‘MATURATION OF HEPATIC EXCRETORY FUNCTION INFLUENCE OF CARBON TETRACHLORIDE AND POLYBROMINATED BIPHENYLS DISSBI‘LZLIOII fer the Degree of Ph D MICHIGAN STATE UNIVERSITY STUART ZENO CAGEN ' = a '0" waning- -41.: .' I LIMex/CJZJ I I‘l/Iichfgm Shite University [$1.2 - _. This is to certify that the thesis entitled Influence Maturation of Hepatic Excretory Function: of Carbon Tetrachloride and Polybrominated Biphenyls presented by Stuart Zeno Cagen has been accepted towards fulfillment of the requirements for Ph.D. degree in Pharmacology Q \ v \ , U Major professor Date June 24, 1977 0-7 639 ABSTRACT Maturation of Hepatic Excretory Function: Influence of Carbon Tetrachloride and Polybrominated Biphenyls by Stuart Zeno Cagen The biliary tract is a major route for elimination of xenobiotics from the body and the ability of the newborn to excrete chemicals into bile is low with respect to adult excretory capacity. The purpose of this investigation was to characterize the maturation of hepatic excre- tory function in rats and to determine the effect of carbon tetrachlo- ride and polybrominated biphenyls on liver excretory function in developing rats. Maturation of the hepatic excretory system was determined in developing rats by measuring the plasma disappearance and hepatic and intestinal (biliary) appearance of intravenously administered ouabain. Cumulative (40 minute) intestinal ouabain content was lower in 15 day old rats than in 21, 25, 35, and 45 day old animals and reached adult levels when rats were 35 days old. Decreased ouabain excretion in young rats resulted in retention of ouabain in plasma when compared to plasma ouabain concentrations in older animals. Biliary excretion of sulfobromophthalein (BSP) is dependent on hepatic uptake from plasma and intrahepatic conjugation to glutathione (GSH). Enzymatic conjugation of BSP to glutathione in vitro was low _ Stuart Zeno Cagen in young rats, however, elimination of BSP and conjugated BSP (BSP—GSH) from plasma was more rapid in adults than in 15 day old rats. Impaired elimination of ouabain and BSP from plasma of rat neonates correlated to low initial concentration of these drugs in liver. The inability of young rats to accumulate BSP and ouabain in liver may be the most important determinant for functional inSufficiency. Studies were undertaken to determine whether the rate-limiting step in the excretion of drugs by liver is age dependent. Bile duct liga- tion and bile salt infusion, treatments that primarily depress and enhance (respectively) excretion of BSP from liver into bile, markedly altered the disappearance of BSP from plasma of adult rats but did not appreciably affect BSP disappearance from blood of 15 day old rats. The effect of bile duct ligation on ouabain transport in 15 day old rats was also not as dramatic as the effect produced in adult rats. Hepatic uptake is rapid in adult rats and overall excretion is limited by a slower rate of transport from liver into bile. The lower rate of uptake in 15 day old rats may limit overall transport function in these animals. Carbon tetrachloride (CC14) depressed hepatic excretory function in adult and developing rats and plasma ouabain concentration was significantly higher and biliary (intestinal) ouabain content was significantly lower in treated animals when compared to controls. Hepatic ouabain was significantly lower than controls in CCl4 treated young rats (decreased uptake into liver), but significantly higher than controls in treated older rats (decreased excretion from liver into bile). CCl4 may disrupt all mechanisms in the drug elimination _ Stuart Zeno Cagen process. Since the rate—limiting step in drug transport changes with age, CCIA may disrupt transport function in accordance with existing age differences. Exposure of developing rats to polybrominated biphenyls (PBBs) did not produce overt toxicity when compared to controls over a 49 day postnatal period. However, prenatal and postnatal dietary exposure to PBBS resulted in elevated liver weight. In 15 day old rats, increased liver weight following PBBs correlated to enhanced ouabain excretion into bile. Liver weight was also elevated in 21, 35, and 49 day old rats treated with PBBs but this effect was not associated with stimulation of ouabain transport in these animals. The mechanism for stimulation of ouabain transport following PBBs in 15 day old rats was increased hepatic uptake of ouabain. The selective stimula— tion in only young rats may be attributed to the relative importance of uptake for overall transport in 15 day old rats. MATURATION OF HEPATIC EXCRETORY FUNCTION: INFLUENCE OF CARBON TETRACHLORIDE AND POLYBROMINATED BIPHENYLS BY Stuart Zeno Cagen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology 1977 cw ACKNOWLEDGEMENTS I would like to express my sincere appreciation to graduate committee members: Drs. Theodore M. Brody, James E. Gibson, Jerry B. Hook, Douglas E. Rickert, and Bernard A. Schwetz for their helpful assistance in the preparation of this thesis. I would expecially like to acknowledge Dr. James E. Gibson for his guidance, encourage— ment, and constructive criticism over the course of my graduate educa- tion. Special thanks are offered to Dr. Jerry B. Hook for his encouragement during my final graduate year. I would like to thank Drs. James S. Bus, John G. Dent, James E. Ecker, and Maurline M. Preache, and Mr. Kevin M. McCormack for their intellectual contributions during my graduate career. The able technical assistance of Miss Cathy Herrmann and Miss Harriett Sherman are gratefully acknowledged. For her endless tolerance and encouragement, I would like to dedicate this thesis to Sherri K. Cagen. These studies were Supported by NIH Research Grant ESOOS60 and by predoctoral fellowship from the Eli Lilly Co. ii AIM TABLE OF CONTENTS Page ACKNOWLEDGEMENTu ii TABLE OF CONTENTS iii LIST OF FIGURES v LIST OF TABLES vii INTRODUCTION 1 Objectives 1 Liver development and maturation of hepatic enzyme systcum 2 Hepatic excretory function during development --------- 5 Hepatic excretion of xenobiotics 12 The importance of bile flow in hepatic drug excretion- 22 Examples of agents that alter hepatic excretory function 27 Polybrominated biphenyls 34 Experimental rationale 37 MATERIALS AND METHODS 39 Animals 39 Hepatic transport of ouabain and sulfobromophthalein in developing rats 39 Influence of experimentally—induced alterations in bile flow on drug transport in adult and young rats 45 Effect of carbon tetrachloride on hepatic transport of ouabain in developing rats 47 Digoxin-mediated inhibition of ouabain transport in adult and developing rats 48 Toxicity of polybrominated biphenyls (PBBs) in developing rats 49 Effect of polybrominated biphenyls (PBBs) on hepatic excretory function in developing rats ------------ 50 Characteristics of stimulation of drug transport in young rats 52 Statistics 53 iii TABLE OF CONTENTS (continued) Page RESULTS 54 Hepatic transport of ouabain and sulfobromophthalein in developing rats 54 Influence of experimentally-induced alterations in bile flow on drug transport in adult and young rats 66 Effect of carbon tetrachloride (CCl ) on hepatic transport of ouabain in developing rats ---------- 82 Digoxin~mediated inhibition of ouabain transport in adult and developing rats 85 Toxicity of polybrominated biphenyls (PBBs) in de- veloping rats 85 Effect of polybrominated biphenyls (PBBs) on hepatic excretory function in developing rats 95 Characteristics of stimulation of drug transport in young rats 111 DISCUSSION 119 Hepatic transport of ouabain and sulfobromophthalein in developing rats 119 Differential effects of bile salt administration, bile duct ligation and hypothermia on biliary function in adult and developing rats 127 Effect of carbon tetrachloride (CCl ) on hepatic transport of ouabain in developing rats ---------- 133 Effect of polybrominated biphenyls (PBBs) on hepatic drug transport in developing rats 138 Characteristics of stimulation of drug transport in young rats 143 SUMMARY AND CONCLUSIONS 146 BIBLIOGRAPHY 150 iv LIST OF FIGURES Figure Page 1 Elimination of (3H)ouabain from plasma of rats of ages ranging from 15—45 days ------------------ 55 2 Hepatic ouabain concentration and totgl hepatic ouabain content with time following ( H)ouabain administration in rats of ages ranging from 15- 45 days 58 3 Elimination of sulfobromophthalein (BSP) from plasma of 18 day old and adult rats -------------- 62 4 Hepatic content of sulfobromophthalein (BSP) with time following BSP administration in 18 day old and adult rats 64 5 Effect of sodium taurocholate (bile salt) on elimination of sulfobromophthalein (BSP) from plasma of 15 day old and adult rats —————————————— 70 6 Effect of sodium taurocholate on sulfobromoph- thalein (BSP) concentration in liver of 15 day old rats 72 7 Effect of bile duct ligation on elimination of sulfobromophthalein (BSP) and ( H)ouabain from plasma of adult and 15 day old rats —————————————— 74 8 Effect of bile duct ligation on elimination of (3H)ouabain from plasma of 15 day old rats ——————— 77 9 Effect of carbon tetrachloride (CC14) on hepatic transport of ouabain in rats of various ages ————— 83 10 Effect of digoxin on elimination of (3H)ouabain from plasma of 15 day old, 21 day old, and adult rats 86 11 Effect of digoxin on hepatic content of (3H) ouabain in 15 day old, 21 day old, and adult rats 89 12 Effect of exposure to polybrominated biphenyls (PBBS) from birth to 49 days after birth on post- natal growth of rats 91 LIST OF FIGURES (continued) Figure 13 14 15 16 17 18 19 Effect of exposure to polybrominated biphenyls (PBBs) from birth to 49 days after birth on. postnatal mortality of rats - Effect of exposure to polyhrominated biphenyls (PBBs) on elimination of ( H)ouabain from plasma of rats of ages ranging from 15-49 days-—--——---- Effect of exposure to polybrom§nated biphenyls (PBBs) on hepatic content of ( H)ouabain in rats of ages ranging from 15-49 days Effect of pre- and/or postnatal exposure to poly- b ominated biphenyls (PBBs) on elimination of ( H)ouabain from plasma of 15 day old rats--—---— Effect of pre- and/or postnatal exposure to poly- b ominated biphenyls (PBBs) on hepatic content of ( H)ouabain in 15 day old rats Effect of digoxin on elimination of (3H)ouabain from plasma of control 15 day old rats and 15 day old rats pre- or postnatally exposed to poly- brominated biphenyls (PBBs) Effect of digoxin on hepatic content of ouabain in 15 day old control rats and 15 day old rats pre- and postnatally exposed to polybrominated biphenyls (PBBs) vi Page 93 99 102 105 108 112 114 Table 10 ll 12 LIST OF TABLES Cumulative hepatic excretion of ouabain in developing rats — — Liver to body weight ratio in developing rats-—-— Hepatic glutathione S—aryl transferase activity in developing rats Disappearance of BSP or conjugated BSP (BSP-GSH) from plasma of 15 day old and adult rats --------- Cumulative intestinal ouabain in sham or bile duct ligated 15 day old and adult rats ----------- Hepatic concentration of ouabain following intra- venous administration in sham or bile duct liga- ted (BDL) adult and 15 day old rats Effect of anesthesia induced hypothermia on ouabain tissue distribution in 15 day old and adult rats Effect of digoxin on cumulative hepatic excretion of ouabain in developing and adult rats --------- Effect of continuous exposure to polybrominated biphenyls (PBBs) on liver to body weight ratio in developing rats Effect of prenatal, postnatal, and combined pre- and postnatal exposure to polybrominated bi- phenyls (PBBs) on body weight gain and Z liver wt/body wt in 15 day old rats Effect of dietary polybrominated biphenyls (PBBs) on ouabain LD50 in 15 day old rats Effect of exposure to polybrominated biphenyls (PBBs) on cumulative hepatic excretion of ouabain in developing rats vii Page 57 61 67 68 79 80 81 88 96 97 98 104 LIST OF TABLES (continued) Table 13 14 15 Page Initial rate of elimination of indocyanine green (ICG) from plasma in 21 day old rats ex- posed to dietary polybrominated biphenyls (PBBs)- 110 Effect of digoxin on cumulative hepatic excre- tion of ouabain in 15 day old rats exposed to polybrominated biphenyls (PBBs) 117 Effect of carbon tetrachloride (0014) on tissue distribution of ouabain in 15 day old rats treated with polybrominated biphenyls (PBBs) ----- 118 viii INTRODUCTION Objectives Functional immaturity of the liver is exemplified by the inability of newborn animals to effectively excrete xenobiotics into bile (Klaassen, 1972). The mechanisms responsible for the maturation of hepatic excre- tory function are not precisely known, however, determination of the development of the excretory system may be complicated by the multifaceted nature of the drug elimination process. Transport of drugs from blood into bile requires a number of steps including specific uptake into the hepatic parenchyma, intrahepatic storage and metabolism, and finally secretion into bile. Thus, the deficiency in the young, for drug elimi- nation may be due to a combination of any or all of these factors (Klaassen, 1975). The low capacity for chemical excretion in newborns has toxicolo- gical consequences, for when compared to adults, young animals may be particularly susceptible to chemical-induced toxicity, including lethality (Klaassen, 1972,1973a). An equally important area of concern is the influence that foreign agents may have on the liver as an excre- tory organ. In adults, hepatic excretory function is well known to be disrupted following exposure to a variety of xenobiotics, but the in- fluence of such substances on liver excretory function during develop- ment has not been extensively studied. 2 Since chemical excretion by the liver is a multifaceted process, the influence of chemical agents on overall drug transport may be a reflection of specific and distinct dysfunction at any transport step. In adults, the rate-limiting step in drug elimination into bile is hepatic secretion. Disruptions in hepatic function, therefore, are most evident when the hepatic secretory mechanisms are altered. Although drug transport into bile is low in the neonate when compared to the adult, the relative importance of the various transport steps has not been elucidated. Thus, the objectives of this investigation were two-fold, both of which relate to hepatic excretory function and dysfunction during develop- ment. The first objective was to characterize the development of hepatic excretory function in rats and determine the relative importance of the various transport steps as a function of age. The second objective of this research focused on chemical-induced alterations in function. In particular, the objective was to determine the effects of carbon tetrachloride and polybrominated biphenyls on hepatic excretory function in both developing and adult rats. Liver Development and Maturation of Hepatic Enzyme Systems During embryonic development, the liver arises as an outgrowth of the endodermal wall of the primitive gut. The original outgrowth gives rise to a hollow hepatic diverticulum which soon differentiates into two parts: an anterior portion which proliferates to become the large glandular mass of the liver and its bile ducts (hepatic bile ducts); and a posterior part which gives rise to the gall bladder and cystic duct. The stalk connecting these two parts to the gastrointestinal tract becomes the common bile duct (Weichert, 1970). 3 In most mammalian species, the fetal liver, once organogenesis is complete, contains two major cellular components; hematopoietic tissue and hepatocytes (Jacquot g£_§1,, 1973). In the very young rat fetus, the hematopoietic component is by far the major one and hepatocytes are dispersed in it. The normal ontogenic pattern is a shift in the rela— tive preponderance of these two tissue types. Thus, at birth, hepato- cytes form a well organized parenchyma including diapersed erythro- poietic cells. In order to maintain homeostasis and permit normal growth, marked variations in many metabolic functions occur in the liver of mammals during development. These changes are particularly dramatic at birth, when the abrupt passage from intrauterine to extrauterine environment occurs, and during the weaning period, when great variations in nutri- tion takes place (Serini and Principi, 1971). Greengard (1974) noted that, with few exceptions, the many enzymes that have been studied in rat liver conform to one of four developmental patterns. The enzymes of one group (termed cluster I) appear to be mandatory for growth and are present in all fetal tissues and decrease in amount toward the end of gestation. More specific to hepatic functions are enzymes associated with the other three clusters. A late fetal cluster (II) emerges be- tween the 17th and let day of gestation and includes the enzymes for the urea cycle and glycogen synthesis. The next cluster (III), which is brought forth during the early postnatal period, involves enzymes needed for gluconeogenesis and xenobiotic detoxication. Finally, the enzymes (cluster IV) involved in fatty acid synthesis and in the subtle regula— tion of amino acid and glucose levels in the blood emerge in the third postnatal week or late suckling period (Greengard, 1971,1974). 4 One aspect of fetal and neonatal development which has been studied in great detail is the enzyme system which acts to metabolize chemicals. The metabolism of drugs and other foreign compounds (xenobiotics) occurs to a considerable extent in the smooth endoplasmic reticulum (microsomes) of liver. Metabolism of drugs by this microsomal system results in more polar and water soluble products and often leads to reduction of pharmacological activity and more rapid drug excretion (Mandel, 1971). Newborn human infants appear to be immature in their ability to metabolize drugs (Done, 1964). This immaturity has been best documented clinically in the case of the "grey baby syndrome" in premature and newborn infants following treatment with chloramphenicol (Burns 22 gl., 1959; Lambden E£H21°: 1960; Weiss g5 El!’ 1960). The antibiotic is extensively metabolized in adult liver and undergoes nitroreduction (Fouts and Brodie, 1957) and glucuronidation (Glazko gghaif, 1950) catalyzed by hepatic microsomal enzymes. However, during the neonatal period, the activities of enzymes catalyzing these reactions are low with respect to adult levels. Low activity in the fetus and newborn for hepatic drug metabolism is now well documented in laboratory animal studies (Fouts and Adamson, 1959; Jondorf g£_§l,, 1959; Jacquot 33.31,, 1973; Telegy, 1973; MacLeod gghgl., 1972) as well as in studies with the human fetus and newborn infant (Rane g£_§l,, 1973). Increases in the activitiy of drug meta- bolizing enzymes to adult levels occurs, for the most part, during the Postnatal and suckling period. 5 Low activities of enzymes in the fetus and newborn may be partially related to the presence of hematopoietic cells in the developing liver (Henderson, 1971). Thus, increases in enzymatic activity would, to some degree, reflect the emergence of the hepatocyte population. Henderson (1971) found that 20% of all liver cells on the third postnatal day were still hematopoietic in rat liver and that the develOping blood cells did not disappear from liver until the third postnatal week. However, Greengard g£_§l, (1972) determined that the mean volume of individual hepatic parenchymal cells undergoes a three-fold rise during late fetal life, declines slightly in the early neonatal period, and doubles be- tween the 12th and 28th postnatal days. From these data, it was calcu- lated that only increases in enzyme concentration of less than two-fold would be attributed to the enrichment of parenchymal tissue at the expense of hematopoietic cells (Greengard, 1972). Thus, MacLeod g; .31. (1972) maintained that the magnitude of change in liver cell type after birth would be insufficient to contribute significantly to the pattern of microsomal enzyme development. Consistent with this hypo- thesis is the observation that smooth endoplasmic reticulum, the intra- cellular site of drug metabolism, is less prevalant in fetal and newborn hepatocytes when compared to adult liver cells (Fouts, 1962; Peters 25_ .31., 1963; Palmer g£_§l,, 1966; Koenzig §£_§l,, 1976). Thus, increases in enzymatic activity reflect maturation of the liver cell. flgpatic Excretory Function During Development Immaturity of the hepatic excretory system in human neonates is exemplified by the condition of newborn jaundice. Jaundice appears in 6 about 50% of newborn infants and can be very pronounced (Lathe, 1974). Newborn jaundice is characterized by high plasma concentration of uncon- jugated bilirubin, a metabolic product of the nonprotein component of hemoglobin (heme). If bilirubin in blood of the newborn is displaced from.p1asma proteins to which it is bound, or if plasma bilirubin concentration is exceedingly high, bilirubin will enter the central nervous system and produce a type of brain damage called kernicterus (Lathe g£_§l,, 1958). The high incidence of newborn jaundice attracted the attention of physicians at the turn of the century and systematic studies were under- taken to identify the etiology of this condition. From these studies, it was generally held that the precipitating factor of newborn jaundice was increased hemolysis at birth. This was suggested by two observa- tions: 1) the high hematocrit at birth usually falls dramatically shortly after birth, and 2) by the occurence of extreme jaundice in neonates with known hemolytic disease (Lathe, 1974). In 1947, however, weech reviewed the subject and could detect no direct relationship between the amount of hemoglobin destroyed and the intensity of hyper- bilirubinemia. As an alternative, weech (1947) suggested that, in general, maturity of the neonate determined the extent of hyperbiliru- binemia. This contention was supported when jaundice was found to be ‘most extreme in premature infants (Hsia ggug1., 1953) and that the severity of jaundice in these infants was inversely related to the length of gestation and birth weight (Billings, §E_§;,, 1954). Since the major route for bilirubin detoxication and excretion is the liver and biliary tract, attention was focused on the inability of the liver, in the young, to excrete circulating levels of bilirubin (Zuelzer and Brough, 1969). 7 While most laboratory animals do not develop neonatal jaundice, many have shown impaired ability to excrete an exogenous load of bili- rubin when compared to the adult (Schenker g£_§l,, 1964; Catz and Yaffe, 1968; Gartner and Arias, 1969; Klaassen, 1976). Moreover, hepatic excretion of xenobiotics that are primarily excreted into bile in the adult, are excreted into bile to a lesser extent in the newborn rat and guinea pig (Klaassen, 1972,1973b; Hwang and Dixon, 1973). In newborn rats, decreased biliary excretion leads to accumulation of certain drugs in plasma and results in high sensitivity, in the young, to drug-induced lethality (Klaassen, 1973a). Excretion of bilirubin and other endogenous and exogenous compounds from blood into bile requires a number of transport steps: compounds are taken up from plasma into the hepatic parenchyma, metabolized within the liver, and finally secreted from liver into bile. Since the ability of the liver to metabolize drugs is depressed in the neonate, the excre— tion of agents that require intrahepatic metabolism prior to biliary excretion may be limited, in the young, by the degree to which the compound is metabolized. In mammalian species, bilirubin is conjugated to glucuronic acid in hepatic microsomes prior to excretion into bile. The reaction is cata- lyzed by the enzyme UDP-glucuronyl transferase. Iguyiggg, the ability of microsomal fractions of liver to conjugate bilirubin to glucuronic acid (UDP-glucuronyl transferase activity) is low in the newborn human (Lathe, 1974), monkey (Gartner and Lane, 1972), guinea pig (Gartner and Arias, 1969), and rat (Bakken, 1969; Catz and Yaffe, 1968; Vaisman 25 21,, 1976) when compared to conjugation capacity in adults. Thus, low 8 hepatic metabolism may be an important mechanism for accumulation of bilirubin in plasma in the young. Depressed rate of drug metabolism may also be a factor in reten- tion of sulfobromophthalein (BSP) in plasma of the newborn. BSP is the prototype used for studying biliary excretion of organic acids. Prior to biliary excretion BSP is enzymatically conjugated in the liver to glu- tathione (Combes and Stakelum, 1960). A number of studies have shown that BSP disappears more slowly from plasma of newborn infants (Mollison and Cutarsh, 1949; Yudkin ggngl., 1949), guinea pigs (Goldstein g; él-: 1965; Whelan g£_§l,, 1970a) and rats (Klaassen, 1973b) than in their respective adults. Inasmuch as the capacity for BSP conjugation to glutathione is lower in the newborn than in the adult (Combes and Stakelum, 1962; Krasner and Yaffe, 1968; Whelan g£.§l,, 1970a) the slower rate of BSP elimination from plasma may be a reflection of the deficiency for BSP metabolism. Other processes involved in hepatic excretion of drugs are also considered to be immature in newborn animals. Hwang and Dixon (1973) examined hepatic excretion of indocyanine green (ICG) in developing and adult guinea pigs and observed the ability of the newborn to clear ICC from plasma into bile was less than in the adults. Since ICC is not metabolized in the passage from blood to bile, this age difference was considered to represent a deficiency in the newborn in the absolute maximum capacity for biliary excretion. Similarly, hepatic excretion of ouabain, ICC, and conjugated BSP (BSP-GSH) is lower in newborn rats ‘than in adults (Klaassen, 1972,1973b). These drugs do not require Inetabolism prior to biliary excretion and thus the depressed rate 9 of biliary excretion cannot be attributed to a deficiency in conjugating capacity. The newborn may also be immature in the ability to take up drugs into liver from plasma. The initial rate of elimination of BSP from plasma, which primarily represents hepatic uptake capacity, is rapid in the adult and in older children (t% = 5.5 min) but is significantly slower (t% = 9.6 min) in human newborns (Wichmen g£_§;,, 1968). Cumu- lative hepatic uptake of bilirubin by liver was reduced during the neo- natal period in guinea pigs and achieved adult capacity at approximately 15 days of age (Gartner and Arias, 1969). Hwang (1975) determined uptake capacity ighgiggg_by measuring accumulation of ICC into liver slices. Liver preparations obtained from newborn guinea pigs could not effectively accumulate 106, however, accumulation of ICC was high in slices obtained from adult liver (Hwang, 1975). In rats, the low rate of clearance of BSP and ouabain in newborns was related to a lesser ability relative to adults in the uptake of these compounds into the liver cell (Klaassen, 1972,1973b). Although the concentration of ouabain in liver of an adult reaches 50 times that of plasma, liver of the newborn rat cannot concentrate ouabain at all (Klaassen, 1972). The ability of the liver to extract ouabain from plasma in rats develops concurrently with the decrease in ouabain toxicity that occurs with development (Klaassen, 1972). Little is known about the cellular mechanisms responsible for hepatic uptake of drugs. However, it has been suggested that a cyto— Tsolic protein, ligandin (Litwack gghgl,, 1971; Levi g£“§1., 1969a,b) may I>lay an important role in hepatic uptake of organic anions (such as 'bilirubin, BSP, and ICG). Hepatic content of ligandin is low in early 10 life and increases with age in rats (Klaassen, 1975), guinea pigs (Levi g£_§1,, 1969a), and monkeys (Levi g£_§1,, 1970). In rats, the increase in hepatic concentration of ligandin appears to correspond with the age related increase in the ability to accumulate organic anions into the liver (Klaassen, 1975). The relative deficiency of ligandin has been suggested to be important in the etiology of unconjugated hyperbiliru- binemia in human infants (Levi g£“§;., 1970). Whether the transport system that carries drugs from the hepatocyte into bile is also immature in young animals is difficult to determine, since its function may be limited by the concentration of drugs available to it. In guinea pigs, however, the maximum biliary transport capacity (Tm) of ICC in newborns is one-third that in the adult (Hwang and Dixon, 1973) even though bile flow rate in the young is the same as the adult value when calculated on a body weight basis (Hwang and Dixon, 1973). The ability of newborn rats to excrete drugs from liver into bile cannot be directly elucidated since it is difficult to obtain bile samples from the small animals. However, DeWblf-Peeters g£_§l, (1972) have described the morphological development of the biliary tract in rats as an indirect means to estimate function. Using electron micro- scopic and histochemical techniques, these authors examined the differen- tiation of the bile canaliculus, the primary site of both bile formation and drug excretion into bile. From 16 until 19 days of fetal age, the canaliculus is forming and is defined by an intracellular invagination of two adjunct cell membranes into one of the two neighboring hepato— cytes. At birth, the canaliculus has a distinct lumen, however, the sstructure is irregular in form. The lumen of the canaliculus widens during the first 3 postnatal days but contains few microvilli. Follow— ing 3 days after birth, the lumen becomes progressively smaller and 11 gradually fills with microvilli leading, at 10 days after birth, to a normal adult canalicular structure (DeWolf—Peeters g£_§l., 1972). These authors suggested that underdeveloped canalicular morphology may be an important factor in different conditions of human neonatal jaun— dice. However, in the rat, 10 days after birth, overall hepatic excree tory function remains depressed (Klaassen, 1972). Therefore, if the adult—like canalicular structure reflects secretory function of the liver of the 10 day old rat, the maturation of hepatic excretory func- tion following postnatal day 10 must be due to the development of uptake and/or conjugation capacity. Since uptake, metabolism, and biliary excretion of drugs may all be depressed in the newborn relative to the adult, it is difficult to ascertain which deficiency in the newborn is most important in the immaturity of overall transport function. The solution to this problem may also be dependent on the animal species and the transported com— pound. Depressed hepatic transport of both BSP and conjugated BSP (BSP- GSH) is apparent in rat neonates relative to adults (Klaassen, 1973b). Newborn guinea pigs, however, are deficient only in the transport of free BSP and are adult-like in their ability to excrete BSP-GSH (Whelan g£‘§1., 1970a). Therefore, even though uptake capacity for bilirubin and ICC is low in newborn guinea pigs (Gartner and Arias, 1969; Hwang, 1975), depressed BSP uptake may not be as important for functional insufficiency as the inability of the newborn to conjugate BSP. In the newborn rat, however, hepatic uptake may represent the primary deficient step. 12 Hepatic Excretion of Xenobiotics The liver occupies an anatomical site that facilitates the elimi- nation of drugs and other environmental chemicals. Blood is directly received from the intestinal tract via the portal vein. Thus, orally administered compounds must first pass through the liver before reaching the systemic circulation. Perhaps the first to point out a possible role of bile in the elimination of foreign compounds was M.J.B. Orfila who, in his work Traite d5 toxicologie generale (1813—1815), pointed to the fact that many metallic poisons are taken up by the liver and are either retained there or are excreted into the bile. Subsequently, Claude Bernard found that copper sulfate, potassium iodide, and turpentine spirits, when injected into the blood, rapidly pass into the bile (Smith, 1973). The role of bile, however, as a route for chemical elimination has not, until relatively recently, received much attention. In the past 25 years it has become increasingly apparent that the biliary tract is a major route for excretion of numerous drugs and other foreign compounds. The extent of biliary excretion of a compound is influenced by two physico—chemical factors: 1) molecular weight, and 2) polarity (Smith, 1973). Evidence in support of the view that molecular weight has an important bearing on biliary excretion comes from two sources. First, from a consideration of the molecular weights of both endogenous and exogenous compounds which are excreted into bile, and secondly, from systematic studies on the relationship between molecular weight and the extent of biliary excretion of variOus groups of chemicals. Brauer (1959) suggested that substances which are highly concentrated in bile are usually organic carboxylic acids with molecular weights of 300 or greater. Sperber (1963) made a similar generalization stating that "the l3 majority of the compounds efficiently secreted by the renal tubules have a relatively low molecular weight (200-400); whereas, the substances excreted into the bile usually have larger molecules (molecular weight above 400)". Based on correlations between molecular weight and the extent of biliary excretion, a threshold molecular weight was determineo for organic anion excretion. Compounds with molecular weights below this limit were only minimally (less than 10% of dose) excreted into bile. The molecular weight threshold varies with species and ranges from about 325 in rats to approximately 500 in man (Millburn g£_gi., 1967; Millburn, 1970, 1976; Hirom g£_§l., 1972). The presence of a strongly polar group on a molecule also appears to be a requirement for extensive biliary excretion to occur. Smith (1973) has suggested that the presence of a potentially ionizable moiety, such as a carboxylic acid or quaternary ammonium group, augments biliary excretion. Such groups allow a molecule to exist at physio- logical pH as water-soluble anions or cations. Occasionally, as in the case of the cardiac glycosides which may be highly excreted in bile, there is no charged anionic or cationic center, but this may be com- pensated for by the presence of one or more water-soluble sugar residues in the molecule (Smith, 1973). A number of compounds are eliminated from plasma and excreted into bile in an unchanged form. These include ouabain (Cox and Wright, 1959), indocyanine green (Wheeler g£_§1., 1958) and some azo dyes (Ryans and Wright, 1961). However, many drugs that are excreted into bile are eliminated in the form of metabolites. With respect to biliary excre— tion, the most important metabolic reactions involve conjugation to certain endogenous substrates. In the liver there have been identified 14 eight conjugation mechanisms which utilize glucuronic acid, sulphate, glycine, glutamine, glutathione, methyl, acetyl, or thio groups as conjugating substrates. The most common of these conjugation reactions involve glucuronic acid and glutathione. In essence, conjugation to these substrates may augment biliary excretion for two reasons: 1) they introduce a polar center into the molecule, and 2) they increase the molecular weight of the compound (Smith, 1973). Based on selective competition studies, it has been suggested that the liver has at least three transport systems for the excretion of organic compounds into the bile (Kupferberg and Schanker, 1968). These include an organic anion transporting system, for compounds such as indocyanine green, bilirubin, sulfobromophthalein and glucuronide conjugated compounds; an organic cation system, for which procaine amide ethobromide has become the prototype; and a third transport system for neutral compounds including cardiac glycosides, such as ouabain. An additional anionic transporting system may exist for bile acids (Alpert 25 gl., 1969; Paumgartner and Reichen, 1975,1976). In 1909 Abel and Rowntree published an important paper showing that a number of phthalein dyes undergo extensive biliary excretion. This observation was of considerable significance since it laid the basis for the development of diagnostic agents for the hepatobiliary system. Graham g£_§1, (1925) conceived the idea of using the substance tetra- iodophenolphthalein, which is extensively excreted in bile and opaque to x-rays, for the x-ray visualization of the gall bladder. Equally signi- ficant, however, was the introduction by Rosenthal and White (1925) of Sulfobromophthalein (BSP) for a simple test of liver excretory function. 4A8 used clinically, the hepatic transport of BSP is evaluated by 15 determination of the rate of disappearance of the dye from plasma. Retention of BSP in plasma has been a good indication of various forms of adult and newborn hepatic disease (Leevy 25w213, 1963). In the time since the introduction of BSP for clinical evaluation of liver function, parallel studies have been undertaken to determine mechanisms of hepatic disposition and biliary excretion of BSP and similar prototype compounds. The movement of drugs from blood to bile may be described in terms of four steps: 1) hepatic uptake, 2) intrahepatic storage, 3) conju- gation, and 4) biliary excretion. The uptake of drugs into liver represents the first transport step for biliary excretion. Krebs and Brauer (1949) demonstrated by means of autoradiography that BSP uptake appeared to be a function of hepatic parenchymal cells and not Kupffer cells. This observation has been verified experimentally with cell preparations in which isolated parenchymal but not Kupffer cells accu- mulated BSP from an incubation medium (Stege g£_§l,, 1975). Although little is known about the exact mechanisms by which drugs are accumulated into hepatic parenchymal cells, the process is very rapid. Compounds that are excreted into the bile show a marked tendency to initially accumulate in liver. Within 5 minutes after an intravenous injection of bilirubin in the rat, over 50% of the injected dose was in the liver (Brown g£_§1,, 1964). For this reason it has been assumed that the initial rate of elimination of drugs from plasma represents ‘hepatic uptake (Paumgartner g£_§1,, 1970; Scharschmidt ggfl§1., 1975). 'With this assumption, Paumgartner and co-workers (1970,1976) and others (Scharschmidt g£_§1,, 1975) have characterized hepatic uptake of organic anions by single injection multiple dose techniques. From the plasma lualf-time of the initial exponential line a rate constant, K l6 (functional clearance), was determined such that K = .693/t%. By exa- mining the removal rate (V; V=K times D; where D = the injected dose) as a function of dose (D) the investigators could express uptake in terms of Michaelis-Menten constants (K.In and Vmax) which demonstrates satura— bility. Moreover, hepatic uptake of bilirubin, indocyanine green, and sulfobromophthalein showed selective mutually competitive inhibition, suggesting these anions are accumulated into liver by the same mechanism (Scharschmidt g5fl§1., 1975). These data are therefore compatible with the existence of a carrier mediated transport process for uptake of organic anions. Uptake of bilirubin was not altered in the presence of the bile acid taurocholate (Paumgartner and Reichen, 1976), and it was therefore suggested that bile acids are accumulated in liver by a separate mechanism. Scharschmidt g£_§l, (1975) discussed the limitations of the single injection multiple dose technique and cautioned that several variables may cOmplicate interpretation of values for K.m and Vmax' Particularly, the rate of hepatic blood flow may be rate-limiting with low doses of the drugs and thus K.In values (but not Vmax) would vary in accordance with hepatic perfusion. It is noteworthy that reduction in hepatic blood flow during hypothermia resulted in diminished uptake of BSP in the anesthetized dog (Brokaw and Penrod, 1949). In addition, enhanced hepatic blood flow following treatment with phenobarbital has been interpreted by some (Neis ggugl., 1976; Branch g£_§1,, 1974) to be a Inajor mechanism for enhanced uptake of compounds following phenobarbital jpretreatment. Thus, under non-saturating conditions (below Vmax)’ 'hepatic uptake may be dependent on the intrinsic capacity of the liver 17 to remove compounds from blood, and also on the load presented to the liver (plasma flow times plasma concentration; Keiding, 1976). The hepatic slice technique has also been utilized to characterize the uptake of compounds into hepatic parenchyma. Using this method, Kupferberg and Schanker (1968) concluded that the glycoside, ouabain, is taken up by the liver by an active process and that uptake is in- dependent of the process which transports organic anions and cations. These investigators noted that accumulation of ouabain into rat liver slices was saturable and that the extent of ouabain accumulation into slices was depressed under nitrogen atmosphere or in media containing metabolic inhibitors or other cardiac glycosides. Ouabain accumulation into slices was not inhibited by p-acetylamdnohippuric acid (anion) or the cation procaine amide ethobromide (Kupferberg and Schanker, 1968). However, inhibition of ouabain uptake was observed following addition of several naturally occurring and synthetic steroids and it was suggested that the cyclopentanophenanthrene steroid nucleus of these compounds may be important in transport specificity (Kupferberg, 1969). In similar experiments, Hwang and Schanker (1973) observed saturable uptake of the cation n-acetyl procaine amide ethobromide into rat liver slices which could be inhibited with a series of metabolic inhibitors or by other cations. However, accumulation of BSP into rat liver slices could not be depressed by metabolic inhibitors (Brauer and Pessotti, 1949). Thus, in contrast to transport of organic cations and cardiac glycosides, organic anions may not accumulate into the liver by an active transport system. An alternative mechanism for uptake of anions was suggested by Arias and co-workers (Levi g£_§l,, 1969b). The authors described an 18 important role of the cytoplasmic anion binding protein, ligandin, for hepatic uptake of anions. The purported mechanism.was that ligandin influences net uptake of organic anions into liver specifically by binding to, and thus regulating anion efflux from the cell into plasma (Arias g£_§l,, 1976). Thus, ligandin was proposed to act as intra- cellular receptor for free ions which had crossed the sinusoidal mem- brane. Several lines of evidence support the contention that ligandin may be important for hepatic uptake of organic anions. 13,21££g_compe- tition for binding to ligandin among many organic anions correlates with ighzizg competition for hepatic uptake (Levi g£_§1,, 1969b). In newborn guinea pig, rat, monkey, and man and in teleosts and elasmo- branchs, absence of ligandin in liver supernatant correlates well with impaired hepatic uptake of BSP and bilirubin (Arias, 1970; Levi g£_§l,, 1970; Levine g£_§l,, 1971). Stimulation of hepatic uptake of anions following administration of phenobarbital is associated with increased hepatic content of ligandin (Reyes g£_§l,, 1971). A close relationship between ligandin and glutathione (GSH)-S- transferase activity in liver cytosol has been proposed on the basis of finding an identical elution volume in gel filtration for both GSH—S- transferase activity and BSP binding (Kaplowitz g£_§1,, 1973). The GSH- S-transferases are a major group of soluble liver proteins and six transferases have been separated from rat liver and are designated E, D, C, B, A and AA in order of their elution from carboxymethyl cellulose columns (Habig g£_gi,, 1974,1976). The GSH—S-transferases are known to have specific enzymatic functions in catalyzing glutathione conjugation with certain xenobiotics including BSP (Habig g£H§1., 1974; Kaplowitz g; 31., 1975; Smith g£_§l,, 1977). GSH-S-transferase B has been shown to 19 be immunologically identical to ligandin (Habig g£H§1., 1974). Thus, the GSH—S-transferase system may have a dual purpose for hepatic drug excretion; by both mediating organic anion uptake and providing a catalytic site for glutathione conjugation (Kaplowtiz ggugi., 1975). The importance of transferase-mediated glutathione conjugation for excretion of drugs into bile has been studied almost exclusively for hepatic elimination of 38?. Glutathione conjugation to BSP was first suggested in 1959 when Combes reported the existence of metabolites of BSP in rat bile. Before this time it was generally believed that biliary excretion of BSP depended only upon hepatic uptake into liver cells and transport from liver into bile. In rat bile, about 70—85 percent of BSP appears in conjugated form (Combes, 1959). By comparing the biliary excretion rate of BSP and conjugated BSP (BSP-SSH), it has become apparent that BSP-GSH is excreted from liver into bile more rapidly than is BSP (Whelan g£_§1,, 1970b). This observation has been subjected to a number of interpretations as to the relative importance of intrahepatic conjugation for BSP excretion. Whelan g£_§1, (1970b) have suggested that the conjugation of BSP facilitates dye transport into bile and moreover, ig.zizg_may be the rate-limiting step in overall transport of injected free BSP. Klaassen and Plaa (1967), however, compared the maximal biliary excretion rate (Tm) for BSP in rat, rabbit and dog, and determined that for each of these species, the theoretical iggyiggg conjugation capacity greatly exceeded the observed ig_yi!g' excretory rate. Thus, biliary excretion was suggested to be rate— limiting (Klaassen and Plaa, 1967). By plotting hepatic concentration of BSP and BSP-GSH against drug excretion rate, Varga g£_§l, (1974) determined that BSP-GSH has a 10—13 fold greater affinity for the 20 biliary transport system than does BSP. These studies suggest that conjugation of BSP to glutathione, although perhaps not rate—limiting, converts BSP to a compound that may be more readily excreted into bile. Although it is not clear whether conjugation is rate-limiting in BSP elimination into bile, the rate of hepatic uptake of BSP-GSH is slower than that for unconjugated BSP (Krebs, 1959; Melter g£_§l,, 1959; Whelan g£“§1., 1970a,b). Since BSP—GSH is excreted into bile more rapidly than is unconjugated BSP, it is likely that hepatic uptake is not rate limiting in overall excretion. The time lag between hepatic uptake and biliary excretion of BSP suggested that the dye was stored within the hepatocyte prior to excre- tion (Wirts and Cantarow, 1942). Following the intravenous admini- stration of BSP to dogs it was shown by Wirts and Cantarow (1942) that the output of BSP in bile continued for over three hours after its virtual disappearance from plasma, indicating that BSP is first rapidly taken up by the liver and then gradually excreted into bile. Wheeler g£_ El. (1960) quantified this phenomenon as the difference between the amount of BSP removed from plasma and the amount appearing in the bile and was termed "the relative storage capacity". The existence of hepatic storage of BSP may be most easily interpreted as a manifestation of the difference between hepatic uptake and biliary excretion of the dye. Thus, rate of uptake of BSP from plasma may be very rapid and storage of the dye within the liver would reflect a slower rate of biliary excretion. The final step for drug elimination into bile is transport from the liver cell into the bile canaliculus. It is generally assumed that 21 substances excreted into bile are actively transported (Schanker, 1968). The evidence for this is indirect and not entirely rigorous. The principle findings suggestive of active secretion are concentration, saturation, and competition. Drugs may be concentrated into bile several hundred, and as much as one thousand, fold above plama levels (Brauer, 1959). Saturation of biliary excretory capacity has been shown for several compounds (Schanker and Solomon, 1963; Wheeler, 1969). The term used to designate the maximum velocity of biliary secretion is the transport maximum (Tm). Finally, specific competitive inhibitors may depress biliary excretion (Wheeler $5.31., 1960; Schanker and Solomon, 1963; Wheeler, 1969). Since hepatic uptake mechanisms are also concentrative, saturable, and specific, it may be suggested that carrier mediated drug transport from liver into bile is a manifestation of carrier mediated drug accumu- lation into liver. However, concentration of drugs in bile may exceed concentration in liver (Kupferberg and Schanker, 1968; Wheeler, 1969; Russell and Klaassen, 1972). In addition, when maximal uptake velo- cities are compared to the steady state excretory transport maximum (Tm), uptake capacity (Vmax) exceeds excretory Tm by a factor as high as 60-fold (Goresky, 1964; Paumgartner, 1974; Paumgartner, 1975; Schar- schmidt ggfl§1., 1975; Paumgartner and Reichen, 1976). Thus, the excretory mechanism may be saturated before saturation of hepatic uptake which suggests carrier mediated uptake and secretion. These data may also demonstrate that uptake is not rate-limiting in overall drug transport from blood into bile. I IIFI I I I 31531.3 ell. 22 The Importance of Bile Flow in Hepatic Drungxcretion The formation of primary bile occurs at bile canaliculi, minute (1 pm in diameter) channels located between 2, or sometimes 3 hepato- cytes. The canaliculi are closed at one end and are connected at the other end to bile ductules which in turn are connected to bile ducts (Popper and Schaffner, 1957; Steiner and Corruthers, 1961). Sperber (1959) suggested that secretion of bile acids intobiliary canaliculi provides an osmotic driving force for water and electrolytes and thereby initiates bile flow. This view is supported by the fact that the choleretic potency of bile acids is roughly proportional to their osmotic activity, and that other osmotically active compounds demonstrate a choleretic effect (Preisig 35 gl,, 1962). There is increasing evidence, however, that excretion of bile acids may not be the only factor responsible for the output of canalicular bile. In the isolated perfused rat liver, bile flow persists when bile acid excretion is minimal or absent (Boyer, 1971; Boyer and Klatskin, 1970). After interruption of the enterohepatic circulation and depletion of the bile acid pool in the rat, bile flow decreased less than did bile acid excretion (Klaassen, 1971a). In studies of the correlation between bile flow and bile acid excretion, a positive intercept appears when bile acid excretion is extrapolated to zero (Erlinger E£;§lx, 1970; Boyer and Klatskin, 1970). Thus, a bile acid independent fraction of canalicular bile flow was postulated. A number of studies have suggested that active sodium transport, Possibly mediated by a canalicular membrane Na-KeATPase, into bile may be the mechanism for the bile-acid independent fraction of canalicular bile flow (Erlinger g£_§l,, 1970; Boyer and Klatskin, 1970; Boyer, 23 1971; Boyer 25.21:: 1976; Layden and Boyer, 1976). The evidence in favor of this hypothesis is, for the most part, indirect, since primary bile (canalicular) cannot, at present, be collected. None— theless, Boyer and co-workers (1970,1976), demonstrated a positive correlation between the activity of plasma membrane Na-KrATPase and canalicular bile acid independent bile flow. Included in these investi— gations was demonstration of bile flow inhibition in the isolated perfused liver preparation following ouabain induced inhibition of Na- KrATPase (Boyer, 1971; Boyer 25.31,, 1976). However, low concentra- tions of ouabain may increase bile flow in the isolated perfused liver preparation (Graf g£_§l,, 1973; Graf and Peterlik, 1976). Graf §£_§1, (1973) have suggested that the Na-KrATPase-dependent mechanism regu- lating bile flow might be located in the sinusoidal side of the liver cell. Bile flow, thus, would be regulated by the effects of the sodium pump on intracellular Na. Bile salt dependent and independent components of canalicular bile flow are not necessarily mutually exclusive. For example, bile salts and certain nonionic detergents activate Na-KrATPase lg giggg (Emmelot g£_§1,, 1966). It has been suggested that bile acids secreted across the canalicular membrane could cooperatively stimulate the secretion of Na+ by a Na—KrATPase pump (Plaa and Priestly, 1977). Following formation of "primary" bile at the canaliculus, secre- tory and reabsorptive mechanisms may result in modifications of bile during passage through the biliary tract. The choleresis produced by secretin is independent of bile acid secretion (Preisig g£_§l., 1962) and of total canalicular bile formation (Wheeler g£_§1,, 1968). 24 Secretin—induced choleresis involves the net addition to bile of a solution rich in bicarbonate and chloride, and the site for this inorganic ion secretion appears to be the lower part of the biliary tree (Wheeler and Mancusi-Ungaro, 1966). Early studies of O'Maille §£_§l. (1966) and of Ritt and Combes (1967) demonstrated that BSP transport maximum (Tm) could be enhanced significantly in dogs by infusion of sodium taurocholate and dehydro- cholate. The effect of these bile salts (acids) on BSP excretion was associated with the increase in bile flow that results from bile acid infusion. Thus, the increase in BSP excretion was attributed to the increase in canalicular bile flow, which, by diluting BSP in canali— cular bile, permitted the excretion of additional dye without exceeding a putative concentration maximum. Increased biliary excretion of pentobarbital metabolites (Knodell and Hollowing, 1976), digitoxin (Greenberger and Thomas, 1973), propylthiouracil (Papapetrores g£_§1,, 1972), and cholecystography contrast agents (Dunn and Beck, 1972) has also been observed following infusion of bile acids. These effects are similar to the correlation between bile flow and BSP elimination that results from treatment with microsomal enzyme stimulators. Microsomal enzyme stimulators such as phenobarbital, pregnenolone—l6a-carbonitrile and spironolactone increase bile flow and drug excretion into bile (Klaassen, 1974a; Zsigmond and Solymoss, 1972); whereas other microsomal enzyme stimulators, such as 3-methyl- Icholanthrene and 3,4-benzypyrene, which do not increase bile flow also do not enhance BSP excretion (Klaassen, 1970). The increase in bile flow following phenobarbital was not due to an increase in bile salt excretion and thus was attributed to enhanced 25 formation of the bile acid-independent fraction of canalicular bile production (Berthelot §£_§l,, 1970; Paumgartner §£_§T,, 1971). Since enhanced drug excretion associated with bile salt infusion would increase bile flow by increasing the bile salt-dependent fraction of canalicular bile, it appears that bile flow pg£_§§_might be a deter— mining factor in biliary excretion of drugs. In support of this contention is the relationship between body temperature, bile flow, and drug excretion. During anesthesia induced alteration of rectal body temperature, a linear relationship was found to exist between body temperature and the rate of excretion of BSP and bilirubin (Roberts g£_§1,, 1967). The maximum biliary excretion rate (Tm) of bilirubin and BSP was 1.5-2.0 times greater in rats whose body tem- peratures were 39°C compared to rats with a 31°C body temperature. Inasmuch as bile flow rates diminished with decreasing body tempera- ture, hypothermia—induced alteration in drug transport was considered to be secondary to altered bile flow (Roberts g£_§l,, 1967). Therefore, these results would be consistent with the hypothesis that bile flow pg£_§g_regulates biliary excretory capacity. This hypothesis was questioned recently when canalicular bile flow, increased acutely with drugs such as theophylline, 4-methyl- umbelliferone and 8-(2,4-dimethoxy-S-cyclohexylbenzoyl)propionic acid (SC-2644), was not associated with enhanced biliary excretion of BSP in the dog (Erlinger and Dumont, 1973; Barnhart g£_§13, 1973; Forker rand Gibson, 1973; Gibson and Forker, 1974; Barnhart and Combes, 1974). It has been suggested that bile flux proper (i.e., water flow) might Inot be the determining factor in excretion of compounds across the 26 canalicular membrane. As an alternative, the Suggestion was made that bile acids exert specific, as yet undefined, effects on the BSP excre- tory mechanism, and that the increase in BSP excretion during bile acid infusion is not the result of the increase in canalicular bile production (Forker and Gibson, 1973; Gibson and Forker, 1974). However, this mechanism would not explain the effects of phenobarbital on BSP excretion since phenobarbital does not enhance bile-salt dependent bile flow (Klaassen, 1971b). The relationship between hepatic drug excretion and bile flow may be more complicated than was previously considered. The importance of bile flow in the elimination of compounds from blood is most easily recognized with bile duct ligation. Acute extra- hepatic bile duct ligation is a commonly employed procedure used to ascertain the relative importance of the biliary route for the elimina— tion of xenobiotics from the body. Moreover, bile duct ligation resulted in enhanced susceptibility to drug induced lethality for particular compounds that are normally preferentially excreted by the liver (Gibson and Becker, 1967; Klaassen, 1973a). Studies undertaken to exclusively quantify drug-induced or age related differences in the uptake of drugs into liver employ the bile duct ligation procedure in order to minimize biliary excretion (Whelan g£_§l., 1970a; Reyes £5 _§l., 1971). Thus, it has been assumed that bile duct ligation would specifically diminish bile flow and drug transport from liver into bile. However, bile duct ligation has recently been demonstrated to influence hepatic uptake of drugs in rats (Yam g£_§1., 1977). Extra- liepatic cholestasis produced by acute bile duct ligation decreased net hepatic uptake of BSP, BSP—GSH, phenol-3,6—dibromophthalein, and 27 ouabain (Yam.g£_§l,, 1977). The effect of bile duct ligation on hepatic uptake of BSP was apparent when determined as early as 2 hours following bile duct ligation. Alteration of uptake capacity following bile duct ligation was returned to normal function after recannulation of the bile duct and subsequent release of bile, suggesting the effect was readily reversible. These results suggest that the mechanism for impaired uptake was not liver damage but rather competition between endogenous bile constituents and transported test drug (Yam g£H§£., 1977). Examples of Agents that Alter Hepatic Excretory Function Microsomal Enzyme Stimulators The liver can respond to increases in functional demand by changes in size, involving cellular hypertrophy and hyperplasia, and by quanti- tative and qualitative changes in cell organelles (Feinman ggugl., 1972). Perhaps the type of response that has received the most attention recently is stimulation of the activity of xenobiotic meta- bolizing enzymes following exposure to many drugs and environmental chemicals (Conney, 1967; Conney g£_§1,, 1967; Atio, 1973; Parke, 1975). The stimulation produced by these chemicals varies in accor- dance with the particular stimulating agent. Gillette §£_§1, (1972) suggested that stimulators of microsomal drug metabolism be classified according to their effect on various components of the system. Pheno— ‘barbital and 3—methylcholanthrene represent two distinct types of inducing agent and these compounds have been utilized as prototypes for characterizing alterations in microsomal drug metabolism. Pheno- liarbital-like stimulating agents increase cytochrome P450, NADPH- <:ytochrome c-reductase concentration, and a wide range of microsomal 28 enzymes; whereas 3-methy1cholanthrene—like agents increase cytochrome P1450 and a more specific group of enzymes but not the reductase. Since the excretion of many compounds into bile is dependent upon intrahepatic metabolism, expOSure to hepatic enzyme stimulators often results in enhanced drug excretion into bile. The increase in drug elimination may therefore be a function of the enhanced rate of meta- bolism. However, many hepatic microsomal enzyme stimulators increase biliary excretion of compounds that do not require biotransformation prior to hepatic excretion. Thus, induction of hepatic excretory capacity may not be merely a reflection of increased biotransformation (Klaassen, 1970). Phenobarbital accelerates the elimination of a variety of com- pounds from plasma into bile (Klaassen, 1970). Enhanced biliary excretion is not dependent on increased biotransformation as it occurs with indocyanine green, phenol-3,6-dibromphthalein disulfonate, ama- ranth and ouabain, compounds which are not metabolized before biliary excretion (Klaassen, 1970). One of the mechanisms by which phenobarbital may enhance excretory function is to stimulate the uptake step for the transfer of compounds from plasma into liver. Acceleration of uptake of BSP from plasma into hepatic storage has been demonstrated following phenobarbital in the rat (Reyes 23 al., 1971), mouse (Fujimoto §£_al,, 1965), and man (Capron g£_gl,, 1975). The reports that an increase in hepatic content of the organic binding protein, ligandin, also occurred following phenobarbital treatment suggested that increased ligandin content in liver might be the mechanism for stimulation of hepatic uptake of BSP (Reyes g£>§13, 1971). In support of this contention was the observation 29 that microsomal enzyme stimulators, 3-methylcholanthrene and 3,4— benzpyrene, increase both ligandin content and BSP uptake in rats (Reyes g£_§1., 1971). Klaassen (1975), however, suggested that in- creases in ligandin content might not be the only mechanism for stimu— lation of hepatic uptake. Increased hepatic uptake of the neutral compound, ouabain, was observed in young rats following treatment with phenobarbital, spironolactone, and pregnenolone-l6a-carbonitrile, however, ligandin does not bind to ouabain and thus stimulation of ouabain uptake could not be attributed to increased ligandin (Klaassen, 1975). Moreover, no direct relationship existed between the ability of these inducers to increase ligandin content and BSP excretion even though BSP avidly binds to ligandin (Klaassen, 1975). Phenobar- bital increases liver blood flow in the rat (Ohnhaus §£_§l,, 1971; Ohnhaus and Locher, 1975; Neis g£_§l,, 1976) and rhesus monkey (Branch g£_§l,, 1976) and it has been suggested that the increase in blood flow alone may contribute significantly to stimulation of uptake capacity (Branch g£_§1,, 1974). The increase in liver blood flow may be attributed to the large increase in liver mass produced by treat- ment with phenobarbital (Branch g£_§l,, 1974; Neis 35, gl,, 1976). Therefore, the mechanism(s) for the stimulation of uptake following 'many microsomal enzyme stimulators is not known but may be attributed in part to changes in hepatic blood flow or, for some compounds, increases in ligandin content (Klaassen, 1975). Another mechanism by which microsomal enzyme stimulators may enhance hepatic drug transport is to increase the rate of canalicular lxile flow. Phenobarbital, when administered for 3—15 days, increases lxile flow'in the rat (Roberts and Plaa, 1967; Klaassen and Plaa, 1968a; 30 Hart g£'§1,, 1969) and rhesus monkey (Redinger and Small, 1973). Since treatment with phenobarbital does not result in increased bile salt excretion (Berthelot $5.21,, 1970; Klaassen, 1971b; Paumgartner g£_§1., 1971), the increase in canalicular bile flow may be attributed to stimulation of the canalicular bile salt-independent fraction of bile flow. Several lines of evidence suggest that the effect of microsomal enzyme stimulators on bile flow is not directly related to their influence on the hepatic drug metabolizing system. Among several compounds that stimulate hepatic mixed function oxidase acti- vity and increase liver mass, only phenobarbital significantly in- creased bile flow (Klaassen, 1969). In the hamster, phenobarbital increased liver weight, hepatic cytochrome P450 content, and produced a proliferation of the smooth endoplasmic reticulum, but did not alter bile flow (Capron 25H31., 1974). In the rat, pentobarbital increased bile flow but did not increase liver weight or cytochrome P450 (Capron, 1974). Although no relationship exists between microsomal enzyme stimu- lation and bile flow, a direct correlation can be made between the ability of enzyme inducers to increase bile flow and enhance drug elimination into bile (Klaassen, 1970,1975; Zsigmond and Solymoss, 1972). Treatment with 3-methylcholanthrene and 3,4-benzpyrene does not result in increased bile flow or enhanced excretion of BSP into 'bile (Klaassen, 1969,1970). However, phenobarbital, spironolactone, land pregnenolone-l6a-carbonitrile increase bile flow and BSP excretion, and, moreover, a positive correlation exists between the ability of ‘theee agents to increase drug excretion and bile flow (Zsigmond and SOIJmmSS, 1972; Klaassen, l969,l970,1974a). .Pregnenolone-l6a- currbonitrile was most effective in increasing both bile flow and 31 excretion of ouabain and BSP into bile (Zsigmond and Solymoss, 1972; Klaassen, 1974a). It is noteworthy that some compounds, such as 3—methy1cholanthrene and 3,4-benzpyrene, enhance hepatic uptake of BSP but do not increase bile flow (Klaassen, 1969; Reyes g£_§l,, 1971). Therefore, phenobar- bital may enhance all components of the hepatic excretory system (hepatic uptake, metabolism, and biliary excretion), but the transport steps may be mutually exclusive and selective stimulation is possible. However, hepatic uptake is not rate-limiting in biliary excretion of drugs and thus selective stimulation in uptake capacity may not be important for enhanced overall excretion following hepatic stimulation. Thus, even though 3—methylcholanthrene and 3,4—benzpyrene enhance hepatic uptake of BSP (Reyes g£“§1., 1971), overall hepatic excretion remains unchanged (Klaassen, 1970). Carbon Tetrachloride Carbon tetrachloride (0014) is an agent widely used to produce experimental liver damage in laboratory animals (Recknagel, 1967). The hepatic damage produced by this compound is not specific and all elements of the hepatocyte are disrupted following CCla, including the endoplasmic reticulum, mitochondria, lysosomes, and plasma membranes. The toxic lesions are thought to be mediated by a metabolite of CCl4 and not CCl4 itself (Slater, 1966). This has been suggested since 1) newborn animals possessing low capacity for drug metabolism are relatively iresistant to CCl4 toxicity (Dawkins, 1963) and 2) hepatotoxicity following 0014 may be enhanced following microsomal enzyme stimulation 'by pretreatment with phenobarbital and DDT (McLean and McLean, 1966). In 1966, Recknagel, Ghoshal and Slater proposed that homolytic cleavage 32 of the carbon-chloride bound of CCl4 resulted in production of free radicals that interact with and disrupt membrane lipids. It was therefore suggested that the mechanism for CCl toxicity was lipid A peroxidation (Slater, 1966; Recknagel and Ghoshal, 1966). In support of this hypothesis were the earlier observations of the protective effect of Vitamin E, dipheny1-p-phenylenediamine (DPPD), and selenium on CCl4 toxicity (Hove, 1948; Gallagher, 1962; DiLuzio and Costales, 1965). These substances act as lipid antioxidants and thus the pro— tection afforded by these compounds against CCl4 toxicity suggested destructive lipid peroxidation was involved in the toxic response. Further support of this hypothesis may be apparent in the functional changes resulting from exposure to CC14. CCl4 affects cellular mem- branes containing lipid-rich material and thereby causes alterations in cellular and subcellular structure and function. Some of the subcellular and biochemical effects of CCl4 on the hepatic parenchyma cell hint at a possible influence on drug transport. A single dose of CCl results in altered permeability of 4 membranes of the endoplasmic reticulum followed by an increased per— meability of the mitochondrial and cellular membranes (Rouiller, 1964; Villela, 1964; Zimmerman, 1968). CCl4-induced uncoupling of oxidative phosphorylation that occurs in mitochondria of the liver (Dianzani, 1954; Dianzoni and Bahr, 1954) results in a depletion of cellular ATP levels (Dianzani, 1976). Depletion in energy supply may result in a decrease in available energy for sustaining active drug transport (Reuning and Schanker, 1971). Another mechanism by which 001 might 4 influence hepatic transport may be through the decrease in hepatic blood flow that has been reported following CCl4 treatment (Rice 35 _§l,, 1967). 33 Hepatic excretory function is depressed following CCl4 poisoning as indicated by retention in plasma of drugs administered secondarily. The effect of CCl4 on hepatic drug transport, however, is not speci- fic. Altered transport of both anions (Brauer and Pessoti, 1949; Plaa and Hine, 1960; Klaassen and Plaa, 1968b; Paumgartner SE 31,, 1970) and neutral compounds (Reuning and Schanker, 1971) by the liver has been demonstrated following 0014 although these compounds are trans- ported by separate mechanisms (Schanker, 1968). Liver slices and isolated perfused livers from CClatreated adult rats showed no diffe— rences in BSP uptake but biliary excretion of BSP was decreased following CCl4 (Brauer and Pessotti, 1949). Retention of BSP in plasma following CCl4 was primarily due to decreased biliary excretion associated with decreased bile flow (Klaassen and Plaa, 1968b). Ouabain (neutral compound) retention in plasma was accompanied by accumulation of the drug in the liver (Reuning and Schanker, 1971). These results may be consistent in that CCl4 appeared to have a specific effect on the transport of drugs from liver to bile and did not appear to alter the uptake of drugs from plasma into liver. However, CCl4 treatment may result in a reduction in BSP uptake in isolated perfused rat livers when measured soon after poisoning (Plaa and Hine, 1960). Furthermore, a decrease in the maximal removal rate of indocyanine green (ICG) from plasma has been observed following CCl4 where hepatic 'uptake was exclusively measured (Paumgartner g£_§1,, 1970). C014 has also been demonstrated to reduce conjugation of BSP to glutathione 33 §§££Eg_(Klaassen and Plaa, 1968b). Thus, treatment with C014 may Iresult in alteration of all steps in hepatic drug transport. Since CC].4 poisoning results in hepatic cell death (Recknagel, 1967), it may 34 be expected that the effect of CCl4 on drug transport would be non— specific. Polybrominated Biphenyls Polybrominated biphenyls (PBBs) are used commercially as flame retardants. The adverse consequences of exposure to PBBs are not completely known. However, a similar class of compounds, the polychlori- nated biphenyls (PCBs), has been extensively studied and PBBs may share many of the biological and toxic properties of PCBS. PCBs are known to cause chloracne (Meigs g£_gl., 1954) and Yusho disease in humans (Karatsune SE gl., 1972). Rodents exposed to PCBS exhibit decreased reproductive function (Kihlstrom g£_§l., 1975), hepatic and renal histopathological changes (Bruckner EEHii-’ 1973), hepatic porphyria, and stimulation of hepatic microsomal enzymes (Vainio, 1974; Goldstein §£.§l:! 1975). Because PCBs accumulate and persist in the environment (Risebrough g£_§13, 1968; Koeman $5.31., 1969) and are distributed on a world wide scale (Risebrough g£_§l., 1968; Hutzinger 33 gl., 1974; Report, 1976), they are considered to be an environ- mental hazard. The production, distribution, and usage of PBBs has not been as widespread as PCBs (Report, 1976) and perhaps for this reason the toxicity of PBBs has not been extensively studied. However, recent interest in the toxicity of PBBs was generated following recognition of accidental contamination of a commercial animal feed supplement with P333. In 1973, 500-1000 pounds of the flame retardant Firemaster BP-6 was accidentally mixed into feed that was widely sold and distri— buted to Michigan farms (Carter, 1976). Firemaster BP—6 is a mixture of IXBBS containing approximately 70% hexabrominated biphenyl (Jacobs 35 g£_§l,, 1976; Rickert gE_§1., 1977). Signs of toxicity in cattle receiving feed containing PBBs (at levels as high as 3000 ppm) in- cluded anorexia, decreased milk production, abnormal hoof growth, decreased growth rate in young animals, and aborted and malformed calves (Report, 1976). The contamination of PBBs to Michigan farm animals eventually lead to the destruction of 30,000 cattle, over 1,000 sheep and pigs, and about 1.5 million chickens (Carter, 1976). It has been estimated that between the onset of contamination in the fall of 1973 and the establishment of quarantine of contaminated livestock in the spring of 1974, over 10,000 Michigan residents were exposed to PBBs through consumption of contaminated milk and meat (Report, 1976). Human exposure has not been associated with any acute ill effects (Report, 1976; Kay, 1977). An important feature of the pharmacodynamics of halogenated biphenyls is the biological persistance of these compounds (Hutzinger g£_§1,, 1972). This may be especially true for biphenyls having six or more halogen moieties. Cumulative seven day excretion of hexachlo- rinated biphenyl was less than 20% of the injected dose (Matthews and Anderson, 1975). Studies with decabromobiphenyl in rats and Firemaster BP—6 (containing mostly hexabrominated biphenyl) in farm animals suggest that brominated biphenyls are also slowly eliminated from the body (Fries and Marrow, 1975; Lee g£_§1,, 1975; Gutenmann and Lisk, 1975; Willett and Irving, 1976). Because of their high solubility in fat and low solubility in water, polychlorinated biphenyls (Matthews and Anderson, 1975) and polybrominated biphenyls (Fries and Marrow, 1975; Lee g£_§1,, 1975; Rickert §£H§1., 1977) may accumulate and be Stored in fat. In particular, halogenated biphenyls accumulate in 36 mammary tissue and are present in milk fat (Fries and Marrow, 1975; Willett and Irving, 1975; Takagi g£_§l,, 1976; Rickert g£H§1., 1977). Human exposure to PBBs has been associated with detectable levels of PBBs in human breast milk (Report, 1976). In laboratory animals, one of the most prominent effects of both P333 and PCBs is induction of a large increase in liver weight and, in addition, stimulation of hepatic drug metabolizing capabilities (Johnstone gghgl., 1975; Goldstein 23 gl,, 1975; Dent g£_§1,, l976a,b). PBBs are several times more potent than PCBs in stimulating microsomal enzyme activity (Farber and Baker, 1974). PBBs and PCBs represent a class of hepatic mixed function oxidase stimulators which exhibit characteristics of both phenobarbital and 3—methylcholanthrene (Al- varez ggngl., 1973; Stonard, 1975; Dent g£_§1,, l976a), two agents which are distinct in their stimulating properties (Sladek and Manner— ing, 1969a,b). Following a single intraperitoneal injection of PBBs to adult rats, the pattern of hepatic microsomal enzyme stimulation changes from phenobarbital—like initially to 3-methy1cholanthrene-like at later times after administration (Dent g£_§1,, 1976b). However, the same treatment of PBBs to young rats resulted in a different pattern of stimulation and appeared to more closely resemble 3-methyl- cholanthrene stimulation initially with a phenobarbital-like effect occurring at later times (McCormack g£_§l,, 1977). Following dietary Exposure of pregnant and lactating rats to PBBs, hepatic and extra- hepatic mixed function oxidase activity was stimulated in 15 day old Offspring when neonates were exposed transplacentally and/or via the mothers milk (Dent g£_§1,, 1977). In summary, these studies demon- Strate a number of important properties of PBBs as hepatic microsomal 37 enzyme stimulating agents: 1) the pattern of stimulation may change with time following exposure, 2) the characteristics of this pattern may be age dependent, and 3) stimulation may occur in developing rodents following pre- and postnatal exposure. Egperimental Rationale Excretion of drugs from blood into bile requires hepatic uptake, intrahepatic metabolism, and biliary excretion. The capacity for drug elimination into bile in young rats is low when compared to adults and functional immaturity may be attributed, in part, to a deficiency in hepatic uptake (Klaassen, 1972,1973b). However, in adult rats, hepatic uptake is rapid and is not rate-limiting for overall drug transport. Therefore, if the maturation of hepatic excretory function is charac— terized by an increase in hepatic uptake capacity, uptake may be the rate—limiting step for drug transport in young animals, and with age, the rate-limiting step for drug elimination may change. In adult animals, overall transport function may be altered follow- ing changes in the rates of l) bile flow; 2) hepatic blood flow; and 3) drug biotransformation. In addition, hepatic drug elimination may be influenced by 4) hepatic content of ligandin, and 5) the presence of endogenous and specific exogenous competitive inhibitors. The first objective of this investigation was to characterize the maturation of hepatic excretory function and to determine the relative importance of hepatic uptake for overall drug transport in developing rats. This was accomplished by comparing hepatic function in developing and adult rats in the control state, as well as in experi- mentally—induced situations that are known to influence drug transport in the adult . 38 This investigation was also concerned with chemical-induced alterations in hepatic function. The second objective of this re— search was to determine the effect of carbon tetrachloride and poly- brominated biphenyls on hepatic excretory function in both developing and adult rats. MATERIALS AND METHODS Animals All animals used were Sprague-Dawley rats purchased from Spartan Research Animals, Inc., Haslett, Michigan. Animals were maintained in clear solid bottomed polypropylene cages at 22°C with a 12 hour light cycle and were allowed free access to food (Wayne Lab Blocks; Anderson Mills, Maumee, Ohio) and water. Timed pregnant or lactating rats with litters of 8-10 offspring were received at least one day prior to experimentation. Adult rats (200-250 gm) were also used. Hepatic Transport of Ouabain and Sulfobromophthalein in Developing Rats Disposition of Ouabain Rats of 15, 21, 25, 35, and 45 days of age were lightly anesthe— tized with ether and injected with [3H]-ouabain (1 mg/kg) via the tail vein. The injection volume was 2.5 ml per kg body weight. Specific activity of ouabain was 116 uCi/mg and was prepared by mixing non- radioactive ouabain (Sigma Chemical Co., St. Louis, Mo.) with randomly labelled (3H)-ouabain (New England Nuclear, Boston, Mass.) in normal Saline. Following ouabain injection, rats were placed under a heat lamp to maintain normal body temperature. Three, 20, and 40 minutes following administration of ouabain, rats were anesthetized with ether, a blood sample was taken (with a heparinized syringe) by 39 40 cardiac puncture and the entire liver and small intestine were rapidly removed. The liver was blotted, weighed, coarsely chopped with a scissors and duplicate samples (100 mg) were placed in scin— tillation vials. Blood was centrifuged at 2500 rpm to obtain plasma and plasma samples (100-200 ul) were placed in scintillation vials. The quantity of radioactivity in the intestine was measured to estimate biliary excretion of ouabain (Klaassen, 1974b). The entire small intestine was homogenized in 5—10 ml distilled water with a PolytronR homogenizer (Brinkman Instruments, Westbury, N.Y.) and aliquots (200 pl) of whole homogenate were placed in scintillation vials. Samples of liver, plasma, and intestine were solubilized at 80°C in 1 ml of a mixture of water, methanol, and Triton X-405 (6:3:1 con- taining 2 moles of NaOH/liter) for 2-3 hours. When solubilization was complete, the samples were acidified by the addition of 0.5 ml 4.4 M HNO3 and counted in 12 ml of toluene—Triton X—lOO (2:1) scintillation cocktail [which contained 2.5 gm PPO; 2,5-diphenyloxazole, and 100 mg dimethyl POPOP; 1,4-bis(2—(4—methyl-5-phenyloxazolyl)benzene; per liter] (Dent and Johnson, 1974). Radioactivity in all samples was determined with a Packard Model 3380 liquid scintillation spectrometer equipped with automatic external standard for quench correction (Packard Instrument Company, Downers Grove, 111.). Radioactivity in blanks for plasma liver, and intestine was negligible. Disposition of Sulfobromophthalein Eighteen day old and adult rats were lightly anesthetized with ether and injected with 120 mg/kg sulfobromophthalein (Hynson, Westcott, and Dunning, Inc., Baltimore, Md.) via the tail vein. Sulfobromo- Phthalein (BSP) solutions were diluted in normal saline for injection 41 volumes of 10 m1/kg (18 day old animals) or 4 ml/kg (adult rats). Following injection of BSP, rats were placed under a heat lamp (for maintenance of normal body temperature). Three, 20, and 40 minutes following BSP injection, blood samples were taken by cardiac puncture with a heparainized syringe and the liver was rapidly removed. Con— centration of BSP in plasma was determined by diluting plasma samples (20—100 ul) with water and 0.1 N NaOH and measuring absorbance at 580 mu in a Beckman dual beam spectrophotometer (Beckman, Instruments, Fullerton, Calif.). Hepatic concentrations of BSP were determined by the method of Whelan g£_§l, (1970b). Liver was blotted, weighed, and coarsely chopped with a scissors. Duplicate 1 gm samples were homo- genized with 1 ml of distilled water in a Polytron homogenizer. BSP was extracted from liver by addition of 5 ml anhydrous acetone and centrifuged at 2500 rpm in an International Centrifuge, model PR—2 (International Equipment Co., Needham, Hts., Mass.). The resulting supernatant was diluted with 0.1 N NaOH and optical density recorded at 580 mu and at 620 mu in a Beckman Dual Beam Spectrophotometer. Readings were carried out at 620 mu and subtracted from values at 580 mu in liver extracts to correct for tissue turbidity. The ex— traction procedure was performed twice and hepatic BSP concentration for each sample was calculated by the sum of BSP levels in both extracts. Final calculations for BSP in plasma and liver were made from standard curves containing BSP in plasma and liver blanks. In Vitro Conjugation of BSP to Glutathione in Developing Rats Liver BSP conjugating activity was assayed in rats 8 to 70 days Of age. Animals were decapitated and the livers were excised, blotted, and coarsely chopped into ice—cold 1.15% KCl. Soluble liver fractions 42 containing this enzyme (Goldstein and Combes, 1966) were prepared by homogenization (Polytron) of liver in 4 volumes of 1.15% KCl buffered to 7.4 with 20 mM Tris-H01. The homogenate was centrifuged at 10,000 x g in a Sorvall model RCZ—B centrifuge (Ivan Sorvall Inc., Newtown, Conn.) for 20 minutes and the supernatant was then recentri- fuged for 60 minutes at 105,000 x g in a Beckman L3—50 ultracentrifuge. The supernatant was assayed for BSP conjugating activity by the method of Goldstein and Combes (1966) as described by Klaassen and Plaa (1967). Varying amounts of soluble supernatant were incubated at 37°C for 5 minutes in a Dubnoff incubator with 20.5 mM reduced glutathione (Sigma Chemical Co., St. Louis, Mo.), 227 uM BSP, and sufficient volume of 0.1 M sodium pyrophosphate buffer, pH 8.2, to bring the total incubation volume to 4.4 ml. The reaction was started by addi- tion of BSP. BSP conjugating activity was measured by recording 5 minute change in optical density (O.D.) at 330 mu from the time of addition of BSP. Increases in O.D. at this wavelength reflect produc— tion of conjugated BSP since BSP~GSH but not unconjugated BSP absorbs light at this wavelength (Goldstein and Combes, 1966). The conjuga— tion of BSP to glutathione also occurs non—enzymatically and thus non- enzymatic activity (changes in O.D. measured in incubations containing substrates but not liver homogenate) were subtracted from values for the total reaction to obtain net enzymatic activity. Reactions were protein dependent for the 5 minute incubation period. Activity of the enzyme was expressed as nmoles BSP conjugate produced/min/gm liver and was calculated by the method of Goldstein and Combes (1966). 43 Plasma Disappearance of BSP and Conjugated BSP (BSP—GSH) in 15 Day Old and Adult Rats Preparation of Conjugated BSP Conjugated BSP (BSP—GSH) was prepared by the method of Whelan gt al. (1970a,b). Reduced glutathione (736 mg) was dissolved in a 40 ml commercial solution containing concentrated BSP (50 mg/ml). The pH was brought to approximately 10.0 with 2 ml of concentrated ammonium hydroxide and the contents were covered and shaken slowly at 37°C in a Dubnoff incubator. Following 2 hours of incubation, 105 ml of anhydrous acetone was added and contents centrifuged at 2500 rpm for 10 minutes. The resulting precipitate was discarded and an equal volume of anhydrous acetone was added to the supernatant. Following recentrifugation, the supernatant was discarded and the precipitate was washed in 87.5% acetone, dissolved in distilled water, quick frozen in a dry ice-ethanol bath, and lypholized overnight (Virtis Freeze-Mobile, Gardiner, Mich.). Confirmation that the resulting powder was conjugated BSP (BSP-GSH) was made by the paper chromato— graphy method of Combes (1959). Standard aqueous solutions of the powder (BSP-GSH) were spotted on Whatman No. 3 filter paper, dried with a hair dryer, and developed by descending chromatography in a solvent system consisting of n—propyl alcohol, distilled water, and glacial acetic acid, 10:5:1 (v/v). Following 12—14 hours, BSP on the chromatographs was identified by exposure to ammonia vapor and was then eluted in distilled water. Rf value for the newly synthe- sized powder (BSP-GSH) was compared to literature values for BSP—GSH (Whelan g£_§1., 1970b), standard BSP solutions, and samples of bile from adult rats administered BSP. By this procedure, the resulting powder was determined to be approximately 95% conjugated BSP (BSP- GSH). 44 Elimination of BSP and BSP-GSH from Plasma of 15 Day Old Rats Fifteen day old rats were lightly anesthetized with ether, injected via the tail vein with 150 nmoles/kg BSP or BSP-GSH (deter— mined colorimetrically) and placed under a heat lamp. BSP and BSP—GSH were diluted in normal saline for an injection volume of 10 ml/kg. Three, 10 and 20 minutes following drug injection, blood samples were taken by cardiac punture with a heparinized syringe. Concentrations of BSP and BSP—GSH in plasma were determined by measuring absorption at 580 mu after dilution of plasma samples-(20-100 pl) with water and 0.1 N NaOH. The rate of elimination of BSP or BSP-GSH from plasma was determined by the method of least squares (Goldstein, 1971) and fidu- cial limits and correlation coefficients determined as described by Goldstein (1971). Elimination of BSP and BSP—GSH—from Plasma of Adult Rats Rats were anesthetized with 3 ml/kg of Equi-ThesinR (Jensen— Salsbery, Inc., Kansas City, Mo.). This anesthetic preparation con— tained 44 ml propylene glycol, 0.97 gm pentobarbital, 4.25 gm chloral hydrate, 2.126 gm MgSOA, and 11 ml ethanol, in 100 ml of distilled water. Anesthetized rats were placed on a 6”x12" board and the femoral artery and vein were cannulated with PE polyethylene tubing. The 50 animals were placed under a heat lamp to maintain body temperature. BSP or BSP—GSH (100 nmoles/kg) was injected through the venous cannula and blood samples (0.3 ml) were drawn into a heparinized syringe through the artieral cannula at 3, 10 and 20 min. Plasma samples were analyzed for BSP or BSP—GSH as described above. The rate of elimination 0f BSP or BSP—GSH from plasma was determined by the method of least 45 squares (Goldstein, 1971) and fiducial limits and correlation co— efficients determined as described by Goldstein (1971). Influence of Experimentally—Induced Alterations in Bile Flow on Drug Transport in Adult and Young Rats Effect of Bile Salt Administration on Plasma Elimination of BSP The effect of simultaneous administration of the bile salt tauro— cholate (sodium) and 120 mg/kg sulfobromophthalein (BSP) on the plasma disappearance of BSP was examined in 15 day old and adult rats. Sodium taurocholate (100 mg/kg) was dissolved in normal saline for an injection volume of 3.2 ml/kg and BSP was appropriately dissolved in saline for injection volumes of either 2.4 ml/kg (adult rats) or 6.8 ml/kg (15 day old rats). The two solutions were mixed in a syringe immediately prior to intravenous injection and control rats (no bile salt) received an additional (3.2 ml/kg) volume of normal saline. Fifteen day old rats were injected with the drug(s) via the tail vein and plasma and liver concentrations of BSP were determined 3, 10, 20, 30 and 40 minutes following injection as described in "Disposition of Sulfobromophthalein". Adult rats were anesthetized with Equi-Thesin and administration of drug(s) and collection of serial (3—40 minute) blood samples taken from each rat as described in "Elimination of BSP and BSP-GSH from Plasma of Adult Rats". Effect of Bile Duct Ligation on Ouabain and BSP Transport Fifteen day old rats were lightly anesthetized with ether and the bile duct was exposed following a midline abdominal incision. Liga— tion of the common bile duct was made with silk sutures and the abdominal wound was closed by means of an autoclip applier (Clay— Adams, Inc., New York, N.Y.). Sham operated animals were used as 46 controls. Following the surgical procedure, animals were placed under a heat lamp for recovery and, after 1 hour, were administered a single bolus, by tail vein injection, of 3H—ouabain (1 mg/kg; specific activity of 200 uCi/mg) or BSP (120 mg/kg). After drug administra— tion, rats were placed under a heat lamp and, at various times follow— ing drug administration, blood samples were taken by cardiac puncture and concentration of drugs in plasma was determined as described in "Disposition of Ouabain" and "Disposition of Sulfobromophthalein". In experiments with 3H—ouabain, hepatic and intestinal ouabain levels were also determined as previously described. In a separate experi— ment, the effect of bile duct ligation on ouabain transport was deter- mined in 15 day old rats injected with ouabain immediately following the Surgical procedure and the rats were not allowed a 1 hour recovery period. The effect of bile duct ligation on (3H)-ouabain (1 mg/kg; spe- cific activity of 155 uCi/mg) and BSP (120 mg/kg) elimination from plasma was also determined in adult rats. Adult animals were anesthe— tized with Equi—Thesin and the common bile duct was ligated with silk sutures. After 1 hour, ouabain or BSP was injected into a PE50 femoral vein cannula. Sham operated animals were used as controls. Serial blood samples were drawn from an arterial cannula (in the femoral artery) at various times following drug administration. Plasma concentration of ouabain (tritium) and BSP were determined as described in "Disposition of Ouabain" and "Disposition of Sulfobromo- ‘ phthalein". After collection of the final blood sample (40 min), liver and intestine were immediately removed (from ouabain injected rats) and ouabain levels in these tissues were determined as previously described. 47 Effect of Pentobarbital-Induced Hypothermia on Ouabain Disposition The effect of hypothermia on the disposition of intravenously administered ouabain was determined in 15 day old and adult rats. Hypothermia was induced in these animals with a single intraperitoneal injection of sodium pentobarbital (30 mg/kg) prepared in normal saline. Control animals received comparable doses of pentobarbital, but were placed under a heat lamp to maintain normal body temperature. Body temperature was continuously monitored by means of a rectal probe attached to a telethermometer (Yellow Springs Instrument Co., Yellow Spring, Ohio). When temperature decreased to about 30-33°C, rats were admini— stered a single bolus injection of 3H—ouabain (1 mg/kg; specific activity in adults, 307 uCi/mg; in 15 day old rats, 206 uCi/mg). Thirty minutes following ouabain injection, the amount of ouabain (tritium) in plasma, liver and intestine was determined as described in "Disposition of Ouabain". Effect of Carbon Tetrachloride on Hepatic Transport of Ouabain in Developing Rats Rats of 14, 20, 24, 32 and 45 days of age received a single ip injection of corn oil (5 ml/kg) or CCl4 (1 ml/kg) prepared in corn oil. The administration of CCl4 across litters was arranged so that both control and CCl4-treated animals were obtained from each litter. Twenty-four hours following administration of CC14, animals were lightly anesthetized with ether and injected with (3H)—ouabain (1 mg/kg) via the tail vein. The specific activity of (3H)-ouabain was 250 uCi/mg. Thirty minutes following administration of ouabain, rats were anesthetized with ether, a blood sample was taken (with a hepa— rinized syringe) by cardiac puncture, and the entire liver and small 48 intestine were rapidly removed. Radioactivity in duplicate samples of plasma, liver, and intestine was determined as described in "Disposition of Ouabain". Digoxin-Mediated Inhibition of Ouabain Transport in Adult and Developing_Rats The effect of simultaneous administration of digoxin (nonradio— active) and 3H-ouabain on hepatic transport of 3H-ouabain was examined in 15 day old, 21 day old, and adult rats. Specific activity of ouabain was 200 uCi/kg (in 15 day old rats), 180 uCi/mg (in 21 day old rats) or 155 uCi/mg (in adult rats). 3H-Ouabain (1 mg/kg) was prepared in normal saline for administration of 5 ml/kg injection volume. Digoxin (0.5 mg/kg) was mixed in 100% ethanol, sonicated for 10 minutes, and, following sonication, diluted further with normal saline for injection volume of 2.5 m1/kg which contained 20% ethanol. The two injection solutions were prepared separately and were mixed in a syringe immediately prior to tail vein injection. Thus, rats injected with 3H-ouabain plus digoxin were administered a total in- jection volume of 7.5 ml/kg which contained 1 mg/kg 3H—ouabain, 0.5 mg/kg digoxin (nonradioactive), and approximately 7% ethanol. Control rats (administered 3H—ouabain but no digoxin) received 7.5 ml/kg injection solution which contained 7% ethanol. Rats were administered drug(s) as a single bolus injection and plasma, liver and intestinal ouabain (tritium) was measured in animals sacrificed 3, 20, and 40 minutes following injection as outlined in "Dispostion of Ouabain". 49 Toxicity of Polybrominated Biphenyls (PBBs) in Developing Rats One day old Sprague—Dawley rats and lactating mothers were ob- tained from Spartan Research Animals, Inc. (Haslett, Michigan) on the morning following parturition. Lactating dams and litters of 10 offspring were housed in clear plastic shoe box cages and were imme- diately placed on diets containing 0 or 50 ppm P833 (in the form of Firemaster BP—6, Michigan Chemical Co., which contains a mixture of PBBs of which 2,2',4,4',5,5'-hexobromobiphenyl comprises about 70%; Jacobs_g£.al., 1976). The PBBs were dissolved in acetone or ether (10 ml/kg diet) and were thoroughly mixed with powdered food pellets (Wayne Lab Blocks) over a 10 minute period. The control diet was powdered food to which only solvent had been added. Analysis of diets for PBBs was made by the method of Rickert g£_§l. (1977) following extraction of PBBs into petroleum ether (Fehringer, 1975). Control diet (0 ppm) contained less than 50 parts per billion PBBs and experi— mental diet (50 ppm) was determined to contain 40.7—50.1 ppm PBBS. Total litter body weights and mortality were recorded at weekly or biweekly intervals from postnatal day 7 to termination of the experiment at postnatal day 49. Litters were weaned on postnatal day 28 and weanlings were continued on the same diet fed to their mothers. In a separate experiment, timed pregnant Sprague-Dawley rats were obtained on day 5 of gestation from Spartan Research Animals and on day 8 of gestation were placed on diets containing 0 or 50 ppm PBBS. After birth lactating rats were continued on respective diets until Postnatal day 15. At birth all litters were normalized to 10 pups. All litters were cross—fostered at birth to give litters born to and nursed by mothers with the following dietary exposures: 0 ppm prenatal, 50 0 ppm postnatal (0—0); 50 ppm prenatal, 0 ppm postnatal (50-0); 0 ppm prenatal, 50 ppm postnatal (0—50); 50 ppm prenatal, 50 ppm postnatal (50-50) Total litter weight was recorded on postnatal day 8 and postnatal day 15. Interaction of PBBs with Ouabain Lethality in 15 Day Old Rats The lethality of ouabain was determined in 15 day old rats whose mothers received dietary PBBs (0, 50, or 100 ppm) beginning at birth and continued until the day of the experiment. Various doses of ouabain (octahydrate; Sigma Chemical Co., St. Louis, M0.) were dis- solved in distilled water and injected intraperitoneally. Mortality was recorded over a 24 hour period and calculation of ouabain LD50 was made by the method of Litchfield and Wilcoxon (1949). Potency ratios were calculated following affirmation that log-probability plots yielded Curves that were parallel. Effect of Pol brominated Bi hen ls PBBs) on He atic Excretor Function in Developing Rats Effect of Exposure to PBBs on Ouabain Transport in Developing Rats The effect of exposure to PBBs on ouabain elimination from plasma and hepatic and intestinal dispostion of ouabain was determined in 15, 21, 35 and 49 day old rats. The animals used in this experiment were exposed to PBBs by two treatment regimens. The first group received 0 or 50 ppm PBBs through the mothers diet from the day of birth to the day of the experiment. The mothers received dietary PBBs on postnatal day l and on postnatal day 28 the weanlings received the same dietary dose of PBBS. Hepatic transport of ouabain was determined when rats Were 15, 21, 35, or 49 days old. Hepatic transport of ouabain was determined in the second group of animals on postnatal day 15. Rats 51 in this group received prenatal and/or postnatal PBBs through the mother's diet (at a dietary dose of 0 or 50 ppm) as described for the cross-fostering procedure in "Toxicity of Polybrominated Biphenyls (PBBS) in Developing Rats". Hepatic transport of 3H-ouabain (1 mg/kg) administered by a single tail vein injection was determined as described in "Disposition of Ouabain". The specific activity of 3H-ouabain was 200 uCi/mg. Effect of PBBs on Initial Rate of Elimination of Indocyanine Green (106) from Plasma in 21 Day Old Rats Twenty-one day old rats whose mothers received dietary PBBs (O or 50 ppm) beginning at parturition (and continued until postnatal day 21) were lightly anesthetized with ether, injected with a single bolus of ICC (Hynson, Westcott and Dunning, Inc., Baltimore, Md.; 40 mg/kg) via the tail vein, and were placed under a heat lamp. One, 5, 10 and '15 minutes following administration of ICC, blood samples were taken by cardiac puncture into a syringe rinsed in sodium oxalate (1.6 mg %). Sodium oxalate was used as the anticoagulant since the sodium bisulfide present in heparin preparations interferes with ICG absor- bance (Cobb and Barnes, 1965). ICC in plasma was quantified by diluting the plasma sample in water and measuring absorbance at 805 mu(Caesar ,§£_§l,, 1961). The rate of elimination of ICC from plasma was determined by the method of least squares (Goldstein, 1971) and fiducial limdts and correlation coefficients were determined as described by Goldstein (1971). 52 Characteristics of Stimulation of Drug Transport in Young Rats Digoxin-Mediated Inhibition of Ouabain Transport in Rats Exposed to Polybrominated Biphenyls (PBBs) Control 15 day old rats, 15 day old rats exposed to PBBS during the prenatal period (50—0; from 8 of gestation to birth) and 15 day old rats exposed to PBBs during the postnatal period (0—50; from birth to postnatal day 15) were injected with 3H—ouabain (1 mg/kg) via the tail vein. In addition to ouabain, the rats were simultaneously injected with digoxin (0.5 mg/kg in 7% ethanol) or with a saline- ethanol mixture (7%). The specific activity of 3H—ouabain was 200 uCi/mg and the experimental procedure was the same as outlined in "Digoxin-Mediated Inhibition of Ouabain Transport in Adult and Developing Rats”. Effect of Carbon Tetrachloride (CC1,) on Tissue Distribution of Ouabain in 15 Day Old Rats Treated with Polybrominated Biphenyls (PBBs) Fourteen day old rats whose mothers were fed diets containing 0, 50, or 100 ppm PBBs continuously from the day of birth were injected intraperitoneally with a single dose of C014 (1 ml/kg). CCl was 4 prepared in corn oil for an injection volume of 5 ml/kg. Twenty—four hours after CC14, animals were injected with 3H—ouabain (1 mg/kg; specific activity of 246 uCi/mg) via the tail vein and placed under a heat lamp. Thirty minutes following ouabain injection, blood was obtained by cardiac puncture, liver and small intestine were removed, and radioactivity in plasma, liver and intestinal homogenates was determined as outlined in "Disposition of Ouabain". 53 Statistics Statistical evaluation of the data was made by Student's £7 test (Steel and Torrie, 1960). The level of significance was chosen as p<0.05. RESULTS ngatic Tgapsport of Ouabain and Sulfobromophthalein in Developing rare The disappearance of ouabain from plasma of developing rats is depicted in Figure l. Rats 15 and 21 days of age retained ouabain in plasma relative to the older animals. For animals older than 21 days of age, differences in the disappearance of ouabain from plasma are less conspicuous and it appears that the curves representing the plasma disappearance of ouabain in 35 and 45 day old rats are Superimposable (Figure 1). In the 40 minute experimental period, the disappearance of ouabain from plasma may be represented by a single line on a logarith— mic—concentration vs. time plot in 15 day old rats; however, in older animals, the curves appear to be biphasic (Figure 1). Following a single intravenous injection of ouabain, cumulative hepatic excretion of the drug, estimated by 40 minute intestinal con— tent, increased with age. Forty minute intestinal ouabain content in 15 day old rats was ll6.6:23.0 ug/kg body weight and was 2.5—3 times less than values obtained from 35 and 45 day old rats (Table 1). The concentration of ouabain in the liver changed with time following a single bolus injection of the drug (Figure 2). In 15 and 21 day old rats, hepatic ouabain concentration and total hepatic Ouabain content (pg in total liver/kg body weight) reached maximum 54 55 Figure l. Elimination of (3H)ouabain from plasma of rats of ages ranging from 15—45 days. Rats were administered (3H)ouabain (1 mg/kg) via the tail vein and following 3, 20 and 40 minutes, blood was obtained by cardiac puncture and plasma samples analyzed for tritium. Each point represents the mean value for 4 rats obtained from 4 litters. Standard errors (not shown for clarity) were approximately 10% of the mean values. ouaum ( no] 100 ml rusuu) 56 100 0 SO 0 O 0 15 DAYS 10 0 2| DAYS 0 25 DAYS 35 DAYS 5 ‘ 45 DAYS 3 IO 20 30 40 TIME FOLLOWING ADMINISYIAYION OI OUAIAIN (MIN) Figure l 57 TABLE 1 a Cumulative Hepatic Excretion of Ouabain in Developing Rats Age (Days) Ouabain Excretion (pg/kg body wt) 15 ll6.6i23.0b 21 l68.0i47.7 25 222.2:29.3 35 350.9:16.0 45 300.0i13.6 aRats were injected with 3H—ouabain (1 mg/kg) via the tail vein and after 40 min the intestine was analyzed for tritium. bValues represent the mean i S.E. for 4 rats obtained from 4 litters. 58 .uaHom onu mo uouoamww onu mono HoHHmEm mos uouuo wumvamum .cBonm uoc ohm mumn uouuo vumvcmum dosz .muouufia q Eouw vosamono mums q pom .m.w H some osu muammounou uuHom somm .aowufluo How commando mums uo>wa mo moaaamw .mooacfla oq mom .ON .m waHBOHH0m mam aflo> HHMu ofiu ma> wa\mfi HV GHmQMSOAmmV monoumHSHEwm mums momm .mzmw mqumH Eoum waflwcmu moms mo mums CH GOfiumuumflcflsvm :HmnmsoAmmV mafiBoHHOM wEflo nufls usouaoo oflmnmoo ofiumao: HmuOU was coaumuusoocoo :Hmnmoo owumaom .N ousmflm 59 mum OUAIAIN comm: (pg/kg low wv ) .---° D3§§§§§§§88 9- . 3 / a v -—o“ 9- a /. C a 40":— ’ «a "‘r¢”"' -o’ o \ —D D:- § § O.D- .... g...— 3 I I fl “ I '0 .‘h ~~~ - o‘c- aw. ’. .9 .—. ("I01 M ) nouvumauo: mum :uvau 't Y. mu Ann ADMINISTRATION on: OUAIAIN (min) Figure 2 60 values 20 minutes following drug administration. In 15 day old rats, 20 minute ouabain concentration in liver was 6.02i0.23 ug/g wet weight tissue which was almost three times the concentration at the 3 minute time point but essentially the same as the 40 minute value. In 21 day old rats, 20 minute hepatic ouabain concentration was 7.40:0.28 ug/g and was higher than hepatic ouabain concentrations at both the 3 and 40 minute intervals. Hepatic ouabain concentration and total hepatic ouabain content in 25, 35, and 45 day old rats were maximum at the earliest (3 minute) time point. The time dependent pattern of ap- pearance and disappearance of hepatic ouabain was similar when calcu- lated on a per gram tissue or on a body weight basis for rats of all ages. Liver to body weight ratios, however, increased with age (Table 2). Plasma concentrations of sulfobromophthalein (BSP) in adult rats were significantly different from values obtained from 18 day old rats. When compared to adult values, significantly higher plasma BSP concen— trations were detected in the 18 day old rats 10, 20, 30 and 40 minutes following a single bolus injection of the dye (Figure 3). The time dependent pattern for hepatic disposition of BSP was also age depen- dent. In the young animals, hepatic BSP content appeared to be maximum 20 minutes following dye administration; whereas, in the adult, maximum values were apparent at the 3 minute time interval (Figure 4). Hepatic BSP content in 18 day old rats was significantly lower than adult values at the early (3 minute) time point, but significantly higher than adult levels 40 minutes following injection of BSP (Figure 4). 61 TABLE 2 Liver to Body Weight Ratio in Developing Rats Age (Days) Liver Wt/Body Wt (%) 15 2.97:0.10“ 21 3.50:0.06 25 4.15:0.06 35 4.64:0.10 45 4.50:0.13 aEach point represents the mean i S.E. for 11 rats obtained from 4 litters. 58 .oafiom oSu mo Houmamfiw wan cmnu Hoaamam mos uouum cummomum .ssosm oo: mum mums uopuo pudendum song .muwuuHH q scum monsoono moon q pom .m.m H some onu muaomoumon usfioa zoom .adflufluu How wouhamco ohms uo>wa mo mmadamw .moosofla oq won .oN .m maflsoaaom vow cfio> Haws onu mH> Amx\wa Hv afimnmsofimmv monoumflafisvm ohms muom .mzow mclma scum wsflwamu moms we woos as coaumnumficflsvm cfimnmaoAmmV wcfisoHHOM .N ouswwm mafia :uHB usouaoo aflmnmso ofiumao: Houou mam coaumhuomoaoo cwmnmso ofiumaom N ouowam “cm—av Z_(-<=O no 203<¢hn=§§¢ Cahu< at: 59 HIPATIC OUAIAIN comm! (pg/kg low wr ) ON 6. ON.— 8— 9: ° 3 . i “>3 at —o“ :3 an ”(and ("I01 M ) nouvumano: mum :uvaau 62 Figure 3. Elimination of sulfobromophthalein (BSP) from plasma of 18 day old and adult rats. Animals were administered BSP (120 mg/kg) via the tail vein and following various times, blood was obtained by cardiac puncture and BSP concentration was determined in plasma. Each point represents the mean i S.E. for 3 rats. Eighteen day old animals were obtained from 3 litters. When standard error bars are not shown, standard error was smaller than the diameter of the point. Asterisk indicates plasma BSP concentration significantly different from values obtained from adults (p<0.05). BSP (mg/100 ml PLASMA) ‘IO SO IO _'|8 DAYOLD ' ' ' 'ADUL'I' 3O 40 TIME (min) 64 Figure 4. Hepatic content of sulfobromophthalein (BSP) with time following BSP administration in 18 day old and adult rats. Rats were administered BSP (120 mg/kg) Via the tail vein and following 3, 20 and 40 minutes, samples of liver were analyzed for BSP. Each point represents the mean i S.E. for 3 rats. Eighteen day old animals were obtained from 3 litters. Asterisk indicates hepatic BSP content significantly different from values obtained from adults (p<0.05). "mm :9 (mg/kg nonvvn) 65 "Ml (min) Figure 4 — I. DAY OLD I n a I Awl' 66 In Vitro Conjugation of BSP to Glutathione in Developing Rats The activity of hepatic glutathione S-aryl transferase (using BSP as substrate) was low in young rats when compared to adults (Table 3). BSP conjugating activity in 21 day old rats was approximately 2 times higher than activity in 1-2 week old animals and appeared to reach maximum levels in 35 day old animals (Table 3). Plasma Disappearance of BSP and Conjugated BSP in 15 Day Old and Adult Rats The disappearance of BSP and conjugated BSP (BSP—GSH) from plasma of 15 day old and adult rats is depicted on Table 4 by the rate of elimination of these drugs from plasma. Each calculated regression line (slope) for plasma drug concentration vs. time demonstrated a signifi— cant correlation (Table 4). The rate of elimination of both BSP and BSP-GSH from plasma was significantly lower in 15 day old animals than in adults. In adult rats, plasma half-lives (t%) for BSP or BSP-GSH were not different from each other (7 min vs 6 min, respectively) but were 2—4 times lower, respectively, than values from 15 day old ani- mals. The rate of elimination of BSP from plasma was almost 2 times greater than removal rate for conjugated BSP in the young animals. Influence of Experimentally-Induced Alterations in Bile Flow on Drpg Transport in Adult and Young Rats Effect of Bile Salt Administration on Plasma Elimination of BSP The disappearance of BSP from plasma was enhanced in adult and, to a lesser extent, 15 day old rats by simultaneous administration of the bile salt taurocholate. Adult rats administered taurocholate plus BSP had significantly lower plasma BSP concentrations 30 and 40 minutes following BSP injection with values of 54% and 45% of control at these 67 TABLE 3 Hepatic Glutathione S-Aryl Transferase Activity in Developing Rats Age (Days) Glutathione Transferasea 8 432 8+ 35 5b 9 228.7: 8.3 10 254.2: 14.2 14 363.7: 8.7 21 772.3i 38.3 35 1385.7i183.3 70 718.8: 17.3 aActivity, designated as nmoles BSP conjugate produced/min/ gm liver, determined in rat liver 100,000 x g supernatant fraction. The reaction mixture contained 227 uM sulfobro- mophthalein and 20.5 mM reduced glutathione in 0.1 M sodium pyrophosphate buffer, pH 8.2. Mean 1 S.E. for 6 rats obtained from 3 litters. 68 TABLE 4 Disappearance Ratea of BSP or Conjugated BSP (BSP-GSH) from Plasma of 15 Day Old and Adult Rats 15 Day Old Rats Rate of Elimination Estimated t% Correlation Coefficient Drug (Slope)b (min) BSP 0.019:0.004d 15 .920 BSP-GSH 0.010:o.007d’e 3o .760 Adults Dru Rate of Elimination Estimated tk Correlation Coefficient g (Slope)b (min) BSP 0.046i0.008 7 .960 BSP-GSH 0.053:o.001 6 .990 aDisappearance rates determined by the method of least squares from plasma samples taken 3, 10 and 20 min following injection of 150 nmoles/kg (15 day old rats) or 100 nmoles/kg (adult rats) of BSP or BSP—GSH. bElimination rate r fiducial limits for 3 (adult group) or 9 (15 day old rats) animals. 3 litters. cSignificant correlation (p<0.05). Fifteen day old animals were obtained from dSignificantly different from rate in adult rats (p<0.05). eSignificantly different from rate of BSP removal (p<0.05). 69 time points, respectively (Figure 5). In 15 day old rat neonates injected with taurocholate, significantly lower plasma BSP concen- tration was detected at only the 30 minute time point and represented a 19% decrease when compared to neonates injected with BSP alone (Figure 5). Hepatic concentrations of BSP in rat neonates injected simulta- neously with taurocholate were significantly lower than BSP concentra— tions in the control group 3 and 10 minutes following bolus injection of BSP (Figure 6). Effect of Bile Duct Ligation on Ouabain and BSP Transport Adult and 15 day old rats whose bile ducts had been ligated 1 hour prior to bolus injection of BSP and ouabain significantly retained these compounds in plasma (Figure 7). When compared to sham operated controls, plasma drug concentrations were significantly higher in bile duct ligated adult rats at all times following BSP injection and at all time points except the earliest (3 minutes) in rats injected with ouabain. Acute bile duct ligation in 15 day old rats resulted in significantly higher plasma concentrations of BSP 10 and 40 minutes following BSP injection when compared to sham operated rat neonates; whereas ouabain concentrations in plasma were significantly higher than control values in bile duct ligated rat neonates 3, 20 and 40 minutes following ouabain injection (Figure 7). Retention of ouabain and BSP in plasma was more pronounced in bile duct ligated adult rats than in similarly treated 15 day old animals. Forty minutes following intra- venous injection of BSP and ouabain, plasma concentrations of these drugs in bile duct ligated adult rats were 513 and 780% of sham operated controls, respectively. In bile duct ligated 15 day old rats, plasma 70 .Amo.ovav ocean mmm monoumfisaavm mums aouw vosHMupo mosam> Eouw usoHMWWHv %Huamofimfiawflm coaumuuaoUCOU mmm mammam moumofluda xmfluoum< .uaflom on» mo umpofimwm onu dosu uoaamam was gonna vumocmum .ssonm uoa mum mama soups unaccoum cosz .muouufia q scum moaflmuno mums mamfifiam vac mow cooumam .mumu elm pom .m.m H some oau musomouaou uofloa zoom .mamwaa CH monashouoe was coaumuuaoucoo mmm .moafiu moowuo> um .vam wa\ma ooav oumaonoOH:Mu moan wa\wfi owav mmm no wa\ma ONHV mmm wouoomwdflawm %Hmooao>muusfi mums mood .mumu oases mom vac moo ma mo «anode Scum Ammmv afloamnonaoaounowasm mo soauwaflafiao so AuHmm oHHnV oumaoaoouoou sswuom mo uoomwm .m madman 71 Eszmm do 20:55.2..23 5:? us: as on on o. ml I s: / film :3 3.. .oino ~23: ollo naO > scum uooquMHw %Hudmofimfiswfim cofiuouocooaoo mmm saunas: woumowwcfl xmfluoum< .muouuwa q Eouw wocfimuno mums q pom .m.m H same onu muaowouawu peace room .mmm new wonaancm mums uo>wa mo moaasom .moaflu mSOHum> on was awo> Haou osu mw> Amx\wa ooav oumaonoousmu moan Amx\wa ONHV mmm Ho wa\ma ONHV mmm wououmwcwavm mums mood .mumu vac %mv ma mo H0>HH as sowumuuaoosoo Ammmv :Honaunaoaounomasm so oumaonoounmu ssfioom we ooommm .o muswwm 73 O? o muzwfim AcmEv mi..— om ON uh<.—O:UO¢=<.— .7st I I nz= u< .GOfiumeH uosv mafia wafisoHHOM noon H no Amussaa HV %Houmflmoaafl afim> Hfimu onu mH> wa\wa av sfimnmsoammV wououmwsfiawm mums mumm .mumu vac how ma mo saunas Scum awmnmooAmmv mo :owumsfiaflao so coaumwwa uoso mafia mo uoomwm .w ouswflm w ouswam 2...: 2.52:0 02.3039. 22: 2. cu m 78 .‘vmeIIII.I:I.llIIIII-.hro a! e/. /Iol 1:25 3:0: 52. 3... I .555 3:0... .52. 3:. I .. 2<1m I I (WISH: Inll ooulm ) Nlnvno 79 TABLE 5 Cumulative Intestinal Ouabaina in Sham or Bile Duct Ligated 15 Day Old and Adult Rats Age Treatmentb Intestinal Ouabain (us/kg B.W.) 15 day old Sham 116.6:23.0c 15 day old Bile Duct Ligated (1 hour) 20.7: 1.9d 15 day old Bile Duct Ligated (l min) 22.7ilO.6d Adult Sham 236.6163.l Adult Bile Duct Ligated (1 hour) 6.8: 0.1d aRats were injected with 3H-ouabain (1 mg/kg) via the tail vein and after 40 minutes the intestine was analyzed for tritium. bCommon bile duct was isolated and ligated following a midline abdominal incision. Animals were injected with ouabain either 1 minute or 1 hour following surgical procedure. cValues represent the mean i S.E. for 3—4 rats. Fifteen day old animals were obtained from 3 litters. dSignificantly different from sham at appropriate age (p<0.05). 80 TABLE 6 Hepatic Concentration of Ouabaina Following Intravenous Administration in Sham or Bile Duct Ligated (BDL) Adult and 15 Day Old Rats Hepatic Ouabain (pg/gm) Age (Days) Treatmentb 3 min 20 min 40 min 15 Sham 2.110.10 6.0:0.2 6.0:0.5 15 BDL (1 hr) l.0t0.3 1.510.2d l.5i0.5d l5 BDL (l min) 2.2i0.l 7.4i0.7 5.0iO.3d Adult Sham —————————————— 0.5i0.0 Adult BDL (1 hr) -------------- 4.6i0.7d aRats were injected with 3H-ouabain (1 mg/kg) and at designated times the liver was analyzed for tritium. Z)Common bile duct was isolated and ligated following a midline incision. Animals were injected with ouabain either 1 minute or 1 hour following surgical procedure. 0 Each value represents the mean i S.E. for 3-4 rats. Fifteen day old animals were obtained from 3 litters. dSignificantly different from sham of the appropriate age (p<0.05). 81 .Amo.ovmv mama uoom + Houfinpmnouson Boom mammoMMHv %Husmoflmflsmwmfi .muouufla m aosm vodflmuno ouoB mHmEHao nHo %mw sooumwm .muMp q How .m.m H some onu mucomoumou o=Hm> nommo .QEMH umon asexufis momma as moumaa mums so musumuoafiou hoop Hashes dampness ou mama umo: m Hows: moomam ponuflo mums was wa\ma omv Hmufinumnouaom Sums %Hammnoufiuoamuuafi wouoonca mums mameasm m .BoHoHuu How wmumfioco mums osflummusfl was Ho>HH .oEmmHa :HE om noumm mom dfio> HfiMu ego oH> Amx\ma HV ofimomDOIm suHB wouoomafi mums mummo m . I . . 1 . . I . . I . mama umwm + m mH+N msa m m +0 0H s o+H m H o+m mm Houflnumnouoom H.maflq.mqa mm.o Hm.nm mm.owm.q ma.osm.mm Houflnumnoudom Aces ABM wx\mav oaaomouaH ASm wx\w:v Hobflq AHE ooa\mav mammHm ouaumuomaoe uaoaumoua zoom com: & sowusnwuumflo aflmnmso oasw< . I . . 1 . . I . . n . mama ummm + m oH+w Hm N ¢H+q NNH q N+m ma 0 0+0 mm HouHAHMQOuaom mn.N Hm.mN m.mHHo.ONH mm.nHm.wm @«om.owo.om Hmuwnumnouaom Aoov Azm wx\w:v wcfiumousH ABm wx\w1v Ho>fiq Ada ooa\mnv mammam undumuomEmH ucoaumouH %mom moo: Q cowusnfiuumflm aflmnmoo mac sac ma mumm uH=v< mom VHO %mo ma CH mowusnfiuumfin osmwflH BaHmnmso no mHEuwcuomem woosvcH mHmonumoc< mo uowwwm m mqm aoum ucouowwflv %Huamoflwwcwfim demo Imposooaoo campmso mammam moumofivcfi xmfiuoum< .usHon onu we nouoEMHv onu nmSu uoHHmEm was soups muowamuw .csoam uo: mum mums hound wuovsmum cmnz .muooufia m aouw voGHMuno mums moms vac %mm Hm was vac zoo smouwfim .momu «In How .m.m H anus onu mosomouaou ucfioa somm .Asfimnmsov Eofiufluu Mom wonzamco moamfimw mammaa mam unsoundm omflvumo %a vosfimuno was uooan .wouscwe oq mam .ON .m wuHBOHHOM mam cHo> HHMu onu mfl> wa\wa m.ov awxowfiw mafia wa\mfi Hv cflmnmsoAmmv Ho wa\wa HV :Hmnmso AmmV vapoumficflaum mums mumm .wumu oases was .vHo hop Hm .vao was ma mo mamman scum samnmdo AmmV we Gomomcflawao so nHNova mo uoowmm .OH ouswflm 0H magmas 3.5 228...... 02.3039. .1: 9 8 2 o. n 9 8 2 o. n I . o o: o II’II’ * 10 m m 2:80... 0:6 * .233 olo ’ o .2331 .23: 05 I 4 z 30 >3 2 ,0 30 >3 3 ’o _ * out." gm 00. /.n NIVIVIIO 88 TABLE 8 Effect of Digoxin on Cumulative Hepatic Excretion of Ouabain in Developing and Adult rats Ouabain Excretion (pg/kg Body Wt)a Age Ouabain + Saline Ouabain + Digoxin 15 days lO7.6ill.6c 48.9: 5.6d 21 days 209.8:37.7 ll9.3i50.7 Adult 288.2:27.6 285.2:39.9 aRats were injected with 3H-ouabain (1 mg/kg) via the tail vein and after 40 minutes the intestine was analyzed for tritium. b igoxin (0.5 mg/kg) was administered simultaneously with H-ouabain (1 mg/kg). cEach value represents the mean i S.E. for 3-4 rats. Fif- teen and 21 day old animals were obtained from 3 litters. dSignificantly different from rats injected with 3H— ouabain alone (p<0.05). 89 .Amo.ovav macaw aHmnoso monouchflawm moan aouw woCHMuno mo=Hm> Eonw ucouomev sfiuaMUHchme ucouaoo sHmnmno oHumdo: mouQUHvsH meuoum¢ .ocHoa mnu mo Hoqume was smnu HoHHmam mos Hound vumvcnom .nsozm uo: was made uouuo vumvcmum sopz .muwuuHH m Scum vochuno mums moon vac mow am can wao mac doouMHm .mumu elm mom .w.m H cmoa ofiu musomonmou uaHom comm .AdenmJOV sdHuHuu How wonhaocm mums uo>HH mo monEMm .wmoscHa 05 can .om .m wcHBoHHow can :Ho> HHou onu wH> wa\wa m.ov :onva mafia Amx\me Hv aHmnmsoAmmv Ho wa\wa Hv :HmnmooAmmv monoudeHavo ohms mood .muon oases was .wHo mow HN .vao mam ma :H aHmnmsoAmmv mo ucooaoo oHuoaox so sHNowHu mo uoomwm .HH oudem HH oHDMHm Asa—.5 10:.qu— gp’OaaOs ui—b 9 81 be a L 2:82.. 1 1 2:32.. 1 1 2:60... 1 1 .233 I .23: I .23: I 9. o... 3 mo. 8. 8. 8. _ 11111 _ 0-111 *vo _ _* \\ . \\ \\ I \\ / \x a: z_ \\ 09 62 I \ . o o _ _ _ o o 802:. m. _ So :3 a _ 8a 08 02 ('w ‘m III/'1') mama mum 91 .H mom so HouuHH pom mums OH mums oumnH .muouuHH QHIOH How muooUHH cHnuHB uanoB one some osu mH usHon 50mm .Honuos ou wow uon oEmw onu oudo cosmos mums mums .wm mow Hmumcumoa .wchmos u< .Honuos wcHumuUMH mo uoHv cw mmmm and on we ucoaoomaa kn mmmm ou womomxo ouo3 mums manow .muwu mo SuBOMw kumcumom man so :uan Rooms made as ou 5H3 aoum Ammmmv magnesia woumfisougaoa 3 35098 mo uoomwm .NH oustm 1““ NH ousmHm 15.—n a uhu< m>Hom ou oudmomxo mo uoommm .MH ouswwm 94 ms «gamma Ira: 8nhs< m> Homm m .mH moo Houmaumoa so vodHEuoumw mOHumu u:MHoB moon ou uo>HHo .udu Hon usmHos owmuo>w Hammouuou mosHm> mam memn HouuHH oHofis o no coszmB mums mumm .auHHn Houmm mxoos N mam H wsHpsw madam GH cosHmw usmHoB %von owmuo>Hw ou nuan on wououmOMImmouo duos muouuHH HH< .mH ems Hmumsumom ou GOHomumow «o w moo Eoum mmmm and om Ho 0 wow mums mum» unmawoum "mucwaumouao mHH.o H om.q mmo.o H HN.m mmo.o H nq.m 00.0 H mH.m oANV u: moom\u3 Ho>HH Hm.m H Nm.qH mm.H H o.mH mm.o H Hq.mH oo.o H NH.~H #3 N q~.o H «H.w mq.o H qw.m om.o H mm.w NH.o H mm.w axs H Amsmv % nHoo uanoS hvom owmuo>< om1om omlo 010m 010 Houmamumm oudoaumoHH mama UHo %ma mH :H oz %vom\u3 Hw>HH N was sHmo u5MHo3 zoom :0 Ammmmv thsoanm wouwsHEounzHom ou ousmonxm Hmumsumom mom loam woanaoo mam .Hmumoumom .Hmumooum mo uoomwm 0H mqm aouw unoHoMMHw %HuamoHMstHm mGOHumuusoocoo onnmvo mammHm moumoncH wauoum< .usHom omu mo HouoEme ofio ammo HmHHmBm mos Houuo pudendum .csoam uoa mum mums Houuo mummsmaw conz .muouuHH q Eoum moaHmHno mumu q How .m.m H some mam muaomonmou ucHoa comm .Achnmsov ESHHHHu How coanmcm monamm mammHQ can ousuocom omHoumo %n monHmHAo mos mOOHn .mquGHE oq was .ON .m wcHBOHHom was :Ho> HHMu wnu mH> Amx\wa Hv chnodoAmmV moumumHaHEem mums mumm .Hmcuoe ou wow uon oEMm ofiu Ouno vosmos ohms moms .mm %d@ Hmumcumoa .wchmos u< .Honuos wsHuMuUMH mo uon :H mmmm Eda 0m «0 ucosoUMHm an mmmm Ou vomomxm ohms mums masow .mmmm anmH Scum wdedmu moms mo mums Mo mammHa Eoum :HmnmsoAmmV Ho GOHuocHEHHo so Ammmmv mHmdofidHn mmoocHaonp%Hom ou ousmoaxm wo uoomwm .qH oustm 100 0' ON «H mmsmsm «cm-5 mi... 0' ON an: 5&8 I in: saga I I I - >40 0' 12 on .naasacoaI a... £25 1.. OOH >10 an /. ..........l / .2... 5.25 :i > Eoum usouomch hHusmon lHanm nHmnmso mo usouaoo oHuwmo: moumonsH meHoum< .uCHoa ozu mo HomoEMHw emu coco uoHHmEm was nouns vumodmum .ssonm uo: mum wuon Houuo wumvcmum nonz .muouoHH q souw vochono mums q pom .m.m H some one wusowoudou udHOd zoom .AsHmnmsov stuHuu How wonkH swam mums Ho>HH mo moHaedm .mouosHE ow was om .m wsHaoHHom was :Ho> HHmu oau MH> wa\ms Hv chnmaofimmV monoumHoHawm mums mumm .Hoguoa map ou mom uoHo mamm omu ouno modems mums moan .wN moo Houmsuwoa .manmos o4 .Honuoa wdHumquH mo oon :H wmmm see om mo uswEoUMHd an mmmm ou womonxm mums mum» manor .mwmv m¢1mH aouw maHmcmu mowm we mums :H chnoooAva mo odousoo oHomaon so Ammmmv wstoLmHn woumsHEouanom ou swamoaxo mo uoowwm .mH ouame 103 mH «mamas 2...: at: ow as a ow as n 2. an «can San-On I cum.— Enncn I 82.5.5 1 . 3... £20 a . >(o 0‘ >19 an >(n n— ..N 11M MOI 3,115" ) vauvno 311nm 104 TABLE 12 Effect of Exposure to Polybrominated Biphenyls (PBBs) on Cumulative Hepatic Excretion of Ouabain“ in Developing Rats Age Dietary PBBs Ouabain Excretion (DaYS) (ppm) (us/kg Body Weight) 15 02 122.3 1 10.7: 15 500 289.5 f 16.0 15 50—00 174.6 1 14.3: 15 0-500 274.1 1 10.88 21 02 178.4 1 5.7 21 50 202.1 1 25.8 35 02 211.6 i 53.5 35 50 206.0 i 31 6 49 0: 343.1 1 15.2 49 50 398.0 1 19.3 aRats were injected with 3H-ouabain (1 mg/kg) via the tail vein and after 40 min the intestine was analyzed for tritium. bConcentration of PBBs in diet from day of birth until day of sacrifice (rats younger than 28 days, weaning age, received PBBs through mother's diet). cPregnant rats were fed 0 or 50 ppm PBBs from day 8 of gestation to postnatal Day 15. All litters were cross- fostered at birth to give litters born to and nursed by mothers with the following dietary exposures: 0 ppm pre- natal, 0 ppm postnatal (0-0); 50 ppm prenatal, 0 ppm post- natal (50-0); 0 ppm prenatal, 50 ppm postnatal (0-50); 50 ppm prenatal, 50 ppm postnatal (SO-50). dEach value represents the mean i S.E. for at least 4 rats obtained from 4 litters. eSignificantly different from 0 ppm PBBs at corresponding age (p<0.05). 105 .Amo.ovmv ouo aouw uosHmuno made> aoum uaoHomew zHusmonHamHm aOHumHucoocoo chnmso mamea mwumochH meHoum< .oon> some mo NOH %Houmstouaam mos AzuHHMHo pom ssozm uodv Houuo euvaMHm .muouuHH q aoum mosHmuno mumu wlo HOM some onu monomouawu udHom somm .AnHmnm30v ssHuHuu How mondem moHdEMm mamMHm mew ousuocsn omHvao he moaHmuno mos vooHn .mounsHE oq was .ON .m wdHBOHH0m mam aHo> HHMu emu MH> wa\wa Hv chnwno Ammv monoumHsHawm mHmB mumm .Aomlomv HMumsumom Ban on .HmuMCoum Sam on “Aomlov HouNSumoa Ema om .HMuMdoum Ema o ”Actomv HmuMSumom and o .HMumsoua Ema om ”Honov Hmumoumom and o .Hmumcoud Ban 0 ”mohomonxo %HmuoHv waHBOHH0m onu nuHs muscuoa kn menus: was ou anon muouuHH o>Hm Ou :uan on monoumowlmmouo oHoB muouuHH HH< .mH %mm Houmsumom ou GOHumOmom mo w %mv Scum mmmm Ema 0m Ho 0 wow mums mums usmdwmum .wumu vHo mac mH mo mammHa Eoum uHmnmsoAmmV mo GOHumsHEHHo no Ammmmv mHsoosaHn woumcHEounzHoa ou oHomOQNo Hmumaumon Ho\vdm loud mo uoowwm .oH oustm 106 wH ouame n :mEv at _ h 0' 00 ON 0— nun-On H—_.J 2 01°“ 0 010 O ../ (”Md 1111001 l5") mvavno OO— 107 prenatal exposure to PBBs (50-0) was least effective in enhancing ouabain elimination from plasma. When compared to plasma concentra- tions in control rats (0-0), rats prenatally exposed to PBBs (50-0) had significantly lower plasma concentration of ouabain at only the 20 minute time point; whereas, postnatal (0—50) and pre— and postnatal (SO-50) exposure resulted in significantly lower plasma ouabain concen— tration 3, 20 and 40 minutes following administration (Figure 16). Similarly, cumulative 40 minute intestinal ouabain content, an estimate of biliary excretion of ouabain, was highest in rats exposed to PBBs postnatally, and was affected by prenatal PBBs exposure to a lesser, but statistically significant, extent when compared to controls (Table 12). Total hepatic ouabain content in these animals is depicted in Figure 17. When compared to values from control (0—0) rats, 15 day old animals exposed to PBBs postnatally (0-50) or pre- and postnatally (50- 50) had significantly higher hepatic ouabain content 3 minutes follow- ing ouabain injection (Figure 17). These increases reflect the effect of P833 on liver weight (Table 10). Rats treated with PBBs during the prenatal period (50-0) had higher hepatic ouabain content 3, 20, and 40 ‘minutes following ouabain injection, however, these increases were not statistically significant. Effect of P333 on Initial Elimination Rate of Indquanine Green (ICG) from Plasma in 21 Day Old Rats The disappearance of ICC from plasma of 21 day old control rats and rats exposed to PBBs is depicted in Table 13 by the rate of ICC elimination from plasma. Correlation coefficient (r2) for the rate of elimination (slope) of ICC from plasma of treated (.86) and control (.75) rats was significant. Elimination of ICC from plasma was 108 .Amo.ovmv Clo Eoum moaHmHno moaHm> Eoum unopowva hHunmoHMchHm chnmso mo ucouaoo oHumnon moumoHvaH meHoum< .osHm> come we NOH mHouma Ionnaam mos A%uHumHo How ssosm uoav Hound pudendum .muouuHH q Eoum moaHMuno mums wlo now Emma man monomoumou uaHOQ zoom .AaHmnmoov ssHuHHu How woumHmcm ohms uo>HH mo moHQEMw .woussHfi oq was ON .m wcHsoHHow was cHo> HHmo onu mH> wa\wfi Hv :HmnmdoAmmV monoumHaHEwm mums momm .Aomlomv Houmaum0d and on .Hmumcoua Ema om “AOmlov Houmcumon and cm .HMumnoua Bee 0 «nonomv Hammoumon and o .Houmsoum 8am on “AOIOV Houmsumom and o .HMumcoua and o "monomonxo %Hmuon waHsoHHow wzu nuHs mnonooa >n wounds can on anon muouuHH o>Hw ou nuHHo um monoum0m1mmouo mums muouuHH HH< .mH how Houmoumoa ou COHumumom mo m zoo Scum mmmm and om no 0 wow opus mums usmamoum .mumu wHo mom mH :H aHmnmsoAmmv mo uaoucoo oHummon co Ammmmv mHhsmamHn voumcHEounhHoa ou ousmomxo Houmcumoa Ho\vam Iona mo uoowwm .nH ouome 4O "mu-in) (DWMNIVOVHO DILVIIII'I Figure 17 110 .Amo.ovnv mmmm and o scum oaonMMHw %HuomonHdemmo .muouuHH m Eoum vosHmuno mumu NH How muHEHH HmHoson H COHumcHEHHm mo oummo .mmumsvm unmoH mo moauoa onu kn GOHuoonaH msHsoHHow sHE mH was .0H .m .H on maOHumuuaooaoo 90H mammHm scum voaHshmuov mos mammHa aoum 60H mo sOHuwaHaHHo mo mums mam use aHo> HHou ozu MH> mouoonsH was Amx\wa oqv UUHQ .uamaHumaxo mo use HHuco :oHHo mo %mv Scum uoHu muosuos :H mmmm mo sOHumuuaoooous 0H 20.0 mused on m.OH OHo.o ammo.o o AcHav muHsHo HmHusmHmAmv mammOHmv magmmv mmmm Mu mammasumm aoHumaHaHHm mo mums .' Ammmmv mHkamnon doomsHaounhHom humuoHQ ou monomxm wumm mHo awn HN sH mammHm scum AUUHV cooum oaHsoaoovaH mo :OHumaHEHHm mo comm HMHuHcH MH mHde lll s;i_gnificant1y greater in 21 day animals whose mothers were fed 50 ppm IPIsBs (Table 13). Animals (21 day old) used in this experiment were e2)cposed with the same dose and treatment regimen of PBBs as were 21 day ()]_d animals used in the experiment depicted on Figure 14 for hepatic 1:1:ansport of ouabain. The initial rate (from 1 to 15 minutes following 21 single ICG injection) of ICC removal from plasma was 0.025:0.010 (rug/min) in control rats (t% = 10.5 min) and was 0.04210.013 (mg/min) 111 rats exposed to PBBs (t% = 7 min) (Table 13). (Hiaracteristics of Stimulation of Drug Transport in Young Rats Digoxin—Mediated Inhibition of Ouabain Transport in Rats Exposed to Polybrominated Biphenyls (PBBs) In comparison with plasma ouabain concentrations in rats injected 1vith only ouabain, simultaneous tail vein injection of digoxin plus ouabain resulted in retention of ouabain in plasma of 15 day old control rats (0-0), and in plasma of rats exposed to PBBs (Figure 18). Plasma ouabain concentrations were significantly higher in digoxin treated rats than in controls (no digoxin): 3 and 40 min following drug administration in rats whose natural and foster mothers received no dietary PBBs (0—0); 3, 20, and 40 min following drug administration in 15 day old rats exposed to PBBs prenatally (50-0); and 3 and 20 min fOllowing drug administration in animals postnatally exposed to PBBs (0-50) (Figure 18). In 15 day old rats treated with all three exposure regimens to PBBs, digoxin-mediated transport inhibition resulted in a decrease in hepatic ouabain content for the entire 40 min experimental Pariod (Figure 19). Significantly lower hepatic content of ouabain was detected in digoxin treated rats 3 min following administration of .Amo.ovav oGOHm aHmnoao ooHoumHsHavm moms Boom woaHmono mosHm> Scum udouomew %HusmonstHm GOHumHoaoosoo onnmoo mEmMHd moumUHwnH meHoum< .uaHom onu mo HoooaoHv onu ammo HoHHoam was Houuo powwomum .asosm uoc mum mums Hoops mumpcmum cos: .muouuHH a aoum mosHouno moon mum How .m.m H duos onu monomoumon udHon comm .AsHmnmsov ESHuHuu you moanmdm moHoEmm mammHm was monooasa oonHmo he wochono mos UOOHQ .wouonHE as was .ON .m wsHBOHHow can nHo> HHmu oau mH> wa\we m.ov conme msHm wa\wa Hv sHmnmdoAmmv no wa\wa Hv cHonmsoAmmv monoumHsHanm mums mumm .Aomlov Hoomauwom Ema om .Hmumdopa and o "Acnomv Hoodoomoa and o .Hmumsoua and 0m “Honey Hooooumom and o .Hmumsoun and o "mousmonxo mHmHoHv wcHBOHHom or» :uHB muonuos >o vomuac was ou upon muouuHH o>Hw ou :uHHo um monoumomlmmouo ohms mumuuHH HH< .mH %ov Houmaumoa ou GOHumumow mo m %mv Eouw mmmm and on no 0 wow mums mums passwoum .Ammmmv mH%so£aHn woumcHEoun>Hoa ou monomxo %HHmumsumoa so loud moon vHo moo mH can mums oHo amp mH Houuaoo mo mamMHa aoum chnmsoAmmv mo :OHumaHEHHo so conmHm mo uoomwm .wH oustm mH mHaMHm 3.5.1: 113 010m 010 (III-01¢ mommhmno P 114 .Amo.ovov oCOHo cHonmso possuchHEmm mums Eopw ousHouno moon> Eouw osooomeu %HusmonHcmHm odoudoo sHmoooo oHumao: moumoHvsH wauoum< .oCHoa onu mo HouoEme was ammo HoHHmEm mos House mommaouw .mBOJm uos sun moon Hoops numvcmum sogz .muoouHH q Boom wosHMHno mums mum How .m.m H some onu musomouaou usHoa zoom .Achnmsov EzHuHHu new woumHmsm mums moHaamm Ho>HH .mouSCHa oq can .ON .m wCHBOHHow new sHo> HHmU ozu oH> wa\we m.ov deowHo msHa Amx\wa HV chomdoAmmV Ho wa\ma Hv chnm=oAm v monouchHEom woos mumm .Aomlov HmuNCOwoa and om .HMomcon Ban 0 Maelomv Hmumcumoa Ema o .Hdpmooun Ema on ”Honov HMudCumoa End 0 .Hmomaoua Ema o “moudmoaxo %HMuoHv wsH30HHow wsu :uHs meSOOE so womusa use ou coon muouuHH o>Hm ou moHHn um monoumOHImmouo mums muoouHH HH< .mH mom Houmcumoa oo GOHumumow mo w %mm Scum mmmm and om Ho 0 com mums mums newswomm .Ammmmv mHzconaHn moHMCHEouanoa 0o vomoaxo %HHMumcquQ Ho Iona mums mHo %mv mH mom moms Houucoo mHo %mm mH CH CHmnmso mo odoucoo oHumamn so onowHw mo uoomwm .mH ousmHm 0v as mmsmem Asap-Q mi; Ow R ~2KOO-fl I I nZ-aallll Z.KO0.0. I I ul§¢ul ouon UON 2.xg.d I I .2335 (1M AOOI 'Illow)uwuvno anus» 116 §1,, 1972). Thus, with the assumption that canalicular structure reflects hepatic secretory function, even the youngest group of rats in the present investigation (15 day old) may already be mature in their ability to excrete ouabain from liver. However, this function may be limited by the amount of ouabain avail— able to it. Although maturation of the hepatic excretory system may be attri— buted, to a large extent, to development of hepatic uptake mechanisms, decreased function of the remaining components of the excretory system ‘may also contribute to overall functional immaturity. This may be suggested from the fact that ouabain accumulates in liver of 21 day old rats to approximately 30% of injected dose (at the 20 minute time interval; Figure 2) and thus, transport from liver into bile may be depressed in these animals. Similarly, accumulation of BSP in liver of 125 18 day old animals may be attributed, in part, to decreased hepatic excretion (Figure 4). In regard to BSP, decreased hepatic excretion may be related to low intrinsic capacity to excrete conjugated BSP or low ip.yiyp_metabolism of free BSP to the conjugated form. lphylppg, hepatic activity of glutathione-S-aryltransferase, the enzyme that mediates conjugation of BSP to glutathione in liver, is low in young rats and increases with age (Combes and Stakelum, 1962; Krasner and Yaffe, 1968; Table 3). Since unconjugated BSP is transported from liver into bile at a slower rate than the conjugated form (Whelan pp 21,, 1970b), accumulation of BSP in liver of 18 day old rats may be partially attributed to low conjugation capacity (Table 3). Thus, the low capacity for hepatic uptake of ouabain and BSP in young rats might not be the only mechanism for retention of these drugs in plasma. In the present study, immaturity of hepatic uptake was most evi- dent in 15 day old rats, the youngest animals studied. Klaassen (1972, l973b,l974b,l975) detected low hepatic uptake of ouabain, BSP-GSH, BSP, and indocyanine green (ICG) in rats as young as 5 days old, and uptake capacity was depressed to the greatest extent in the youngest animals. Thus, relative to the adult, where hepatic uptake mechanisms are extremely rapid and not rate-limiting, the ability of young rats to accumulate drugs in liver from plasma may be markedly reduced. It is noteworthy therefore, that the time dependent pattern of hepatic dispo- sition of ouabain was exactly opposite in 15 day old rats when compared to adult rats (25-45 days old; Figure 2) even though plasma concentra- tions of ouabain were decreasing with time following ouabain admini- stration in both groups (Figure 1). This suggests that the transport 126 step influencing the elimination of ouabain from plasma in 15 day old rats is different from that in the adult. In young rats, elimination of ouabain from plasma correlates with ouabain uptake into liver. Thus, uptake may be the primary transport step for the elimination of ouabain from plasma of young rats (Figures 1,2). Whereas in the adult (25-45 day old), elimination of ouabain from plasma parallels elimina- tion of the drug from liver (Figures 1,2) and suggests biliary excre- tion of ouabain is the essential transport step (Meijer g£_§1,, 1976). Conceptually, this may indicate that, in contrast to the situation in adult rats, ouabain uptake into liver of young rats may limit overall ouabain transport. This hypothesis would be consistent with the single, as opposed to biphasic, exponential plot that may be drawn for the plasma disappearance of ouabain in 15 day old rats (Figure l). Klaassen (1973b) demonstrated that decreased biliary excretion of BSP in 7 day old rats was primarily due to a slow rate of BSP uptake. The conclusion was based on the observation that both BSP and con- jugated BSP (BSP—GSH) were retained in plasma of seven day old rats when compared to plasma concentrations in the adult (Klaassen, 1973b). Since the deficient BSP conjugating system in young rats (Combes and Stakelum, 1962; Krasner and Yaffe, 1968; Table 3) would not account for decreased biliary excretion of BSP-GSH, it was suggested that retention of BSP in plasma of newborn rats was primarily due to low hepatic uptake mechanisms (Klaassen, 1973b). In agreement with this hypothesis are the results depicted in Table 4. The rate of elimination of BSP and BSP—GSH from plasma of 15 day old rats was slower than the re- spective rate in adult rats (Table 4). It is noteworthy, however, that BSP-GSH (t% = 30 min) was removed from plasma at a slower rate than was 127 BSP (t% = 15 min) in 15 day old rats, whereas in adult rats, the rate of elimination of BSP (t% = 7 min) and BSP-GSH (t% = 6 min) from plasma was the same (Table 4). The slower rate of removal of BSP-GSH from plasma of 15 day old rats may reflect the importance of hepatic uptake in these animals. Although BSP-GSH is excreted from liver into bile more readily than is BSP (Whelan gpngl., 1970b), BSP uptake into liver is faster than uptake for BSP—GSH (Krebs, 1959; Meltzer gp_gl,, 1959; Whelan g£_§l,, 1970a). Since the rate of elimination of BSP and BSP— GSH from plasma of adult rats was the same, the lower affinity of BSP- GSH for hepatic uptake was not important in these animals during the 20 minute experimental period (Table 4). These studies therefore support the contention that hepatic uptake may be the rate-limiting step for overall drug transport in the newborn rat. Since uptake is not rate— limiting for transport in adults, these results provide a basis for further investigations for the possibility that biliary function in the newborn is not only quantitatively but also qualitatively different from function in the adult. QQifferential Effects of Bile Salt Administration, Bile Duct Ligation _§pd Hypothermia on Biliary Function in Adult and Develqping Rats Administration of the bile salt taurocholate significantly en- lnanced the elimination of BSP from.p1asma of adult rats (Figure 5). AAlthough the same treatment produced a slight reduction in plasma BSP <2oncentrations in 15 day old rats, this effect was negligible when