samples of perfusion medium were analyzed without dilution. The entire lipoprotein layer was diluted to 10 ml with 0.9% NaCl. Tissues (approximately 100 mg in weight) were homogenized in 2 ml 0.9% NaCl. 0.5 ml of these mixtures were added to 1.0 m1 of 0.15 N KOH in 85% dimethyl Sulfoxide (DMSO) and extracted with 2x5 ml hexane. Each of these hexane layers was back extracted into a fresh 0.5 ml 0.9% NaCl in 1.0 ml KOH/DMSO mixture to remove contami- nating metabolites. This KOH/DMSO layer was combined with the initial layer and an aliquot placed in a scintillation vial. Hexane layers for each sample were combined in plastic scintillation vials and the hexane allowed to evaporate. Ten ml of ACS was added to each hexane B(a)P- containing vial and 15 ml to each metabolite-containing vial. The radioactivity in vials was quantified by liquid scintillation spectro- metry. Quenching was corrected by external standardization. This procedure resulted in minimal contamination of the hexane extract with B(a)P metabolites and accounted for 90-95% of the 3H-B(a)P in perfusion 3 medium samples to which known amounts of H-B(a)P had been added. 57 3 b. Separation and quantification of H-B(a)P and individual metabolites in methanol extracts Samples of perfusion medium (1.0 ml) or lung tissue of known weight (approximately 100 mg) were immediately added to 4 ml of ice-cold methanol containing 0.08% butylated hydroxytoluene. The lung samples were homogenized at 0°C with a Polytron homogenizer (Brinkman Instruments, Inc., Westbury, NY), the precipitated material separated by centrifugation, and the methanol layer removed. The pellets were sub- jected to two additional methanol washes of 4 ml each. The methanol layers were combined, filtered through TE 36 filter membranes (Schleicher and Schuell, Inc., Keene, NH), evaporated under nitrogen, and stored at -20°C until analyzed. The evaporated samples were resuspended in 400 u1 of methanol and 200 p1 analyzed by high pressure liquid chromatography (HPLC) according to the method of Selkirk §t_al, (1974). Separation of B(a)P and its metabolites was achieved using an HPLC system (Waters Assoc., Inc., Milford, MA) with a precolumn of C18/Corasil (Waters Assoc., Inc.) and a Bio-Rad ODS-10 column (4x250 mm, Bio-Rad Laboratories, Richmond, CA). Gradient elution was used with a constant flow of 1.5 ml/min. Initially, the solvent was 65% methanol in water and altered in compo- sition by a gradient controller (Waters Assoc., Inc., Model 660 solvent programmer, program No. 7) over 45 min to a final concentration of 85% methanol in water. The absorbance of the effluent from the column was monitored throughout with a Waters Model 440 detector at 254 nm. However, detec- tion of B(a)P and metabolites in the samples was not possible by this 58 method due to interference from the biological matrix. Therefore, the effluent was collected into separate scintillation vials every 30 sec and the radioactivity determined by liquid scintillation spectrometry. Peaks of radioactivity corresponding to the retention of authentic B(a)P metabolites (a kind gift of IIT Research Institute, Chicago, IL) were summed and the concentration of each determined assuming the specific activity of metabolites to be the same as that of the administered labelled B(a)P. This method of analysis was able to account for ap- proximately 80% of the total radioctivity remaining in the system at the end of the perfusions. All concentration values have been corrected to reflect this loss from sample handling. After correction for recoveries, each of these methods of B(a)P analysis gave equal results for samples analyzed by both methods. I. Chemicals 3—Methylcholanthrene was obtained from Pfaltz and Bauer, Stamford, CT. Corn oil (MazolaR) was obtained from Best Foods, Englewood Cliffs, NJ. [G-3HJ-Benzo(a)pyrene (17.4-26 Ci/mmole), [u-‘4c1-sucrose (477 mCi/mmole) and aqueous counting scintillant (ACSR) were obtained from Amersham Corp., Arlington Heights, IL. Soluene-350 was purchased from Packard Instrument Co., Inc., Downers Grove, IL. Non-radioactively labelled B(a)P was purchased from Aldrich Chemical Co., Milwaukee, WI (99+%; gold label). Bovine serum albumin (fraction V) was obtained from Miles Laboratories, Elkhart, IN (PentexR) and Schwarz-Mann, Orangeburg, NY. Sodium heparin, and human serum protein standard were from Sigma Chemical Co., St. Louis, MD as were nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), ethylene diamine tetraacetic acid, 59 disodium—calcium salt (EDTA), and butylated hydroxy-toluene (BHT). Tris-HCl buffers were prepared from tris-(hydroxymethyl)aminomethane (Fisher Scientific Co., Fair Lawn, NJ) and 0.2 N hydrochloric acid. Reagent grade D-glucose, sucrose, magnesium chloride (MgClZ), potassium chloride (KCl), potassium hydroxide (KOH), sodium bicarbonate, and sodium chloride (NaCl) were obtained from Mallinkrodt, Inc., St. Louis, MO. Dimethyl sulfoxide (DMSO) was obtained as an analytical grade solvent from Mallinkrodt, Inc. while other solvents (acetone, chloro- form, hexane, and methanol) obtained from various manufacturers were of "distilled in glass" quality. Absolute ethanol was obtained from Aaper Alcohol and Chemical Co., Louisville, KY. J. Statistical Analysis Results are expressed as means i S.E.M. Student's t-test for unpaired observations was used to compare means in experiments with only control and treated groups. One-way analysis of variance, completely random design, was used to detect differences between more than two groups. The least significant difference (lsd) test was used to compare the means for preplanned comparisons, while Tukey's w-procedure was used for unplanned mean comparisons. Where unequal variances were noted, appropriate tests were performed on transformed data (logarithmic trans- formation). P<0.05 was chosen as the level of significance (Steel and Torrie, 1960). RESULTS A. Broken Cell Studies Al. Determination of Apparent Enzyme Kinetic Parameters and Intrinsic Free Clearance In initial experiments the time and microsomal protein concen- tration dependence of AHH activity were determined in microsomal pre- parations of liver and lung homogenates from control and 3MC pretreated rats. The results of these assays are presented in Figures 6-9. For each, time and protein concentration dependence of AHH activity were determined at the highest and lowest B(a)P concentrations used in the substrate concentration dependence assays. For the most part, product formation increased linearly with time and microsomal protein concen- tration. The incubation times and microsomal protein concentrations chosen for the B(a)P concentration dependence assays were in the linear portions of the time and protein concentration dependence curves. In Table l are presented the actual conditions under which the substrate concentration dependence assays were run. Under these conditions 10% or less of the substrate was normally metabolized. In no case was greater than 20% of the B(a)P metabolized. The B(a)P concentration ranges for determination of substrate dependence were selected to span approxi- mately 0.2 and 2.0 times the approximate Km value determined in preli- minary experiments. 60 61 Figure 6. Incubation time and microsomal protein concentration depen- dence of microsomal liver AHH activity of control rats. Rats were pre- treated with corn oil, livers removed, microsomes prepared, and the amount of products formed determined at the indicated time and micro- somal protein concentrations as described in the text. Assay volume was 0.5 m1. Microsomal protein concentration was 0.022 mg/ml during time assays. Protein concentration assays were run at 4 min incubation time. Results are from a single experiment. 62 LIVER-CONTROL 0.2 pM 20 pM 80 Time (min) 160 120 1.0 pmol B(a)P metabolites formed 80'- 0.5 40 10 20 10 20 Protein (#9) Figure 6 63 Figure 7. Incubation time and microsomal protein concentration de- pendence of microsomal liver AHH activity of 3MC pretreated rats. Rats were pretreated with 3MC, livers removed, microsomes prepared, and the amount of products formed determined at the indicated time and microsomal protein concentrations as described in the text. Assay volume was 0.5 ml. Microsomal protein concentration was 1.08 pg/ml during time assays. Protein concentration assays were run at 2 min incubation time. Results are from a single experiment. 64 LIVER-3MC 0.04 pM Time (min) f 30 20 pmol B(a)P metabolites formed 10' - Protein (p9) Figure 7 Figure 8. Incubation time and microsomal protein concentration de- pendence of microsomal lung AHH activity of control rats. Rats were pretreated with corn oil, lungs removed, microsomes prepared, and the amount of products formed determined at the indicated time and microsomal protein concentrations as described in the text. Assay volume was 0.5 m1. Microsomal protein concentration was 0.526 mg/ml during time assays. Protein concentration assays were run at 4 min incubation time. Results are from a single experiment. 66 LUNG-CONTROL 0.1 M 2.0 M 4, y a- P Time (min) 0.8 f 40 r 0.6 - . 30 f pmol B(a)P metabolites formed 0.4 0.2 . . j . . . 200 400 200 400 Protein (pg) Figure 8 67 Figure 9. Incubation time and microsomal protein concentration de- pendence of microsomal lung AHH activity of 3MC pretreated rats. Rats were pretreated with 3MC, lungs removed, microsomes prepared, and the amount of products formed determined at the indicated time and microsomal protein concentrations as described in the text. Assay volume was 0.5 ml. Microsomal protein concentration was 0.152 mg/ml during time assays. Protein concentration assays were run at 2 min incubation time. Results are from a single experiment. pmol B(a)P metabolites formed lUNG-SMC 401' 30. 20 V 10 40' 30' 20 10 0.1 pM 68 1.1 pM Time (min) 80 F 60-- 4o- 20* 100 200 Protein ()1 9) Figure 9 L 100 L 01b 200 69 TABLE 1 Assay Conditions for Determination of Apparent Enzyme Kinetic Constants of Hepatic and Pulmonary Microsomal AHH Activity1 Control 3MC Lung Liver Lung Liver Incubation time 4 4 2 2 (min) Microsomal protein 614:17 20:1 209i18 0.98:0.05 (pg/ml) B(a)P concentration 0.1-2.0 2.0-20.0 0.1-2.0 0.04-0.55 range (uM) 1In initial experiments at the extremes of the B(a)P concentration ranges, linear dependence of B(a)P metabolism on incubation time and microsomal protein concentration were observed (Figures 6-9). The times and protein concentrations indicated were within those linear dependence regions. Microsomal protein is presented as mean i S.E.M. of the three substrate concentration dependence experiments. 70 In Figures 10 and 11 are presented the results of the B(a)P concentration dependence assays for the microsomal metabolism of B(a)P by livers and lungs, respectively, from control and 3MC pretreated rats. These Lineweaver-Burk plots appeared linear. Using the activity and B(a)P concentration data from such microsomal incubations the apparent enzyme kinetic constants, Km and Vmax’ were calculated (Table 2) using the weighting procedures of Wilkinson (1961). The microsomal preparation of control lung had a lower Km for B(a)P than did that from control liver. However, when organs from 3MC pretreated rats were used the situation was reversed; liver had a Km value four times less than that of the lung microsomes. Pretreatment of the rats with 3MC did not alter the Km of lung micro— somes. On a per milligram of microsomal protein basis liver micro- somes had a much greater activity than those of lung in both control and 3MC pretreated rats, being about 140 and 60 times greater, respectively. When the data were expressed in terms of the metabolic activity of the whole organ, liver was by far the organ of greater metabolic capacity having Vmax values 2500 and 1200 times greater than lung in control and 3MC pretreated rats, respectively. However, 3MC pretreatment had a greater effect on lung than liver, stimulating this organ's metabolic activity 8 times while raising liver activity only half as much. Under first-order conditions, Cl'int is probably a better index of the metabolic capacity of the organ than the Vmax alone, since it also takes into account the affinity (Km) of the degradative enzyme for its substrate (see footnote 4 to Table 2). As shown in Table 2 the 71 Figure 10. Lineweaver-Burk plots of the substrate concentration de- pendence of liver and lung microsomal AHH activity of control rats. Details of the treatment procedures, incubations, and assay techniques are described in "Methods". Data points represent the mean i S.E.M. of three experiments. The lines were drawn from the mean calculated values of% and V determined by the method of Wilkinson (1961). S, substrate (B( (a)P)x concentration; v, velocity of reaction; AHH, arylhydrocarbon hydroxylase. 72 30 r- uvea-comam .1- C '3 '5 s 2 1/v .2 “ '5 E E .5 E v -2 -1 V 5 01“") LUNG-CONTROL soot A ., .5 in o 2 E 2 200b ‘- o. l/h :5 . r’ o E E S E v L 100 -o -4 -2 2 4 o a 10 V5 OM") Figure 10 73 Figure 11. Lineweaver-Burk plots of the substrate concentration de- pendence of liver and lung microsomal AHH activity of 3MC pretreated rats. Details of the treatment procedures, incubations, and assay techniques are described in "Methods". Data points represent the mean i S.E.M. of three experiments. The lines were drawn from the mean calculated values of% and V determined by the method of Wilkinson (1961). S, substrate (BTalP) concentration; v, velocity of reaction; AHH, arylhydrocarbon hydroxylase. 74 LIVER-3MC .’ 0.30 r b 0 1 0 50.9:- 03.5.: L $0.53. .25. l/v V 20 25 15 (fiNVU an LUNG - 3MC p o 5 o 4 L n p O O 3 2 £20... 93......- 3063. .05: Vv V Figure 11 75 TABLE 2 Apparent Enzyme Kinetic Parameters and Intrinsic Free Clearance for Microsomal B(a)P Metabolism Control 3MC Lung Liver Lung Liver Km (on) 0.22:0.08 5.5:1.1 0.23:0.03 o.os4:o.oo7 Vmax2 0.012:0.003 l.7:0.5 0.107:0.003 6.8:0.4 Vmax3 0.16:0.05 409:100 1.3:0.1 1524:256 Ci'm4 o.9:o.4 73:3 o.o:1.2 28,257t3006 (ml/min) 1 Metabolic activity of microsomes from lungs and livers of corn oil and 3MC pretreated rats was assessed by the method of Van Cantfort gt_al. (1977) and analyzed by the procedure of Wil- kinson (196117' Values represent mean i S.E.M. of three experi- ments. Km, Michaelis-Menten constant; Vmax, maximal velocity of reaction; Cl'int’ intrinsic free clearance. 2nmol B(a)P metabolites formed/min/mg microsomal protein. 3nmol B(a)P metabolites formed/min/organ. 4 1 _ C1 int - Vmax/Km 76 Cl'int of lungs of control rats was 0.9 ml/min which increased to 6 m1/min when the rat was pretreated with 3MC. Similarly, livers of control rats had a Cl'int of 73 m1/min, while that of 3MC pretreated rats was 28 liters/min. Accordingly, it appears that in both control and 3MC pretreated rats liver has a much greater Cl'int than lung. A2. Effect of Added Bovine Serum Albumin (BSA) on Microsomal B(a)P Metabolism Binding of substrates to plasma proteins reduces the free fraction of the compound in the blood and, thus, may decrease the amount of substrate available for metabolism. Attempts were made to measure the protein binding of B(a)P directly by two conventional methods, equilibrium dialysis and column chromatography. However, in the former method, equilibrium could not be reached even after 6 days, while in the latter method, the B(a)P bound avidly to the column material. Instead, the influence of binding was assessed by determining the effect of added BSA on the microsomal metabolism of B(a)P. This is shown in Figure 12 for the metabolism of B(a)P by microsomes of livers from control rats. No change in AHH activity was observed with increasing concentrations of added BSA up to 1 mg/ml. At that concentration activity began to de- crease with increasing BSA concentration. This effect was also examined at two BSA concentrations for each of the organs and pretreatments (Table 3). The concentrations of added BSA were those present in the enzyme kinetic assays (1 mg/ml) and used in isolated organ perfusion medium when present (32 mg/ml). B(a)P concentration was 0.2 pM, the nominal initial concentration in the perfusions. Activity in microsomes of livers from control rats was 77 .mem Foaexm we“ cmgp mmm_ mm=Fo> .z.m.m um; meocsm vemvcmum psocpwz mucwoa apes .mpcmewemaxm mass» mo .z.m.m n xpw>wpom cams mew m»_:mwm .2: o._ mm; covpmcpcwocou avam .emc05umze cw umaweumwu mm amnescmumv xpw>wpum Azzon mo mcowpmep -cmucou mcwmmmsocw saw: nmumnzocw mew: mpmc _ochoo we msm>w_ sage mmEOmoeuPz .mpmg Foepcou we xpv>wuom Iz< Fmeomoeowe Lm>w_ co cwE:n_m Esemm wcw>on venue we mocozpmcw use .N_ mszmwa 78 49. .l J 9’? ‘0 5L n :7 1 _L 1 $ . ('0 N I-, o 3 d d d o (ugegmd Bw-ugw / peuuo; gown) MANDV HHV BSA Concentration (mg/ml) Figure 12 79 TABLE 3 Effect of Added BSA on the Microsomal Metabolism of B(a)P1 Concentration of Added BSA (mg/m1) 1 32 AHH Activity 2 Tissue Treatment (pmol B(a)P metabolites formed) Ratio min . mg microsomal protein Liver Corn Oil 53:5 7.9:2.4* 0.14:0.03 Liver 3MC 3,660:50 5,210:290* l.4:0.l Lung Corn Oil 3.1:0.8 3.5:1.l l.l:0.l Lung 3MC 44:1 45:2 l.0:0.0 1B(a)P metabolic activity of microsomal preparations was determined according to the method of Van Cantfort et a1, (1977). Values are mean : S.E.M. of three experiments. BSAITbovine serum albumin. B(a)P concentration was 0.2 uM. 2Ratio of activity at 32 mg/ml added BSA to that at 1 mg/ml added BSA. *Significantly different than activity at 1 mg/ml added BSA by Student's t-test (p<0.05). 80 significantly reduced by increasing the BSA concentration while activity in liver microsomes from 3MC pretreated rats was slightly, but signi- ficantly increased. Activity in lung microsomes from both control and 3MC pretreated rats was not altered. A3. Organ B(a)P Clearances Predicted from Results of Broken Cell Studies Using the estimates of Cl'int calculated from the enzyme kinetic parameters (Table 2) and of f8 from the BSA concentration de- pendence experiment (Table 3; equation 5), B(a)P clearances for rat liver and lung at normal organ flow were predicted using equation 3. Estimates of normal organ flow were taken from the studies of Roth and Rubin (1976a) and Sapirstein gt_al, (1960). Normal hepatic flow was estimated to be 10 ml/min and normal pulmonary flow to be 45 ml/min for the size rats used in these experiments. 3MC pretreatment does not increase total hepatic flow (Nies at_al,, 1976). No corrections were made for recovery of microsomal enzyme activity. As shown in Table 4, in control rats lung was predicted to clear 0.97 m1/min, while liver would clear 4.6 ml/min. In 3MC pre- treated rats, pulmonary clearance was predicted to increase to about 7.0 ml/min, while hepatic B(a)P clearance would be equal to hepatic flow at 10 m1/min. B. Isolated Organ Studies Using isolated, perfused livers and lungs of control and 3MC pre- treated rats, the influence of changes in flow, metabolic capacity and blood binding components on the disposition of B(a)P was evaluated. 81 TABLE 4 Hepatic and Pulmonary Clearance of B(a)P at Normal Organ Flow as Predicted from Microsomal Metabolic Activity Clearance (ml/min) Control 3MC Lung Liver Lung Liver 1 Predicted 0.97:0.14 4.6:O.5 7.0:O.8 10.0:O.1 1Clearance predicted according to the perfusion-limited model (equations 2 and 3) using values of Vmax and Km from Table 2, the estimates of f3 from Table 3, and normal organ flows of 10 ml/min for liver and 45 m1/min for lung (Sapirstein gt_al., 1960; Roth and Rubin, 1976a). Values are mean clearance TT' values : "S.E.M." based on the sum of the variances of the individual variables relative to their means, i.e.: 2 2 2 2 S S . S S _-C1 = Cl int + 6 fB + organ wt xCl 7h]. '7? Xorgan wt int B n u _ 2 1/2 S.E.M. - (S Cl/N) where 52 represents variance, 7 the mean, and N the number of values used in the composite variance determination. 82 B1. Effect of Altered Flow on B(a)P Clearance Various parameters were measured to assess the success of the isolated perfused organ preparations. These are presented in Table 5 for livers and Table 6 for lungs. No differences were detected between the various groups for organ to body weight ratios. The values for this parameter in this study are similar to those reported in the 5-HT clear- ance study (Wiersma and Roth, 1980) and are not significantly different from organ to body weight ratios of organs not perfused (data not pre- sented). Initial inflow pressure (PIN) tended to increase with in- creasing flow and was the same for lungs from control and 3MC pretreated rats perfused at the same flow. Similar tendencies were seen in iso- lated livers, however, a significant increase was observed only for the high flow group of those livers from 3MC pretreated rats. Overall, the change in inflow perfusion pressure (AP) occurring during the experi- ments was similar at all flows for both isolated livers and lungs. However, a significant difference occurred between the pressure increase in lungs of 3MC pretreated rats perfused at 45 ml/min and that of con- trol lungs perfused at 10 or 20 ml/min. In Figure 13 is presented the time course of the disappearance of B(a)P from the perfusion medium reservoir for isolated livers of 3MC pretreated rats perfused at three different flows. As seen in this semilogarithmic plot, disappearance was log-linear at each of the three flows tested, indicating that B(a)P removal occurred by a first-order process. This same pattern was observed in the isolated lungs of 3MC pretreated rats (Figure 14) as well as organs from control rats. The pharmacokinetic parameters ke’ Co and Vd were determined from these Effect of Flow on Isolated Rat Liver Parameters1 83 TABLE 5 Liver Wt . Flow B1le Flow P AP Treatm°"t (ml/min) B°%;)Wt (pl/min) (chHZO) (cm H20) Corn 011 7 4.13:0.30 2.7:0.8 3.1:o.5§ 1.9:o.2 1o 4.26:0.29 3.9:1.o 3.7:o.4a 2.1:0.6 2o 4.08:0.19 4.3:1.3 4.6:0.8 3.2:0.8 3MC 7 4.43:0.18 l.9:0.6 4.1:o.4§ 2.8:0.9 10 4.32:0.15 2.8:0.6 3.5:O.6b 2.3:O.6 20 4.46:0.26 3.1:1.1 7.8:l.l 2.4:1.o 1 Isolated livers of control (corn oil) and were perfused at the indicated flows with at 37°C in a recirculating manner. of five livers. MC pretreated rats H-B(a)P for 45 min Results are mean : S.E.M. Means in a column with different superscripts are significantly different (P<0.05), as judged by analysis of variance as described in "Methods". PIN , initial inflow per- fusion pressure; AP, change in perfusion during the experi- ment (P FINAL - P IN)' l 84 TABLE 6 Effect of Flow on Isolated Rat Lung Parameters Lun Wt Flow nim- P AP Treatment (ml/m1") 0(g) (MIIHQ) (mHg) Corn 011 10 0.63:0.06 5.7:o.2§’g o.5:o.3§ 20 0.58:0.01 6.1:0.2b’c 0.6:0.1a b 45 0.63:0.04 7.3:o.4 ' 2.3:o.7 ' 3MC 10 0.59:0.02 5.1:o.sg 1.3:o.6§'g 20 0.61:0.03 6.9:0.2c l.7:0.8b’ 45 0.64:0.03 8.8:0.4 4.9:1.5 Isolated lungs from control (corn oil) and 3MC pretreated rats were perfused with benzo(a)pyrene at 37°C for 1 hr at the indicated flow with recirculating medium. Results are mean : S.E.M. of 4-6 lungs. Means in a column with different superscripts are significantly different (P<0.05). Data analyzed by analysis of variance as de- scribed in "Methods". PIN: initial inflow perfusion pressure; AP, change in pressure during the experiment (PFINAL - PIN)“ 85 Figure 13. Effect of altered flow on the disappearance of B(a)P from the reservoir of isolated perfused rat livers. Livers from 3MC pre- treated rats were isolated and perfused in a recirculating manner as described in "Methods". At 0 min 20 nmol 3H-B(a)P was added to the reservoir. Results are mean concentration : S.E.M. of 5 isolated livers at each flow. B(a)P Concentration (pmol / ml) 100 6 17"‘1TIITTr 0| 41 r [II 86 Flow (ml/ min) 10 20 30 40 50 Time (min) Figure 13 87 Figure 14. Effect of altered flow on the disappearance of B(a)P from the reservoir of isolated, perfused rat lungs. Lungs from 3MC pre- treated rats were isolated and perfused in a recirculating manner as described in "Methods". At 0 min 20 nmol 3H-B(a)P was added to the reservoir. Results are mean concentration : S.E.M. of 4-6 isolated lungs at each flow. B(a)P Concentration (pmol/ml) 88 50 10 T Il—rll Flow (ml/ min) 1'} 1o . I 20 A 45 01 V l :1 1 . 1 1 1 10 20 3O 40 50 60 Time (min) Figure 14 89 semilogarithmic plots and are summarized in Table 7 for the isolated livers and Table 8 for the isolated lungs. Significant differences occurred between the various groups for ke’ but not for C0 or Vd. 3MC pretreatment increased ke at each of the flows tested in isolated lungs. Such a treatment difference was observed in the isolated livers only at the highest flow tested. Using such pharmacokinetic data from each isolated organ, the clearance of B(a)P was calculated using equation 6. These clearances (corrected for clearance by the apparatus) are presented in Figures 15 and 16 as a function of the flow to the organ. Clearance of B(a)P by isolated lungs of control rats was very low (Figure 15), attaining a value of only l.0:0.l ml/min at a flow of 45 ml/min. Between 10 and 45 ml/min no significant increase in B(a)P clearance occurred. Thus, clearance of B(a)P by isolated lungs of control rats was independent of flow. In contrast, the greater the flow to isolated livers of control rats, the greater was the clearance of B(a)P, reaching a value of 10.9:1.l ml/min in liVers perfused at 20 m1/min. Indeed, it appears that B(a)P clearance increased linearly with flow. When the animals were pretreated with 3MC pulmonary B(a)P clearance was influenced by flow (Figure 16). Clearances at each of the flows tested were significantly different from one another. As in the livers of control rats, the clearance of B(a)P by isolated, perfused livers of 3MC pretreated rats increased linearly with flow. B2. Effect of Altered Metabolic Capacity on B(a)P Clearance Broken cell studies of AHH activity demonstrated that pretreat- ment of rats with 3MC increased the metabolic capacity (Cl'int) of both Effect of Flow on Pharmacokinetic Paramet Elimination by Isolated Livers 90 TABLE 7 ers of B(a)P Flow k C V Tre°tment (ml/min) (mifi‘l) (pmoT/ml) (mi) Corn oil 7 o.o39:o.oozog C 214: 5 93.8:2.6 10 0.063:o.003g . 207:13 97.8:6.0 20 0.084:0.006 156:19 135:14 3MC 7 o.osa:o.oo2g’2 243: 9 82.8:3.2 1o 0.072:0.006d’ 209: 8 96.1:3.5 20 0.lll:0.008 167:17 125:13 1 Isolated livers from control (corn oil) or 3MC pretreated rats were perfused for 45 min with B(a)P as described in "Methods". 20 nmol of 3H-B(a)P was added to the perfusion medium reservoir at 0 min. Abbreviations: constant (slope of disappearance curve); C centration determined by extrapolation of ke, first-order elimination rate 2 , initial B(a)P con- he disappearance to the ordinate; Vd, apparent volume of distribution (dose of B(a)P/C ). Results expressed as means : S.E.M. of five livers. Means w th different superscripts are significantly different. Data analyzed by analysis of variance (P<0.05). 91 TABLE 8 Effect of Flow on Pharmacokinetic Parameters of B(a)P Elimination by Isolated Lungs Flow k C V Treatme"t (ml/min) (mifi'1) (pmol/m1) (mi) Corn oil 10 0.011:o.002§ 199:33 113:15 20 0.014:0.002a 189:12 107: 7 45 0.017:0.001 155: 8 122: 5 3MC 10 0.051:o.002: 205:19 102:11 20 0068:0002C 195:11 103: 5 45 0.078:0.005 159:11 129: 9 1 Isolated lungs from control (corn oil) or 3MC pretreated rats were perfusgd for one hour with B(a)P as described in "Methods". 20 nmol of H-B(a)P was added to the perfusion medium reservoir at 0 min. Abbreviations: ke, first-order elimination rate constant (slope of disappearance curve); C , initial B(a)P con- centration determined by extrapolation to ghe ordinate; V , apparent volume of distribution (dose of B(a)P/C0). Resu ts expressed as means : S.E.M. of 4-6 lungs. Data analyzed by analysis of variance. Means with different superscripts are significantly different (P<0.05). 92 Figure 15. B(a)P clearance by isolated livers and lungs of control rats perfused at several flows. Isolated organs were perfused in a recirculating manner and the clearance of B(a)P determined as described in "Methods". Each organ was perfused at only one flow. Results de- pict the mean clearance : S.E.M. by 4-6 organs. Squares represent clearances by isolated livers, circles represent clearances by isolated lungs. Standard errors of the mean as indicated or less than symbol s1ze. Clearance of B(a)P (ml/min) 14 12 10 93 liver lung 10 20 30 40 50 Flow (ml/ min) Figure 15 94 Figure 16. B(a)P clearance by isolated livers and lungs of 3MC pre- treated rats perfused at several flows. Isolated organs were perfused in a recirculating manner and the clearance of B(a)P determined as de- scribed in I“Methods". Each organ was perfused at only one flow. Results depict the mean clearance : S.E.M. by 4-6 organs. Squares represent clearances by isolated livers, circles represent clearances by isolated lungs. Clearance of B(a)P (ml/min) 14 12 10 / 10 95 Lung 20 3O 40 Flow (ml/min) Figure 16 50 96 rat liver and lung (Table 2). To determine whether such increases in metabolic activity affect B(a)P clearance, B(a)P clearance values were compared at normal organ flows (Table 9). B(a)P clearance by lungs of control rats was low at normal pulmonary flow, but was increased by 3MC pretreatment. Hepatic clear- ance was high for livers from both control and 3MC pretreated rats perfused at normal flow. Thus, increased metabolic capacity enhanced the ability of lung, but not liver, to clear circulating B(a)P. The clearance values at normal organ flow determined in the isolated lungs were not significantly different from those predicted according to the perfusion-limited model (Table 4) as judged by Stu- dent's t-test. For livers, a small, but statistically significant difference between the observed and predicted clearances was obtained. However, even though the hepatic clearances were not the same as those predicted, in control rats hepatic B(a)P clearance was greater than pulmonary, whereas in 3MC pretreated rats lung and liver clearances were about equal. B3. Effect of Altered Flow or Metabolic Capacity on B(a)P Metabolite Production A large number of different metabolites of B(a)P were produced by the isolated, perfused lungs of both control and 3MC pretreated rats (see Figure 17). Many of these and B(a)P as well could be detected in both the lung tissue and the perfusion medium. In Figure 17 is pre- sented the elution pattern from the HPLC column of a mixture of authen- tic B(a)P and metabolites, as detected by their absorbance at 254 nm, and the elution pattern of an extracted perfusion medium sample 97 TABLE 9 B(a)P Clearance by Isolated Rat Livers and Lungs Perfused at Normal Organ Flow Control 3MC Lung Liver Lung Liver 1 01%;5738:) 1.0:0.1 5.6:0.2 8.8:0.5 7.4:0.6 1Clearance was calculated as the product Vd - ke. Normal organ flows are 10 m1/min for liver and 45 ml/min for lung. Results represent mean clearance : S.E.M. of 6 lungs or 5 livers. Vd, apparent volume of distribution; ke, first-order elimination constant. 98 Figure 17. Elution pattern of B(a)P and its metabolites separated by high pressure liquid chromatography using gradient elution. The upper panel represents the elution pattern of a mixture of B(a)P and B(a)P metabolite standards detected by UV absorbance at 254 nm. Full scale on the ordinate equals 0.02 absorbance units. The lower panel repre- sents the elution pattern of a methanol extract of a perfusion medium sample. Details of the separation are described in "Methods". Frac- tions of the eluent were collected every 30 sec and the amount of radio- activity determined by liquid scintillation spectrometry. Full scale on the ordinate equals 8,000 dpm/fraction. The peak labelled "polar meta- bolites" contains polyhydroxylated and conjugated B(a)P metabolites. The identity of metabolite U is unknown. Phenol peak 1 contains pri- marily 9-hydroxybenzo(a)pyrene and other B(a)P phenols which coelute and phenol peak 2 contains primarily 3-hydroxybenzo(a)pyrene among other phenols. Abbreviations for the other B(a)P metabolites are: 9,10-DHD, 9,10-dihydrodiol (9,10-dihydroxy-9,l0-dihydrobenzo(a)pyrene); 4,5-DHD, 4,5-dihydrodiol (4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene); 7,8-DHD, 7,8- dihydrodiol (7,8-dihydroxy-7,8-dih drobenzo(a)pyrene); 1,6-Q, benzo(a)- pyrene-1,6-quinone; 3,6-Q, benzo(a pyrene-3,6-quinone; 6,12-Q, benzo(a)- pyrene-6,12-quinone. 99 .9 g a g E On I 39$ g 992 2 5 3: 5‘ also :3 PHENOI. 1 PHENOI. 2 B(a)P 2’- 73-0140 A254 lll llllllllhmmmlullllll llullmlllllllllmulllnllmllull llllllllll J L I 1 a so 0 20 30 40 ;, L i: C Time (min) Figure 17 100 chromatographed under identical conditions. The elution of B(a)P and metabolites of experimental samples could not be detected by their absorbance at 254 nm and were therefore detected by determination of the radioactivity appearing in the column effluent. Most of the peaks of radioactivity corresponded in retention time to the standards. The two exceptions were the first peak ("polar metabolites"), which is most likely a mixture of conjugated and/or polyhydroxylated metabolites, and a peak which eluted after the dihydrodiols, the identity of which is unknown (metabolite U). In lungs from both corn oil and 3MC pretreated rats, increas- ing flow increased the rate at which B(a)P was removed from the recir- culating perfusion medium and increased the rate at which total B(a)P metabolites appeared in the medium (Figure 18). In addition, pretreat- ment of the animals with 3MC also increased the rates at which B(a)P disappeared and metabolites appeared in the perfusion medium. Thus, at the end of the perfusion period the medium of lungs from 3MC pretreated rats perfused at the higher flow had the lowest B(a)P concentration and the highest metabolite concentration. To determine whether these changes in total metabolite produc- tion reflected altered production of the same metabolites or the produc- tion of different metabolites, the pattern of appearance of the indivi- dual B(a)P metabolites in the perfusion medium was determined. These results are presented in Figure 19. Lungs from control rats produced individual metabolites in parallel with the rate of total metabolite production. For the most part, the concentration of each metabolite (or group of metabolites) increased with time at both flows. An increase in 102 22m l w_ mcamwa Eev 9:: 00 _o._Eou 09 DON ([ua / lewd) uouauueauog 101 .masocm Lace we» mo comm Low muz .Ponsxm any we mem we» cusp mmw_ age: to umumowccw mew mcwws asp mo mcocem ucmucmwm .mcowpmgucmocoo ouTPonmumE Fancy “commeamc mFOQEAm umppww .mcowpmgpcmocou ofimvm ucmmmLamL m_onezm :mqo .A:WE\FE mev 30F» saw; an ammsmgma mmczF pcmmmcawe mmgmzom e_wzz AcwE\FE e_v zoFe 30, um nmmsmsma mm=:_ so» coepmcucmucou cows asp ucmmmcam; mwpuemu .mswu oLmN pm A_E oo—v Lwo>gmmmg we» on venue mew: aflmvmuz eo Foe: om .Auzmv mcmccpcmfiocopxgpms .m to Apoepcoov _wo :Loo cue: umuomtpmsa mam; mama . ope sow; use 30p um ummseema mm:=_ pm: umumFOmw mo Lwo>cmmme we» cw mmue_oamume ofiwvm _auop ace oflmvm mo cowpmcucmocoo .wp acumen 103 Figure 19. The appearance of individual B(a)P metabolites in the reservoir of isolated lungs perfused at low and high flow. Rats were pretreated with corn oil (control) or 3-methylcholanthrene (3MC). Circles represent results from lungs perfused at low flow while squares represent results from lungs perfused at high flow. Standard errors of the means are as indicated or are less than the size of the symbol. Note that the concentration scales vary. Metabolite U was not de- tected in the perfusion medium of lungs from control rats. N=3 for each of the four groups. Abbreviations are the same as those in the legend of Figure 17. coucauunou (pt-din” coucmmou (pal—n CW (anon-ll 104 '0“! “TM"! com-at 50p 23- 60 3MC Minis) TIM! (min) TIMI (ml Figure 19 Win. l—dldl W (NIH, coucsunmou (pa-11.» 105 1,6-0 cannon. TIM! (ml Figure 19 (continued) couceuumu (pan-n CONCENTRATION (pull-a) coucammou (pa-did) 106 3,6-0 1 on F cameo: F 3741c S TIMI Inch) TIME (min) Figure 19 (continued) 107 flow produced an increase in the rate at which each compound appeared, but no novel metabolites were detected. For some metabolites such as 7,8-B(a)P dihydrodiol (7,8-DHD), increased flow resulted in a large in- crease in the amount of metabolite produced, while for others, such as B(a)P-1,6-quinone (1,6-Q), the increase was modest. At both flows the group of polar metabolites was the largest fraction of the total meta- bolites in the perfusion medium. A different picture was seen in the production of individual metabolites by perfused lungs from 3MC pretreated animals. In contrast to the control lungs, the appearance of individual B(a)P metabolites in the perfusion medium of lungs from 3MC pretreated rats did not parallel the pattern of total metabolite appearance. For each of the individual metabolites, except the group of polar metabolites and metabolite U, the pattern consisted of an initial rise in concentration to a peak followed by a decline. The time of occurrence of the peaks was generally similar (20-30 min) for most of these metabolites. However, for the B(a)P-1,6- quinone the peak occurred much earlier at the higher than the lower flow (approximately 10 and 40 min, respectively). For the group of polar metabolites and metabolite U there was an initial linear increase in concentration followed by a tendency to plateau during the latter half of the one hour perfusion of lungs from 3MC treated rats. As in the control lungs, the predominant metabolites were those which eluted with the group of polar metabolites. The effect of a change in flow to the lungs of 3MC pretreated rats varied with the particular metabolite. For the dihydrodiols, quinones, the first phenol peak and the polar metabolites, increasing 108 flow produced an increase in the rate at which these metabolites ap- peared and a higher peak concentration in the perfusion medium. In addition, for the dihydrodiols, quinones, and first phenol peak, in- creasing flow also resulted in a more rapid decline of the metabolite concentration during the latter part of the perfusion of lungs of 3MC pretreated rats. In fact, despite the differences in the concentration vs. time curves for all these metabolites, if the concentration had been determined only at the 60 min time point no difference in concentration would have been apparent between those lungs perfused at low flow and those perfused at high flow. Increasing flow did not alter the rate of appearance of either metabolite U or the second peak of phenol metabo- lites. Figure 19 also permits comparison of the peak concentration of the individual metabolites in the perfusion medium of control lungs with that of lungs from 3MC pretreated rats perfused at the same flow. Despite the observation (Figure 18) that 3MC pretreatment produced a higher concentration of total metabolites in the perfusion medium, the peak concentration of many individual metabolites from control lungs was as high or higher than that from lungs of 3MC pretreated rats. In fact, the highest concentration of the 9,10-dihydrodiol (9,10-dihydroxy-9,10- dihydro B(a)P) observed in the perfusion medium of control lungs was almost twice the peak concentration seen from lungs of 3MC pretreated rats perfused at the same flow. Both flow and 3MC pretreatment affected the concentration of B(a)P and metabolites in the lung tissue at the end of the perfusion. These concentration values are presented in Table 10. In lungs from The Concentration of B(a)P and Methanol-Extr Metabolites in Isolated, Perfused Lungs 109 TABLE 10 actable Concentration (pmol/g) Treatment Control Flow (ml/min) 10 45 10 45 Polar Metabolites 220:31 445:121 605:172 523:141 4,5-0H0 6:1 10:2 13:5 10:5 7,8-DHD 15:1 26:9 l.8:0.7 3:1 9,10—0HD 8.6:0.4 12:2 l.8:0.7 0.7:0.3 Metabolite U 7:6 4:2 4:1 2.9:0.8 1,6-Q 22:7 26:9 11:7 6:2 3,6-Q 45:18 53:24 23:12 16:6 Phenol 1 29:7 39:11 10:6 21:8 Phenol 2 28:16 109:29 28:2 21:9 B(a)P 2090:178 999:196 46:35 12:3 1Lungs were perfused for 1 hr with 100 ml of recirculating medium. B(a)P and metabo- lite concentrations were determined in methanol extracts of the Initial B(a)P concentration was 200 pmol/ml. lungs as described in "Methods and Materials". mean : S.E.M. of groups of 3 lungs. 2 20 mg/kg, i.p., 24 and 48 hrs prior to sacrifice. Results depict 110 control rats an increase in flow resulted in an increase in the concen- tration of several B(a)P metabolites and a decrease in the concentration of B(a)P within the tissue. In contrast, the lungs from 3MC pretreated rats perfused at high flow generally had similar metabolite concentra- tions to those perfused at low flow. However, in 3MC pretreated rats, B(a)P concentration was lower in lungs perfused at the greater flow. When perfused at the same flow, lungs from 3MC pretreated rats had either equal or lower B(a)P metabolite concentrations as compared to control rat lungs after 60 min of perfusion. The notable exception to this was the group of polar metabolites, for which tissue concentrations were higher in lungs from 3MC pretreated rats. 3MC pretreatment also resulted in a greatly reduced concentration of B(a)P in the lung tissue. The ratio of concentrations of B(a)P and its metabolites in the lung tissue and perfusion medium after 60 min of perfusion were calculated, and these lung/medium ratios (L/M) are presented in Table 11. In general, the L/M value in control lungs was greater for the less polar metabolites, such as the phenols, than for the more polar dihydro- diols. However, the group of polar metabolites had a greater L/M than the dihydrodiols in control lungs. These trends were not apparent in lungs from 3MC pretreated rats. Flow did not affect the L/M of B(a)P or its methanol extractable metabolites. Pretreatment with 3MC did lower the L/M of both B(a)P and some of its metabolites (the quinones, phenols, and "polar metabolites") whereas the L/M of other metabolites (the dihydrodiols) tended to be increased by 3MC pretreatment. 111 TABLE 11 The Ratio of B(a)P and Metabolite Concentratiops in Lung Tissue to that in the Perfusion Medium Lung/Medium Concentration Ratio (pmol/g/pmol/ml) Treatment Control 3MC2 Flow (ml/min) 10 45 10 45 Polar Metabolites 33:2 28:5 12:3 9:2 4,5-DHD 10:3 7:2 21:13 70:54 7,8-DHD 19:2 15:4 82:44 18:8 9,10-DHD 10:2 8:2 21:15 3:1 Metabolite 0 N03 N03 7:1 4:1 1,6-Q 49:12 30:7 8:4 13:5 3,6-Q 28:5 15:5 8:4 7:3 Phenol 1 55:11 40:15 14:7 36:17 Phenol 2 85:52 133:38 46:3 41:13 B(a)P 18:2 17:3 7:6 4:2 1Lungs were perfused for 1 hr with 100 ml of recirculating medium. Initial B(a)P concentration was 200 pmol/ml. B(a)P and metabo- lite concentrations were determined in methanol extracts of the lungs as described in "Methods and Materials". Results depict mean : S.E.M. of groups of 3 lungs. 220 mg/kg, i.p., 24 and 48 hrs prior to sacrifice. 3N0 = level in perfusion medium was below detection limits, therefore calculation of L/M was not possible. 112 B4. Effect of Altered Perfusion Medium Composition on B(a)P Clearance and Metabolism The influence of altered perfusion medium composition on B(a)P disposition was examined in isolated, perfused livers of 3MC treated rats. All perfusion media contained red blood cells at a packed cell volume approximately one-half that of normal rat blood. Variations in medium composition were in the additions to the buffer component. One group of livers was perfused with medium containing red blood cells and buffer alone. A second group of livers was perfused with medium which included serum lipoproteins isolated from 100 m1 of blood. This re— sulted in perfusion medium containing 43.3 mg of lipoprotein protein and 126 mg of lipoprotein lipid per 100 ml of medium. The third group was perfused with medium containing added serum albumin at a final concentration of 3.2 g protein per 100 ml. As in the flow dependence studies, several parameters were measured to assess the liver preparations. As shown in Table 12, the post-perfusion weight of the isolated livers was not influenced by the composition of the medium. The liver to body weight ratios of these perfused livers were not significantly different than those of livers from 3MC pretreated rats that were not perfused (data not presented). Livers perfused with medium containing albumin had significantly lower inflow pressure than livers of the other groups. The increase in per- fusion pressure that occurred during the experiment was not affected by the medium composition. No significant difference in bile flow was observed between the livers perfused with media containing serum albumin or the lipoprotein fraction. In the group of livers perfused with 113 TABLE 12 Effect of Perfusion Medium Composition on Isolated Rat Liver Parameters - - RBC's RBC's Perfu510n MedTum RBC's ’. ’. . . ’ Buffer w1th Buffer w1th Compos1t10n Buffer Albumin Lipoproteins Liver Wei ht W76) 5.31‘0.1 4.9i0.1 5.1i0.2 PIN (mmHg) 4.4:0.3 2.5:0.7* 3.9:0.3 AP (mmHg) 3.6:1.0 4.0:l.2 4.2:0.8 Bile flow (pl/min) 4.8:0.6* 7.2:0.3 6.9:0.5 1Isolated livers from 3MC pretreated rats were erfused at 10 ml/min in a recirculating manner at 37°C with H-B(a)P for 45 min as described in Methods. Each perfusion medium contained washed human red blood cells (RBC's) at an hematocrit of 20%. Results are mean : S.E.M. of 4 or 5 livers. Means with an asterisk are significantly different (P<0.05) than the other means in the same row. PI , initial inflow perfusion pres- sure; AP, change in perfusion pressure during the experiment (PFINAL - PIN); RBC's, red blood cells. 114 medium containing only red blood cells and buffer, bile flow was 30% less than that of the other groups. Figure 20 depicts the disappearance of B(a)P from the perfu- sion medium reservoir with time. B(a)P removal appeared to be first- order irrespective of the type of perfusion medium used. Pharmaco- kinetic parameters derived from such plots are presented in Table l3. No differences were observed in the apparent volume of distribution of B(a)P. Differences among the three groups occurred, however, in the first-order rate constant of elimination and in B(a)P clearance. Clearance by isolated livers perfused with medium containing only red blood cells and buffer was low (l.8:0.2 ml/min). The highest B(a)P clearance was by the group of livers with albumin in the medium (8.5:0.9 ml/min), while livers perfused with medium containing added serum lipo- proteins had an intermediate clearance (5.l:0.5 ml/min). Appearance of B(a)P metabolites in the perfusion medium paralleled the differences in clearance. As demonstrated in Figure 2l, livers perfused with red blood cells and buffer released the least amount of metabolites into the medium, while those perfused with albumin- containing medium had the greatest metabolite production. Livers per- fused with medium containing serum lipoproteins were intermediate in metabolite appearance. Biliary excretion of B(a)P was low by all three groups of livers (Table l4), amounting to only about 0.006% of the amount of B(a)P administered. In contrast, significant amounts of B(a)P metabolites appeared in the bile. In livers perfused with albumin about 2% of the 115 Figure 20. Disappearance of B(a)P from the reservoir of isolated livers perfused with media of varied composition. Isolated livers of 3MC pretreated rats were perfused at 37°C in a recigculating manner at a constant flow of l0 ml/min. At 0 min 20 nmol of H-B(a)P were added to the reservoir. Samples were taken at various times thereafter and analyzed for B(a)P content. Results are the mean concentration i S.E.M. of 4 or 5 liver preparations. Circles represent data from livers perfused with red blood cells (RBC's) and buffer; squares, data from livers perfused with RBC's and buffer containing serum lipopro- teins; triangles, data from livers perfused with RBC‘s and buffer con- taining serum albumin. 116 IA - ub--.- p ------b nu nu nu :a nu :4 al I 300 *- A_E\_OE& cat—ozcoucou *on 50 20 10 Time (min) Figure 20 117 TABLE 13 Effect of Perfusion Medium Composition on 1 Pharmacokinetic Parameters of Isolated Rat Livers . . . RBC's, RBC's. Perifiriéfi'éiiiir‘w“ 33%?- Buzfsnmizt“ 513mm ke (min'1) 0.027:0.007a 0.087:0.002b 0.055:o.004c vd (ml) 83.6:22.l 97.8:10.8 94.1:8.8 c1 (ml/min) l.82:0.ZOa 8.45:0.87b 5.07:0.48c 1Isolated livers from 3MC pretreated rats were perfused with 3H—B(a)P at TD ml/min in a recirculating manner with perfusion medium of the indicated composition. Pharmacokinetic para- meters were determined from individual B(a)P disappearance curves as described in "Methods". Results are mean 1 S.E.M. of 4 or 5 livers. Any two means in the same row with different superscripts are significantly different (P<0.05) by one-way analysis of variance. ke, first-order elimination rate con- stant (slope of disappearance curve); V , apparent volume of distribution (dose of B(a)P/initial B(a P concentration deter- mined by extrapolation of the disappearance curve to the ordi- nate); Cl, clearance (ke x Vd); RBC's, red blood cells. 118 Figure 2l. Appearance of B(a)P metabolites in the reservoir of isolated livers perfused with media of varied composition. Isolated livers of 3MC pretreated rats were perfused at 37°C in a recgrculating manner at a con- stant flow of 10 ml/min. At 0 min 20 nmol of H-B(a)P were added to the reservoir. Samples were taken at various times thereafter and analyzed for total B(a)P metabolite content. Results are the mean concentration i S.E.M. of 4 or 5 liver preparations. Circles represent data from livers perfused with red blood cells (RBC's) and buffer; squares, data from livers perfused with RBC's and buffer containing serum lipoproteins; triangles, data from livers perfused with RBC‘s and buffer containing serum albumin. 119 // O O O 0 4. .J 9‘ .I A_E\ .083 co_.o..:oucou 2:23.22 mfim 20 3O 4O 50 10 Time (min) Figure 21 120 TABLE 14 The Effect of Perfusion Medium Composition on the Amount of B(a)P and B(a)P Metabolite Appearing in the Bile of Isolated Livers . . RBC's RBC's Perquion Medium RBC's ’. ’ . . ’ Buffer with Buffer with Comp051tion Buffer Albumin Lipoproteins B(a)P (pmol) 0.8:0.2 l.3:0.l l.3:0.4 B(a)P Metabolites 92:29a 451:27b 303:40c (pmol) l Isolated livers from 3MC pretreated rats were perfused at l0 ml/min with the indicated medium containing 3H-B(a)P in a re- circulating manner for 45 min. Bile produced was analyzed for B(a)P and metabolite content as described in "Methods”. Results are mean amount i S.E.M. for 4 or 5 livers. Means with different superscripts are significantly different (P<0.05) than the other means in the same row. lZl B(a)P dose was excreted into the bile as metabolite during the 45 min perfusion period. This was significantly greater than the amount of metabolite excreted by the serum lipoprotein group. Perfusion of livers with only red blood cells and buffer resulted in the lowest biliary excretion of B(a)P metabolites. BS. Other Causes for Altered B(a)P Clearance In some cases B(a)P clearance by isolated organs was greater or less than suggested by the microsomal kinetic data. Several possible causes for such differences were investigated in isolated organs. a. Reversible clearance of B(a)P by isolated lungs Initial estimates of B(a)P clearance by isolated lungs based on the enzyme kinetic parameters of Vadi gt_al, (l976) suggested that B(a)P clearance would be very low in lungs from control rats. The values observed in the isolated lungs were much higher. Despite the closer agreement of the predicted clearance based on the AHH kinetic parameters reported earlier (Table 2), the reversible nature of B(a)P 14C- clearance was investigated in isolated lungs perfused with both sucrose and 3HB(a)P in a single pass mode. Following attainment of steady-state effluent concentrations of B(a)P and sucrose in isolated lungs from control and 3MC pretreated rats (Figure 22), the concentrations fell as the lungs were perfused with medium lacking sucrose and B(a)P. Since the distribution of sucrose is limited to the extracellular space, it served as a marker for this compartment. The B(a)P and sucrose in this compartment should have identical washout curves as the medium lacking B(a)P and sucrose per- fuses the organ. Indeed, in a lung from a 3MC pretreated rat the wash- out curves for sucrose and B(a)P were almost superimposable (Figure 22, 122 .Puz .m::F u2m mnp cw &wp ucm mczp Fogpcou as» cw cowumcucmocoo zopmcw ms» to &om mm; afimvm mo cowpmcpcwucou mumpm Anowpm och .mcowumgpcmucou afimvm pcmmmcamc mmpucwo :mao .mcowpmcgcmucoo mmocuam “cmmmcgwg mwpocwu vmmopo .covumggcmucou mumpmuxummum to “smegma mm nmucmmmca mew mapsmmm .omcvscmumc mmoguam cam .mmHVFonmpme mflmvm .mflmvm mo mcowp -mcucmocoo mzp use cm—qsmm armsoacwpcou mm: Ezwume ucmspmmm ms» .mmogosm vcm aflmvm mcwaQF Ezvcme mamm any cuwz gamma mm: cowmamgmg .umcrmupm mew; mcowumcucmucou “cmafimwm mumpm-znmopm cog: .A~E\mn eo.ov mmocuamuue use As: oomv mAmeuzm mcwcwmpcoo ucm mppmo coopn em; mcwxumy Ezwvme Lm_:mmg sup: muoe mmmauwpmcwm m cw ummawgma mew: mum; umammgumca 02m Lo Apocpcoov _vo :gou sexy mm:=_ umpmpomH .mmcz— ummeomw seem mmogusm wcm anmvm we xzpwmw use .Nm mcamwm 123 3-MC Control “sisal aims Apoeis ’0 %) uoyouuaauog 2O 15 “3 Figure 22 Time( min) 124 right panel), suggesting that B(a)P which entered the organ was irre- versibly removed. In contrast, the efflux of B(a)P from a control rat lung was slower than that of sucrose, suggesting that B(a)P removal into this lung was, at least in part, reversible. b. Concentration dependent B(a)P clearance Since isolated organs from 3MC pretreated rats were perfused with B(a)P at an initial concentration near or greater than the apparent Km value of AHH in these organs, clearance may be different at lower concentrations. Therefore, B(a)P clearance was measured in iso- lated organs of 3MC pretreated rats perfused at initial B(a)P concen- trations of 200 or 5.6 nM. This lower concentration is l0 times less than the Km value for liver microsomes of 3MC pretreated rats (Table 2), the lowest Km measured. As seen in Table 15, no difference was observed between the B(a)P clearance values obtained at the two initial B(a)P concentrations in either organ. c. Non-uniform distribution of B(a)P B(a)P clearance, as observed in isolated perfused livers of 3MC pretreated rats, was less than flow to the organ (Table 9). However, the perfusion-limited model, using enzyme kinetic parameters, predicted that B(a)P clearance by this organ should be equal to hepatic flow (Table 4). One possibility for this difference was that the liver was perfused in a non-uniform manner. This was assessed by determining the concentration of 3H in various sites throughout the liver following l5 min of perfusion with 3H-B(a)P. No significant difference was ob- served in the concentration of the label between any of 23 sites in the perfused liver (Figure 23). 125 Figure 23. Distribution of 3H within isolated, perfused livers. Iso- lated livers from 3MC pretreated rats were perfused at l0 ml/min with H-B(a)P (20 nmol, 40 uCi) for 15 min. Small tissue samples (approxi- mately 50 mg) were removed from the sites indicated, digested, and the radioactivity content determined. Results are the mean nCi/g i S.E.M. of three perfused livers. (This view of the liver is of the inferior aspect; the hepatic portal vein appears at the top of the drawing; the left liver lobe is on the left.) 126 Figure 23 127 TABLE 15 Concentration Dependence of B(a)P Clearance inIIsolated Livers and Lungs of 3MC Pretreated Rats Initial 2 3 B(a)P Conc. N ke_1 Vd C}:?;;?:§ (nM) (min ) (M1) Lung 200 6 0.051i0.002 102:11 4.6i0.4 5.6 3 0.053i0.004 110:22 5.4i1.1 Liver 200 5 0.072i0.006 96.1:3.5 6 4:0.6 5.6 3 0.083i0.007 81.4i8.5 6 3:1.1 1Lungs and livers were perfused at constant flow (10 ml/ min) with regular perfusion medigm at 37°C in a recircu- lating manner. At 0 min enough H-B(a)P to produce the indicated concentration was added to the reservoir. Samples of perfusion medium were taken periodically and analyzed for B(a)P content. Pharmacokinetic analysis of the data was performed as described in "Methods". Re- sults represent means i S.E.M. of N perfused organs. 2First-order rate constant of elimination (slope of dis- appearance curve). 3Apparent volume of distribution (dose of B(a)P/initial B(a)P concentration determined by extrapolation of the disppearance curve to the ordinate). 128 d. Intra-hepatic shunts Another possibility for hepatic clearance less than predicted was that a shunt was diverting a portion of the flow from the portal vein into the venous outflow of the liver and bypassing the sinusoids. As indicated in Table l6 only about 7 of 20,000 microspheres (0.035%) injected into the inflow to the liver appeared in the outflow. Therefore, shunting of flow around the liver sinusoids is unlikely. C. Association of B(a)P and B(a)P Metabolites with Blood Fractions B(a)P in rat blood is not uniformly distributed among the various blood components. In arterial blood drawn 20 min after i.v. admini- stration of 3H-B(a)P (ll7 nmol/kg) to rats, the concentration of B(a)P was ll.2:2.2 pmol/ml blood (Table l7). Thus, by this time less than l% of the administered B(a)P remains in the blood. Of this B(a)P, 60% was associated with the serum (the serum recovered accounted for 46% of the blood volume) indicating an approximately equal distribution of B(a)P between red blood cells and serum. Although the lipoprotein fraction represented only 20% of the serum volume, 75% of the B(a)P in the serum was associated with the fraction containing them. Thus, 45% of the B(a)P in rat blood was associated with the fraction containing serum lipoproteins. After 20 min of circulation, the blood concentration of B(a)P metabolites (25.6:2.6 pmol/ml) was more than twice that of B(a)P. Seventy percent of these metabolites were recovered along with the serum, indicating that the B(a)P metabolites were preferentially distri- buted into serum. In contrast to B(a)P, however, only 8% of the meta- bolites in the blood were associated with the serum fraction containing 129 TABLE 16 Fraction of Microspheres Appearing in the Effluent of Isolated, Perfused Livers of 3MC Pretreated Rats Source Fraction2 (%) Cannulas and tubing 0.64:0.08 Liver 99.32:0.08 Effluent perfusion medium 0.035:0.02l Total l00.00:0.0l 120,000 Sr-85 labelled microspheres (l5 micron diameter) were injected into the inflow can- nula of isolated livers perfused at l0 ml/min in a single pass mode. Radioactivity was de- termined by gamma scintillation counting for all components of the system exposed to the microspheres. N=4. 2Fraction of total cpm. 130 TABLE 17 Association of B(a)P and Metabolites with Blood Fractions1 Portion Associated (% of Total) Blood Component B(a)P2 B(a)P Metabolite2 Blood l00 100 Serum 59.l:l0.6 69.3:5.7 Serum Lipoprotein 44.8: 8.9 7.79:0.86 Fraction 1ll7 nmol, 2 mCi/kg 3H-B(a)P dissolved in rat serum was injected into the tail vein of conscious rats. Under ether anesthesia, blood was drawn from the abdominal aorta l9.5 min after administration. Blood was allowed to clot, serum prepared and the serum lipoprotein frac- tion isolated as described in "Methods". B(a)P was separated from metabolites by hexane extraction. Re- sults are expressed as the percent of the total amount in the blood 1 S.E.M. N=5. 2Concentrations of B(a)P and B(a)P metabolite in the blood were ll.2:2.2 and 25.6:2.6 pmol/ml, respectively. l3l lipoprotein. Thus, B(a)P metabolites do not concentrate within the serum lipoprotein fraction. 0. B(a)P Elimination from the Blood of Conscious Rats Dl. Assessment of Animals Body weight remained constant or was slightly reduced by the cannula implantation and pretreatment regimens (Table l8). At the time of B(a)P administration, however, body weights among the four groups of animals were not significantly different. During the B(a)P pharmaco- kinetic determinations the animals moved freely about their cages and consumed both food and water. Hematocrit decreased during the course of blood sampling from 45:l% at the beginning of the experiment to 4l:l% at 60 min and was further reduced to 34:l% 300 min after B(a)P administra- tion. This pattern was observed in all rats and was affected neither by 3MC pretreatment nor by route of B(a)P administration. 02. Disappearance of B(a)P from Blood Figure 24 depicts the disappearance of B(a)P from the blood of rats. The same general pattern was observed for all animals irrespec- tive of pretreatment or the route of B(a)P administration. From 2 to 60 min after B(a)P administration there was a sharp decline in blood B(a)P concentration which slowed to a more gradual decline between l and 5 hours. These blood B(a)P concentration data were subjected to pharmaco- kinetic analysis as described in "Methods". 03. Effects of 3MC Pretreatment As demonstrated in Figure 24, 3MC pretreatment reduced the B(a)P concentration at which the terminal phase of the concentration 132 TABLE 18 Effect of Cannula Implantation and Pretreatment on Rat Body Weights Pretreatment Corn 0il 3MC Route of B(a)P . . . Administration i.a. i.a. i.v. h.p.v. Initial weight (g) 226:4 253:18 260:l3 257:8 Final weight (g) 232:4 225:ll 257:ll 243:7 N 6 7 8 4 l Cannulas were implanted in anesthetized rats as described in "Methods". Following a 48 hr recovery period, pretreatment with corn oil or 3MC commenced and continued for 2 days at which time B(a)P pharmacokinetics were determined. "Initial weight" refers to body weight at cannula implantation, while "final weight" refers to weight at the time of B(a)P pharma- cokinetic determination. Values are mean weights : S.E.M. Abbreviations: i.a., intra-arterial; i.v., intravenous; h.p.v., intra-hepatic portal vein. 133 Figure 24. Elimination of B(a)P from the blood of conscious rats. Corn oil (control) and 3MC pretreated rats were given 3H-B(a)P (117 nmol, 2 mCi/kg) in rat plasma by various routes. Periodically, blood samples were removed and B(a)P concentration determined as described in "Methods". Data points represent mean B(a)P concentrations : S.E.M. of the number of animals indicated in Table 18. If no S.E.M. is apparent, it was less than the symbol size. Open symbols represent corn oil pretreated rats, while closed symbols represent 3MC pretreated rats: circles, i.a. administration; squares, i.v.; triangles, h.p.v. B(a)P Concentration (%dose/ ml blood) 1.000 0.500 0.050 0.0l 0 0.005 0.001 0.1 DOLE 134 V II'UII [Tr jj h 1- D Time (hrs) Figure 24 135 decline began. This was reflected as a significant increase in the pharmacokinetic parameters Vd and ClTB (Table 19). Vd increased 4 times, while total body clearance of B(a)P increased from 15 ml/min to 48 ml/min. AUC” was decreased to 37% of that in control rats while the first-order rate constants were not significantly affected. Significant decreases in tissue B(a)P concentration 5 hr after B(a)P administration (Figure 25) were observed in liver and fat, while B(a)P concentration in lung doubled. B(a)P concentrations in kidney, spleen, and muscle were not significantly affected by 3MC pretreatment. 3MC pretreatment did alter the appearance of B(a)P metabolites in the blood (Figure 26). The peak metabolite concentration was not in- creased by 3MC pretreatment. However, the time to peak concentration was reduced. Despite this change, no differences were observed in the AUCco of these metabolites (Table 20). No significant alterations in B(a)P metabolite tissue concentration resulted from 3MC pretreatment (Figure 27). 04. Effects of Varied Route of B(a)P Administration The effect of varied route of B(a)P administration was ex- amined in 3MC pretreated rats to evaluate the role of liver and lung in the total body disposition of B(a)P. As shown in Figure 24, varying the route of B(a)P administration altered the blood B(a)P concentration at which the terminal phase of concentration decline began. Pharmaco- kinetic analysis of this data (Table 19) demonstrated that there was no significant difference in the first-order rate constants for either the initial or terminal phase of B(a)P concentration decline. However, 136 .z_m>wuommmmm .pF ucm .o— .m mcompmamm op mcwugouom umpmpzofimu aos< mam .mppu .m>m .mzogm :umm cw mFmepcm mo Lmnsac .2 "me?“ mpwcwmcw op ogmN eogm m>g=u mew“ .m> comumcucmucom mfimvm mec: mmgm .su=< ”mocmgmmpo Anon _mpou .mHFQ "compsnvspmwm mo mE=Fo> ucmgmmmm .u> mmmmgm mocmgmmmammvo Poe?» mo maopm .m mmmmzm mocmgmmmammvu _mvacw mo mmopm .a "mFonexm ucm mco_pmw>mgnn< .mo.ovm .Apmmu amp »5 comwgmmeou :mms ”cmwmmn eoucmg a—mum_meou .mm:m_gm> mo mvmapmcm xmznmcov ucmcmmmmu zpucmowmmcmwm mam mgmuumF umvgumgmmzm ucmgmmmwu saw: 30: m cw mcmms oz» acm .mmsogm uzm mg» 2H .Acmwmmm mmgvmmcz .pmmuup m.ucmu:umv covumgumwcwsmm mo muse; mamm an umummgp -mcm ozm :mgu pcmcmmwpu appcmowmwcmwm mm:_m> _ocucou mpocmm mxmrgmum< .mcowpmcwsgmmmu 2 mo .z.m.m A :mme mgm mp_:mmm .quoe ammo pcmsugmmsoo 03p m mcvmz mmcvecmumu mcmz mmmpmsmgmm uwpmzwxoumsmmcm .mmcwecmpmn cowumcpcmucoo mflmvm ucm eve com mcm N cmmzpmn czmgmcuwz mgmz mm—QEmm voan uwmowgmm .mpao: umpmurmcw mg» xn mammpm cw um>Pommvu Am¥\woe N .Foe: uppv mAmvmuz cm>wm mgmz mum: maovumcou m P e m N m z mmm.onmm._ mom.oamN.F m_m.on_m.m .m_.0hmu.m AcwmwwMowexv Nam=< . - . . - . . - . . - . me am om+m mm n.mo m+m mm m_ m+m me *_ _+m m_ ACLE\PEV N _m . - . . - . . - . . - . n ma e+~ mm a.mm ¢+m N: am N+_ o_ *N_ o+Nm N “by N > eooo.onmmoo.o mooo.onm¢oo.o mooo.onmmoo.o Nooo.onmmoo.o A_-c:ev a eoo.onpmo.o Noo.onemo.o Npo.ow¢mo.o moo.onkmo.o AF-=WEV a .>.a.; .>.w .m.P .m.P cowpmgumwcw5u< . . aflmvm mo mpaom 02m Pwo cgoo pcmEummgpmgm Pmumm msowumcoo c? cowuwmommwo mamvm mo mcmmemgmm muwamcwxoomEngm mp m4m _mpgom ovumamgumgpcw ..>.m.; mmzocm>mcucw ..>.w .mecmucmumgpcw ..m.w ”meowpmw>mgnn< .mgmgpo mg» mo pug» some mgmmmwm mcsr com mpmmm _mcwcgo mzu pmcp mpoz .Amo.ovmv mcowpmgpcmocoo :mme ucmgmmmwu xpucmmmmvcmwm muocmm mxmwgmpm< .z.m.m n mcowpmcpcmocom cmme mcm mp_:mma .mvaememm mcowpmspcmocom mfimvm mcm .mm>oem: mmzmmwp .mmmwmwcumm mam: mmmc mg» cowpmgpmwcwsmmupmom ewe oom p< .cowpmcpm -wcwemm mo mmuzog mzowgm> xn mammpm um: cw mm>_ommmm Amx\wue N .FoE: NFFV mfimvmuz =m>wm mcmz mum: mmpmmcpmgm uzm mam A_ogpcoov _vo ccoo .mcmmgo pm: cw :owpmgacmucou mhmvm m .mN mesmwm 138 >2:5. L «0.0 .. v0.0 . 00.0 .4 00.0 . 0—.0 . 2.0 .mN 85m: 829% D 00.0 . 0—.0 N—.0 - - / l I: 3.: g M L 3.6 / I . H e A m .26 . a... 3.: E x, in. l, I. W m m 11 w L .38 FL [IL 43... ... ._. a .11..-. _ . 88 4 .86 rILII. E ... .26 * .3: 5.6 0 °. C 2 d (B/uop g) uogtouuo: N 0 111D .[1 0.5.. m 0. °. N °. 95 (Blosopg) uogmuuaauog d(o)g 139 Figure 26. Concentration of B(a)P metabolites in the blood f conscious rats. Corn oil (control) and 3MC pretreated rats were given H-B(a)P (117 nmol, 3 mCi/kg) in rat plasma by the indicated route. Periodically, blood samples were withdrawn and the concentration of total B(a)P meta- bolites determined as described in "Methods". Data points represent mean concentration : S.E.M. Lack of S.E.M. indicate S.E.M. less than symbol size. N as in Table 18. Abbreviations: i.a., intra-arterial; i.v., intravenous; h.p.v., intra-hepatic portal vein. (%otdose/m| blood) B(a)P Metabolite Concentration 05 0.3 0.2 0.l 140 Control , i.a. 0.1 * 0.l ’ i 5 4' 5 Time (hrs) Figure 26 db N h) ‘1- Vi J l A 2 3 4 5 Time (hrs) 141 TABLE 20 Area Under the Arterial Blood B(a)P Metabolite Concentration vs. Time Curves (AUCm) Route of AUCco Pretreatment Administration N (% dose-min/ml blood) Control i.a. 6 225:21 3MC i.a. 7 364:63 3MC i.v. 8 240:25 3MC h.p.v. 4 344:77 Control (corn oil) and 3MC pretreated rats were given 3H—B(a)P (117 nmol, 2 mCi/kg) in plasma by the indicated routes. B(a)P metabolite concentration of blood samples drawn between 2 and 300 min after B(a)P administration was determined and the AUC0° from zero to infinite time determined by pharmacokinetic ana- lysis. Results are mean : S.E.M. of N rats. 142 .:Pm> _mpcom Urummm; never ..>. m. ; .mao:m>m:p:F ..>. .Pmrgmpgm mcpcr ..m.w ”mcompmw> -mcmn< .mcmcpo mgu mo pogo Eogw mammmwu xmcuvx com umom _mCCuLo msp pmcu mpoz .Amo.ovmv mcowpmcucmucou :mms pcmgmmmwm z—ucmuwmwcmwm muocmm mxmwgmum< .mF anmH cw mm 2 .z.m.m I mcovumgpcmucou cmme mam mp_=mmm .umcwEmemo mcowpmcucmucou mpwronmums mfimvm ncm .um>oEmL mmammwp .mmuvmwgumm mam; mum: msp cowpmgumwcwEmmu “mom :Fe oom u< .cowumcpmv:05mm Lo mmpzog msowcm> ma mamm_m um: cw om>Pommpu Amx\pue N Foe: n—Pv a? cm>wm mcmz mum: mmummcu -mcm 02m ucm Apocucomv Fwo cgou .mcmmgo pm: :F coppmgpcmocoo mmFFMQmumE mAmvm .NN mczmwm 143 @%W Due @%W D“; o’.‘.‘ o u: n E .2. . us» .74 4 to . to m U .m w m D . mm M .3 .2...” :3 :3 m. w. a . .m m. 6.. .1526 :3 . 3 m . a u . S .o. m. >0! 0.392 %N.— 60013 WN— n J g g on W _UL w. I: w A 3 .: Nd / 48.0 . 0.0 ._ ‘0 . v.0 . . L r I... .4. r... . . . a II 405 . D5 #3 .3 L9 .3 Good >23! A ’0‘ a N.— h.’—d g“.— (Blosopx) uauouuoauog otgloqbtiw JON 144 differences were observed in Vd and ClTB. Both parameters were doubled when B(a)P administration was varied between the intra-arterial and intra-hepatic portal vein routes. Values following intravenous admini— stration were intermediate to those of the other two routes. Although a trend toward a decreasing AUCco for B(a)P following i.a., i.v., and h.p.v. routes of administration was apparent, no significant difference in this parameter was detected. In 3MC pretreated rats, the fraction of the dose escaping first-pass removal by lung and liver was calculated from AUC0° values (Table 21). 79% of the B(a)P dose escaped extraction on the first pass through the liver. Lung allowed 70% of the dose to pass. Thus, the first-pass extraction of B(a)P by these two organs jg_vj!9_was about equal at 21% for liver and 30% for lung. While no significant changes occurred in blood B(a)P metabo- lite concentration by varying the route of B(a)P administration (Figure 26; Table 20), altered tissue B(a)P and metabolite concentrations were observed (Figures 25 and 27). Following i.a. and i.v. B(a)P admini- stration to 3MC pretreated rats, the highest tissue B(a)P concentration 5 hr after B(a)P administration occurred in the lung (Figure 25). However, following h.p.v. administration the highest B(a)P concentration was detected in fat. For those tissues in which significant changes in B(a)P concentration were observed due to varying the route of admini- stration (lung, liver, kidney, and muscle), the tissue concentration following h.p.v. administration was less than that following i.v. administration which was, in turn, less than that following i.a. administration. Such differences were not seen in each organ. In 145 TABLE 21 The Fraction of the Dose Available (f) and the Fraction Extracted (E) by Liver and Lung of 3MC Pretreated Rats IQ_Vivo 1 E2 Organ f Liver 0.79 (0.49-1.2) 0.21 (-0.20-0.51) Lung 0.70 (0.41-1.6) 0.30 (-0.6-0.30) 1Calculated according to equations 12 and 13 using data from Table 19. Values in paren- theses are the 95% confidence limits for the ratio (Goldstein, 1964). Values of f can actually be no greater than 1.0. 2Extraction = l - f. Values in parentheses are the 95% confidence limits for the ratio. Values of E can be no less than 0. 146 contrast, an increase in lung B(a)P concentration was observed following i.v. administration. No significant changes in B(a)P concentration due to route of B(a)P administration were detectable in fat or spleen. Few differences occurred in tissue B(a)P metabolite levels due to varied routes of B(a)P administration (Figure 27). Significant reductions in metabolite concentration were detected in lung between the i.v. and h.p.v. groups, while in liver metabolite concentration was lower following i.v. administration than when it was given i.a. Highest B(a)P metabolite concentrations occurred in the kidney irrespective of the route of B(a)P administration. E. Comparison of Organ Extractions Based on Results from Broken Cell, Isolated Organ, and Conscious Rat Studies In order to compare results from broken cell, isolated organ, and conscious rat studies, the ability of liver and lung to remove B(a)P from the circulation was expressed as extraction ratios (Table 22). These values represent the fraction of the B(a)P entering the organ that was removed on passage through the organ. For broken cell studies, the predicted clearances, and in isolated organs, the observed clearances were divided by the flow to obtain the extraction ratio, i.a.; E = 01/0 (16) Extractions for liver and lung jn_vj!9_were determined from equations 14 and 15, respectively. As demonstrated in Table 22, predictions from broken cell studies agree well with the results observed in isolated perfused organs. Extraction by lung in conscious 3MC pretreated rats is similar to that observed in the isolated lung. However, extraction by liver jn_vivo was less than in the isolated liver. 147 TABLE 22 Extraction of B(a)P by Rat Livers and Lungs at Normal Organ Flow as Predicted from Enzyme Kinetic Data, Observed in Isolated Organs, and Determined Ig_Vivo Extraction1 Control 3MC Lung Liver Lung Liver 2 Broken cell data 0.022:0.003 0.46:0.05 0.16:0.02 l.0:0.01 Isolated organ53 0.022¢o.002 0.56:0.02 0.20:0.01 0.71:0.06 Conscious rats4 --- --- 0.30 0.21 1Extraction (E) is that fraction of the B(a)P in the blood that is removed on passage through the organ. 2E = predicted Cl/normal organ flow. Clearance (Cl) was predicted using mean Cl'int/g values from Table 2, the weights of the isolated organs, estimates of f from Table 3 and normal organ flows of 10 m1/min for livers and 45 ml/min for lungs. Values represent mean extraction : "S.E.M." The "S.E.M." was calculated as described in legend to Table 4. 3E = 01/0. 4E = l - f, where f is the fraction of the dose available to the systemic circulation following first-pass removal by the organ. DISCUSSION According to the perfusion-limited model of metabolic organ clear- ance, the ability of organs to clear circulating xenobiotic compounds depends on the metabolic capacity of the organ, the fraction of the compound free in the circulation, and the flow to the organ. Altera- tions in these factors may change clearance depending on the particular clearance and flow relationship of the organ for that compound. The purpose of the experiments in this thesis was to examine how these factors influence the clearance of a model xenobiotic compound, B(a)P, by two organs known to be involved in B(a)P metabolic elimination, liver and lung. A. Broken Cell Studies Al. Determination of Apparent Enzyme Kinetic Parameters and Cl'int Experiments utilizing broken cell preparations demonstrated that in both control and 3MC pretreated rats liver has the greater specific (Vmax/mg microsomal protein) as well as total microsomal meta- bolic activity (V per organ) toward B(a)P. This was not true for max affinity (Km). In control rats, pulmonary AHH had a greater apparent affinity than hepatic AHH; with organs from 3MC pretreated rats this situation was reversed. The composite term, Cllint’ paralleled altered metabolic activity. However, the magnitude of the difference bewteen liver and lung metabolic capacity was increased. 148 149 Apparent enzyme kinetic parameters, V ax and Km, for the m microsomal metabolism of B(a)P by liver (Alvares §t_al,, 1968; Hansen and Fouts, 1972; Zampaglione and Mannering, 1973; DePierre gt_al,, 1975; Robie et_al,, 1976) and lung (Vadi et 21,, 1976) of control and 3MC pretreated rats have been previously reported. The Km values deter- mined in these studies were for the most part considerably greater than those which were obtained in the experiments of this thesis. For example, Zampaglione and Mannering reported Km's for the hepatic micro- somal metabolism of B(a)P to be 14.6 and 2.9 uM for control and 3MC pretreated rats, respectively. In contrast, the values determined in these experiments were 5.5 and 0.05 nM. These differences could be due to a number of causes, however, two are potentially the most important. In all but one of the studies cited above, the assay for the production of B(a)P metabolites involved detection of fluorescent material. Even if care was taken to differentiate the fluorescence of B(a)P from that of hydroxylated metabolites, such as in the study of Robie et_al, (1976), the amount of product produced could be underestimated if (1) some products produce less fluorescence than others, (2) subsequent metabolism removes the ability to fluoresce, or (3) some metabolites are not fluorescent. Metabolite production could be overestimated if the major metabolite generates a greater intensity of fluorescence than the standard used to quantify the results. In contrast to these assays of B(a)P metabolites, the radiometric assay used in this investigation measured all of the metabolites produced by quantitative and selective removal of the unreacted substrate from the incubation medium. Thus, no 150 matter which metabolite(s) was produced, it was quantified, even if it underwent further metabolism. Another difference in our experiments was the amount of micro- somal protein present in the assay mixture. In contrast to previous studies, where in most cases liver microsomal protein was present at an assay concentration of 0.5-2.0 mg/ml, we used at least 50 times less (see Table 1). In this way the ratio of free substrate concentration to enzyme was more favorable for the determination of initial rates. By decreasing the microsomal protein concentration (and thus the other microsomal components) in the assay, the number of non-enzymic binding sites was also reduced. The interaction of B(a)P with these sites might have reduced the free concentration of B(a)P at the enzymic sites (Hansen and Fouts, 1972; Robie §t_al,, 1976). Therefore, it is likely that the situation in our assay was closer to the requirement of Ni- chaelis-Menten kinetics that the available substrate concentration be much larger than the enzyme concentration (Segel, 1975). In addition to the above causes, differences could result if the rates of reaction used to calculate Vmax and Km were not initial rates. In our studies, the velocities obtained reflect initial rates since under the conditions examined the rates of reaction were linear with both time and microsomal protein at the highest and lowest B(a)P concentrations used (Figures 6-9). Since only about 10% or less of the B(a)P was metabolized a large substrate-enzyme concentration ratio was maintained throughout the time of incubation. The changes observed in the Km values between the two organs and those due to the 3MC pretreatment suggest several possibilities 151 about the nature of these microsomal enzymes. Cytochrome P450-contain- ing enzymes exist in several forms (Guengerich, 1979). Since the liver and lung enzymes of control rats have such disparate Km values it is likely that these two organs possess different constitutive forms. Induction by 3MC appears to cause an increase in the amount of the constitutive form in lung since the Vmax is increased while the Km value is unaltered. In liver, on the other hand, 3MC pretreatment appears not only to increase the amount of enzyme (increased V ) but also to max result in the production of a new form (or forms) since the affinity of the enzyme is so greatly enhanced. Similar explanations have been proposed for the differential induction of rat liver microsomal enzyme activities by phenobarbital (PB) and 3MC. For example, Dent gt_al, (1980) found that rat hepatic benzphetamine N-demethylase activity was enhanced by PB pretreatment, but not 3MC. In contrast, biphenyl 4- and 2-hydroxy1ase, AHH, ethoxy- coumarin-O-deethylase and ethoxyresorufin-O-deethylase (EROD) activities were only slightly enhanced by P8, while 3MC pretreatment markedly en- hanced these enzyme activities. Thus, PB and 3MC increased the activity of different forms of cytochrome P-450 enzymes; PB inducing those cyto- chromes P-450 with benzphetamine N-demethylase activity and 3MC inducing a different set of activities associated with the appearance of cyto- chrome(s) P-448. Similar results were reported by Ryan gt_al, (1979) and Warner and Neims (1979) for rat liver enzyme activities purified from PB and 3MC (or B-naphthoflavone) pretreated rats. In the latter study, apparent differences were also observed in the Km values for EROD activity by two of the cytochrome P-4505. These results suggested 152 that induction may produce enzyme form(s) of differing affinity as well as activity. Another explanation for the decreased Km value in liver fol- lowing 3MC pretreatment is possible. Since the Km values determined in these experiments are only apparent Km's, it may be that exposure to 3MC alters the enzymic site or structure of the existing enzyme so that the active site is more accessible. However, since time is required for microsomal enzyme induction to occur (for synthesis of new protein; Conney et_al,, 1960), it seems likely that 3MC pretreatment induces the production of new forms of the enzyme. Reported values for the specific AHH activity (Vmax) of liver microsomes vary considerably. The data appear to belong to two groups. A group of lower values (e.g., Matsubara gt_al,, 1974; Lake gt_al,, 1973) were from studies in which the fluorometric assay method was used while the group of higher values included those studies in which other assay methods, such as high pressure liquid chromatography, were used (Prough gt_al,, 1979; Yang gt_al,, 1975). The values of Vmax for AHH determined in these studies were comparable to those of the second group for microsomes from 3MC pretreated rats and intermediate for microsomes of control rats. Since the induced values were similar and control values variable, the differences in values from control rats for the various studies were probably due to exposure to agents in the housing environment. Similar results were observed for AHH Vmax values from lung microsomes. The intrinsic free clearance (Cl'int) depends upon both the Vmax and Km of the enzyme. The V portion of the term reflects the max 153 amount of enzyme present in the organ, whereas Km reflects the affinity of the enzyme for the B(a)P. Both of these qualities are important for removal of the B(a)P from the circulating blood under first-order con- ditions. Thus, the intrinsic free clearance provides a better overall estimate of the metabolic capacity of the intact organ than does enzyme activity (i.e., Vmax) alone. This is important when changes occur in both components such as occurred with AHH following 3MC induction in rat liver. A2. Determination of fB Direct determination of the free fraction in either the micro- somal incubations or the perfusion medium was not possible. To estimate the effect that BSA protein concentration had on the ability of the microsomal enzymes to metabolize B(a)P and, therefore, of organs to clear circulating B(a)P, the relative metabolic activities of the micro- somal preparations were compared at two concentrations of added BSA protein (Table 3). If the affinity of BSA for B(a)P was greater than that of the microsomal enzyme, then added BSA might be expected to decrease the rate of microsomal B(a)P metabolism.1 The microsomal pre- paration with the highest Km (5.5 pM) and, therefore, the lowest affi- nity was from control rat liver. Indeed, increasing the concentration of BSA reduced metabolism by these microsomes seven-fold (Figure 12; Table 3). By contrast, if the affinity of the microsomal enzymes was greater than that of the BSA and dissociation time was not limiting, protein bound B(a)P may have acted as though it was free in solution and the metabolic activity might have been unaffected. Accordingly, in the other microsomal preparations, each of which had a lower apparent Km 154 value than control liver microsomes, the rate of B(a)P metabolism was not reduced by increasing the BSA concentration. This suggested that the affinity of these microsomal enzymes for B(a)P was greater than that of BSA. The microsomal preparation with the very lowest Km was from livers of 3MC pretreated rats. Increasing the BSA protein concentration actually increased slightly the metabolism rate by these microsomes. The reason for this cannot be determined from these data, but it could have been that binding of B(a)P to BSA may in some manner facilitated delivery of this substrate to the high affinity enzyme(s). A3. Prediction of B(a)P Clearance Utilizing the Cl'int and fB estimates from these broken cell experiments the clearance of B(a)P by rat liver and lung was predicted using equation 3 (Table 4). This exercise suggested that clearance was enzyme-limited in lungs of control rats and flow dependent in livers from both control and 3MC pretreated rats and in lungs from 3MC pre- treated rats. A_prjgrj, these predictions should not be expected to be quantitatively exact. Several assumptions have been made which may not apply in all situations. For example, the model assumes that the driving force for clearance is diffusion into the tissue. This is primarily a function of the concentration gradient between the blood and tissue. Any barriers to diffusion are not accounted for. The model also assumes that the substrate undergoes the metabolism described by the Cl'int term; if other processes (e.g., biliary excretion) are also occurring clear- ance will be underestimated. Limiting cofactor concentrations jn_gjvg_ or low recovery of microsomal metabolic activity will also introduce 155 error into the calculation. In contrast to the procedure of Rane gt_al, (1977), the predictions of Table 4 are not corrected for any of these limiting factors. However, this would not result in substantially different clearance values. For example, if recovery of AHH activity were only 50% doubling the Cl'int values, predicted clearances for liver and lung would have been 6.7 and 1.7 ml/min in organs from con- trol rats and 10 and 9.5 ml/min, respectively, in organs from 3MC pretreated rats. In these predictions the use of AHH activity ratios to estimate fB is a likely source of error. B. Isolated Organ Studies Isolated rat livers and lungs were used to examine the effect of changes in flow and metabolic capacity on the ability of these organs to remove circulating B(a)P. In addition, the effect of altered perfusion medium composition was investigated in isolated livers. The influence of flow and metabolic capacity on metabolite production was examined in isolated lungs. The results of these experiments demonstrated that isolated, per- fused lungs as well as livers can be used at physiologic flows to investigate removal of xenobiotic compounds from the circulation. In prior experiments lungs had been perfused at flows much less than normal pulmonary flow. As seen in Table 6 increasing flow did not increase the post-perfusion weight of lungs, despite increased resistance (increased inflow perfusion pressure). These weights (normalized to body weight) were not significantly different than those of lungs which were not perfused. The fact that there was a slight tendency for the perfusion pressure to increase during the course of the perfusions suggested that 156 some edema formation may have occurred. Since post-perfusion lung/body weight ratios were not significantly increased, however, edema formation was minimal. More than in any other group, isolated lungs of 3MC pre- treated rats perfused at 45 ml/min developed significant increases in perfusion pressure. Those lungs in which perfusion pressure increased to greater than twice the initial perfusion pressure were not included in the study. As demonstrated in Tables 5 and 12, isolated livers were also successfully perfused. No significant changes were observed in either liver to body weight ratio, bile production, or increase in perfusion pressure during the flow dependence experiments. 81. Organ Flow and B(a)P Clearance The relationship between flow and B(a)P clearance in isolated lungs differed from that in isolated livers. Clearance of B(a)P by lungs of control rats was independent of flow throughout the range of flows examined (Figure 15). In contrast, clearance by lungs of 3MC pretreated rats was flow dependent (Figure 16). Hepatic clearance in both cases was limited by flow. In all cases, changes in clearance with flow were associated with altered ke’ not Vd (Tables 7 and 8). Flow limited metabolic clearance of highly extracted compounds by liver has been demonstrated for other agents (Branch gt_al,, 1973; Shand et_al,, 1975; Pang and Rowland, 1977b; Pries at al,, 1981; Roth and Rubin, 1976b). However, only recently has flow dependence been investigated in extrahepatic organs such as lung (Wiersma and Roth, 1980; Roth, 1982). Organ blood flow may influence the relative role these organs play in the total body elimination of circulating compounds. For 157 example, normal organ flow in control rats is about 45 ml/min in lungs (i.e., cardiac output) and 10 ml/min for livers (Sapirstein gt_al,, 1960; Roth and Rubin, 1976). The data presented in Table 9 suggest that in the resting state the liver is the predominant organ of B(a)P dispo- sition ig_givg_in control rats. In 3MC pretreated rats, however, clearance of B(a)P by these two organs is probably about equal at flows occurring ig_!1!g,q~ . Physiologic alterations, however, may change these relation- ships. For example. exercise increases cardiac output (pulmonary flow) and decreases hepatic flow (Chapman and Fraser, 1954). Thus, in exercising control rats total body B(a)P clearance may diminish as liver clearance decreases with decreasing flow. No change would be expected in pulmonary clearance since it is not flow dependent. In 3MC pre— treated rats. exercise would decrease hepatic B(a)P clearance as flow decreased, but it would increase pulmonary removal as cardiac output increased. Therefore, in this latter condition lung may predominate in total body B(a)P clearance despite the fact that the liver has far more enzymic capacity. 82. Metabolic Capacity and B(a)P Clearance Pretreatment of the animals with 3MC elevates AHH activity in many organs and thus potentially increases clearance of some xenobiotic agents by organs jg_1139, The Cl'int’ which is determined by the metabolic capacity of the tissue (Rane §t_al,, 1977), is a measure of this potential. As seen in Table 2, 3MC pretreatment increased the Cl‘1nt of lung and liver about 6 and 400-fold, respectively. In iso- lated lungs this stimulation of enzyme activity is reflected as an 158 increase in B(a)P clearance at each flow tested. However, in the iso- lated livers no significant increase in B(a)P clearance results from this increased capacity. These results suggest that extrapolation of results of enzyme induction studies utilizing broken cell assays to the situation jg_vjvg_ should be made with caution. Extrapolation of increased metabolic activity in broken cell preparations to increased metabolic function by the whole organ is useful only under certain circumstances. As the perfused lung data indicate, markedly increased metabolic activity will cause substantial increases in organ metabolic clearance only when that clearance (i.e., extraction) is initially low. 0n the other hand, when clearance (extraction) is already high, as is B(a)P clearance in livers from control rats, increased metabolic capacity may not result in in- creased clearance if clearance is limited by flow. For the most part, extrahepatic organs have low metabolic capacity toward xenobiotic compounds due to a paucity of enzyme in their tissues. Liver, on the other hand, often has both a high specific and total metabolic activity toward xenobiotic compounds. Therefore, an increase in the level of xenobiotic metabolizing enzymes in organs may result in a shift in the relative contributions of hepatic and extra- hepatic clearance to total body clearance as the ability of extrahepatic organs to extract circulating compounds is enhanced. Increased intracellular binding may alter clearance by an apparent increase in C1 Stegmann and Bickel (1977) found that int' hepatic imipramine Cl , as measured in isolated livers, was a measure int of tisSue uptake as well as metabolism. However, this is not a likely 159 cause for increased B(a)P clearance by lungs from 3MC pretreated rats since (1) use of enzyme kinetic parameters of microsomal B(a)P metabo- lism accurately predicts B(a)P clearance, suggesting that metabolism alone is sufficient to account for B(a)P clearance; (2) tissue B(a)P concentrations are lower in isolated lungs of 3MC pretreated rats (Table 10); and (3) B(a)P does not appear to any great extent in the effluent of an isolated lung from a 3MC pretreated rat that has been loaded with B(a)P (Figure 22), suggesting that B(a)P within the lungs is metabo- lized. Therefore, it appears that increased B(a)P clearance by isolated lungs of 3MC pretreated rats is due to their increased metabolic capa- city. B3. Binding to Blood Components and B(a)P Clearance Analysis of the distribution of B(a)P among three fractions of rat blood (Table 17) indicated that B(a)P was not uniformly distributed within the blood, but was preferentially associated with certain blood components. 0f the 60% of the B(a)P found in the serum 75% was asso- ciated with the fraction containing serum lipoproteins. The remaining 15% was likely associated primarily with serum albumin. These results are similar to those obtained by Shu and Nichols (1979) using human 3 blood. Vauhkonen gt_al, (1980) also examined_the distribution of H in rat blood at various times after injection of 3 H-B(a)P. However, they neglected to separate B(a)P from its metabolites making it difficult to interpret their findings. Since B(a)P was preferentially associated with certain blood components, relative changes in their concentration in blood or perfusion medium may affect B(a)P clearance. Whether 3MC 160 pretreatment alters the relative composition of blood components or the distribution of B(a)P between them is not known. The composition of the perfusion medium affected the dispo- sition of B(a)P by the perfused livers (Figure 20; Table 13). Livers perfused with medium containing erythrocytes and buffer alone were least able to clear circulating B(a)P. Inclusion of serum lipoproteins in the medium enhanced B(a)P clearance 2.5 times, while the presence of serum albumin resulted in a clearance about 4 times greater than when buffer alone was present. Changes in clearance could have occurred by an alteration in the free concentration of the compound in the blood (Wilkinson and Shand, 1975). For example, in isolated livers in which the albumin concentration of the perfusion medium was decreased, phenytoin clearance was increased. A concomitant increase in the free fraction was observed (Shand gt_al,, 1976). Opposite results were obtained in this study, however, since inclusion in the perfusion medium of blood components capable of binding B(a)P decreasing its free concentration, increased the ability of the livers to extract circulating B(a)P. Possible models to explain our results include: (1) involvement of receptors, (2) rates of dissociation and transit times, (3) anatomical barriers to efficient extraction, and (4) increased solubility. (1) In their investigation into the hepatic extraction of taurocholate and oleate, Forker and Luxon (1981) and Weisiger et_a1, (1981) suggested that albumin mediates the extraction of certain com- pounds from the perfusion medium of isolated livers by way of an albumin receptor on the surface of the hepatocyte. In both studies, as the 161 concentration of albumin in the perfusion medium increased, the free concentration of the substances decreased. However, the clearance of taurocholate was unchanged while that of oleate increased. Weisiger gt 31, (1981) demonstrated that hepatocytes have binding sites with high albumin specificity and suggested that hepatic extraction may be en- hanced by the interaction of albumin carriers with these sites. Similar high affinity sites for serum lipoproteins are found on cells throughout the body (Goldstein and Brown, 1977). Remsen and Shireman (1981) compared the uptake of lipoprotein-associated B(a)P by cultured lung fibroblasts which possessed these receptors with genetically receptor- deficient fibroblasts. They found that the uptake of lipoprotein associated B(a)P by these cells in culture was not enhanced by the presence of serum lipoprotein receptors on the cells. This suggested that the involvement of lipoprotein receptors may not be needed to explain the enhanced extraction of B(a)P in our study. Furthermore, these investigators found that the presence of lipoproteins in the culture medium inhibited the cellular uptake of B(a)P suggesting that in a system containing cells and lipoproteins, lipoproteins will retard the uptake of B(a)P by cells. (2) Another explanation for the effect that these blood compo- nents which bind B(a)P have on its hepatic extraction involves the relationship between the rate constants for dissociation and the transit time of the bound complexes within the liver. In this model (see Figure 28), the free form of B(a)P within the blood or perfusate is that por- tion of the B(a)P available for extraction. Due to its extremely low aqueous solubility nearly all the B(a)P in the perfusion medium is present in bound forms (Tipping 33 21,, 1980). The supply of the free, 162 A.co?pmcm_axm co: pxma mmmv .Anfiwpmo .mN mczmwa 163 mm mczmaa mmmfi ".0 333 c.0325 wh>UOh