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H L2/f77 >^4- INSUI EIC1FNT CQ6U7rrP^GLUCUR0NIDAT10N ACTIVITY: A POSSIBLE FACTOR IN POLYCIIl.OKI NATED BIPHHKVl (PCB) T^KICITY EDWARD J. CALABRESE, Division of Public Health, University of Massachusetts, Amherst, MA 01003, U.S.A. SUMMARY Considerable evidence indicates that phenolic and biphenolic compounds are detoxified and excreted primarily via conjugate glucuronidation in man. Since fetuses, neonates and certain enzyme deficient adults have significant functional deficiencies. in their capacities to excrete toxic compounds via conjugate glucuronidalion, it is predicted that these groups of individuals arc biochemically predisposed to accumulate polychlorinated biphenyls (PCB's), a widely distributed and highly toxic environmental contaminant. INTRODUCTION Since the late 1960's when polychlorinated biphenyls (BCD's) were first recogni/.cd as a potentially widespread environmental contaminant, hundreds of research papers have been published concerning their structure, industrial uses, presence in the environment, toxicity to numerous animals from insects to man. tissue storage, metabolism and excretion (1,2). As a result ol such data accumulation, regulatory agencies arc now trying to approach the problem of standard setting with respect to PCB's. Standards for air, water and food arc necessary if the total human exposure is to be regulated and controlled. One important component in standard setting is a consideration of the individuals within the population who may be at high risk to the pollutant because of various genetic, physiological, psycho logical and behavioral traits. Individuals lacking the ability to detoxify and excrete PCB's represent such a high risk group since they will be unable to prevent undue accumu lation of toxic levels of PCB's within the body. It is the intention of this paper to: (1) establish the theoretical basis for why individuals lacking a sufficiently functional conjugate glucuronidation process may tend to accumulate unusually high levels of PCB's; (2) identify and quantify (where possible) those groups of individuals com posing this high risk group; and (3) stimulate clinical trial of the proposed hypothesis. GLUCURONIDATION IN THE EXCRETION OF TOXIC SUBSTANCES Mammals, including rabbits, dogs, and humans, have been reported to excrete PCB's. in part, by conjugate glucuronidalion and/or sulfonntion (1,3). The urinary excretion of biphenyl and 4 chlorobiphcnyl has been studied in rabbits. Biphenyl glucosiduronic acid and 4-hydroxybiphcnyl were isolated from urine. Rabbits fed 4-chlorobiphcnyl excreted 4-(p-chlorophcnyl) phenol and 4 chlorobi phcnyl glucosiduronidc. Twice as much 4-chlorobiphenyl as biphenyl was excreted as the glucosiduronic acid derivative. It was suggested that other low chlorinated biphenyls are excreted in a similar manner (3). In dogs injected with 2,4,4 '-trichloro-2 'hydroxydiphcnyl ether, nearly 100% of the material excreted in their urine and fcccs over a 5-day period appeared as the glucuronide or sulfate conjugate. Human adults similarly exposed excrete 65% in urine and 20% in feces alter intravenous injection. It is excreted as a free compound or as glu curonidc (1). Other evidence suggests that some chlorinated biphenyls are hydroxylated by species such as the rat and pigeon. No evidence of reductive dechlorination was observed in cither the trout, rat or pigeon (4). During the excretory process foreign substances (such as PCB's) usually first undergo several metabolic transform ations including oxidations, reductions, and hydrolysis. It often happens that following an oxidative, reductive or hydrolytic reaction, conjugation of the foreign substance with either glucuronic acid, sulfate, glycine, cysteine, methyl or acetyl occurs. Of these reactions, glucuronic acid con jugation is probably the most important in the animal kingdom since it occurs extensively in man and all labora tory animals with the exception of the cat. In the cat, glucuronic acid conjugation is not entirely absent, but occurs at a low level compared with oilier mammalian species. Conjugations are generally regarded as detox ification mechanisms, for compounds which undergo these reactions are converted into products which arc usually less toxic and more rapidly excreted than their precursors (5 8). The widespread occurrence of glucuronic acid con jugation may be due to the facility with which glucuronic acid can be produced in the body from carbohydrate sources and the variety of chemical groups to which glucuronic acid can be transferred enzymatically. The other conjugation mechanisms arc more restricted in their occurrence and this is probably because of the limited availability of the conjugating agents such as glxcinc anu cysteine (via glutathione) and to the small ntimbei ot chemical groupings to which conjugating agents such a sulfate, glycine, cysteine, methyl, and acetyl can be trait.-, ferred. The activity and amount of the transferring cn/\ me concerned in these conjugations arc probably also limiting factors. Glycine conjugation is confined mainly to aromatk carboxyl groups, sulfate to phenolic hydroxyl groups an. STLCOPCB4015522 DSW 0 3 1 5 6 0 coniug.uion to some aromatic Hydrocarbons and halogen Rtcd hydrocarbons, and mclhylalion to certain hydroxyl and nmino groups, and acetylation to some amino and hydrazine groups. However, glucuronic acid conjugation can occur with several types of hydroxyl, amino, and carboxyl groups and with sulfhydryl groups (5 8). The effect of conjugation of a substance with glucuronic acid is to produce a strongly acidic compound which is more water soluble at physiological pH values than the precursor. The majority of foreign substances arc ultimately cleared from the body via their excretion in the urine and bile, most often in the form of polar conjugates such as glucuronidcs with the avenue of excretion of the glucuionidc probably varying with the species or animal considered (5, (>). GROUPS AT HIGH RISK TO PCB's A. Fetuses and Neonates: Immature Fnzyme Detoxification Systems It is apparent that individuals lacking the ability to form either conjugate glucuronidcs or sulfates or both will be at high risk with respect to toxic compounds of a phenolic nature. The occurrence of low levels of glucuronidc for mation in the cat has been suggested as an explanation of the widely observed phenomenon in veterinary literature (9 12) that phenolic compounds should not be used on or around cats because of increased incidence of toxicity in this species. Furthermore, intravenous injections of phenol in cals, pigs, dogs and goats revealed that cats were at least 2 times more sensitive with respect to fatality caused by the phenol (If). Toxicity in this study was related to the partial deficiency in the cat to conjugate phenol with glucuronic acid. The author concluded that the cat is more likely to be poisoned by acute doses of phenol and is more likely to become chronically affected with phenol toxicity due to the inability to rapidly form and excrete glucuronidcs. Biochemical studies with the cat have also indicated that conjugate ghicuronidation is the major pathway for the detoxification and excretion of phenol (e g., more than twice as effective ns sulfonation) (If). Because cats are hypersusceptible to phenolic like compounds as a result of their diminished capacity lo form glucuronidcs, it is strongly suspected that human embryos, fetuses, and neonates (2 to f months old) which also arc deficient in a functional conjugate ghicuronidation system will also be predisposed to the toxic effects of phenolic compounds including PCB's (8,14,15). Usually hy the age of 2 to f months, adult levels of most enzyme systems arc achieved (8.14,15). Unfortunately, this `'developmental immaturity*' in the unborn and the very young may predispose them to the toxic effects of certain substances since they may be unable to detoxify and excrete them as quickly as necessary (lb). It bus already been pointed out that human adults do excrete, in part. PCB's via conjugate ghicuronidation. Clinical experience has shown that infants may respond differently to doses of drugs which .ire easily tolerated by older children and adults. Presum ably this is because older children and adults have fully functioning enzyme detoxification systems while the mechanisms in the scry young have broad significance since the absence of such mechanisms prevents the prompt elimination of toxic substances from the body. A further problem for the very young with regard to PCB's is that in addition to having a difficult time excreting PCB's they may also consume more PCB's per unit of body weight than at any other time during (heir life span. For example, samples of human milk from two cities in California contained aver age PCB levels of 60 ppb and 100 ppb, respectively. Based on a daily milk intake of 150 ml/kg, breast fed infants in California would ingest about 9 /<g/kg/day of PCB's. A range of 1-3 /rg/kg/day has been suggested as a "reason able" level for an Acceptable Daily Intake (2,16). One g/ kg/day has been reported as being 100 times less than the lowest "no effect*' level in animal studies (17). Thus, in children the margin is reduced to about a factor of 10 if excretory activity is equivalent with adults. However, as a result of the immature enzyme systems, the safety factor of 10 may be considerably reduced. How much is presently unknown. A tragic example of toxicity arising from the inability to form glucuronidcs has been reported. The drug chlor amphenicol, which is known to be metabolized in humans by the action of the glucuronic pathway, caused the death of more than 30 premature babies who had been treated with the antibiotic for infectious diseases. Theoretically, the premature babies were not able to conjugate the drug with glucuronic acid and thus the drug could only be slowly excreted and so (ended to accumulate in the body to toxicproportions eventually leading to death (15). An additional problem encountered by many neonates is that approximately five percent of the mothers of normal infants secrete milk which inhibits the activity of glucuronyt transferase (and thus the glucuronidation process) by more than 20 percent via the action of a steroid present in the breast milk. Inhibition of this glucuronv! transferase has been reported for up to 49 days after birth. Clincally these children have been reported to develop unusually severe neonatal jaundice. This condition develops because glucuronide formation which assists in the elimination of bilirubin (breakdown product of hemoglobin) is partially inhibited. Cow's milk does not contain sufficient amounts of this steroid lo affect a noticeable inhibition of the glucuronidation process. Consequently about 5 per cent of the neonates breast fed would be expected to have (heir ability to excrete PCB's impaired (18). Administration of the antibiotic novobiocin has also been associated with un conjugated hyperbilirubinemia in infants (19). Novobiocin is a noncompetitive inhibitor of glycuronyl transferase activity in vitro (20). Thus, children receiving concomitant exposure to novobiocin and PCB's would be expected to have their ability lo detoxify and excrete PCB's impaired. B. Individuals with Genetic Deficiencies and Modified Liver Function After the neonatal period, a broad range of conditions is associated with the improper or incomplete development of the glucuronidc conjugation system. The usual physio logical problem associated with these conditions is the inadequate -detoxification and cxcrclion of bilirubin. This DSW 031561 STLCOPCB4015523 spectrum extends from the frequently occurring "mild" condition known ns Gilbert's syndrome to the very rare, but severe and often fatal Criglcr Najjar syndrome (21), Since the clinical cITccts associated with these syndromes arc considered to be caused by metabolic disturbances ol the glucuronidc conjugation scheme, it is expected that PCB elimination in these individuals would be impeded. The population incidence of Gilbert's syndrome has been variously reported as 1 in 200 males (23). 7 percent based on examinations of 100 medical students (24) and 6 percent of 297 healthy individuals 1252 healthy National Blood Transfer Service donors, and 47 healthy medical students (197 males and 102 females)] with no difference in frequency between males and females (25). Concomitant exposure to PCB's and other drug-like chemicals may potentiate the toxic effects of PCB's. For example, rats and monkeys given SKF525A (/l-dicthylaminoelhyl 2, 2 diphenylpcntanoate, a non-specific inhibitor for many of the microsomal enzymes especially hepatic microsomal enzymes) during the initial 24 hours of ex posure to PCB succumbed rapidly as compared to the control group (22). Of possible significance is the fact that SKF525A inhibits the proper functioning of the glucuronic pathway (15). Individuals with liver infections may also be at high risk with respect to PCB's. For example, depression of glucttronidc synthesis has been observed in humans with infectious hepatitis (15). liven though PCB like pollutants require a functional conjugate glucuronidation system for their excretion, this does not imply that such substances arc merely passive molecules in this process. For example, synthesis of micro somal enzymes such as glucuronyl transferase can be stimulated by drugs such as phcnobarbital. Phenobarbital treatments have been successfully employed with patients witli a partial defect in bilirubin conjunction in order to reduce the levels of bilirubin in the blood (26,27,28,29). Thompson et al (30) demonstrated similar results with oral administration of dichlorodiphcnyl irichloroethane (pp'DDT). Although it has been suggested that the ben eficial cfTccts of phcnobarbital or DDT arc caused by their induction of glucuronyl transferase in the liver (31), con clusive evidence still remains to be demonstrated (28,32,33). Finally, supportive evidence by Allan cl al (22) has recently showed that phcnobarbital treatment prior to PCB exposure may reduce PCB toxicity by stimulating liver enzyme induction in rats. With regard to public health implications, it is still not resolved whether or mil people with a partial glucuronyl transferase deficiency may accumulate significant quantities of PCB-likc substances. These substances seem capable of inducing microsomal enzymes which lead to their (PCB's) metabolism and excretion. This constitutes a built-in safety feature w hich should help to prevent these individuals from accumulating PCB like chemicals in their bodies. In contrast, individuals with the Criglcr-Najjar syndrome seem to have an absolute deficiency of glucuronyl trans ferase, and thus do not have such a built-in compensatory safety system. However, it is not known how effective sucli a system is in those with the partial enzyme deficiency. It should also be noted that substances which require the activity of glucuronyl transferase for metabolism and excretion and which do not afTect microsomal enzyme induction would be expected to accumulate within the bodv of individuals who have any type of deficiency of glucuronyl transferase. REFERENCES 1. Goldberg I,. (Full The toxicity of polychlorinated polvcsdic com pounds and related chemicals. Cruica! Reviews in joxiculuci 2, (4). 445. 1974 2. Sclikot) IJ. (I'd) Polychlorinated biphenyl cm ironmcntai nnpau a review of the panel on hazardous trace substances. I nvirnn Research J, 249, 1972 .7. Rlnck Wl), Cornicb HU Metabolism of biphenyl and 4 chlnr obiphcnyl in rabbil. J lliol Chcin. 274 (12). 7701. 1959 4. Hut/ingcr O. Nash DM, Safe S, Dcfreitas ASVV. Nortsirotn RJ, Wildish DJ, Zilko V. Polychlorinated biphenyls: metabolic behavior of pure isomers in pigeons, rats and brook trout. Science 178, 712. 1972 5. Adamson Rlt, Davies DS. Comparative aspects of absorption, distribution, metabolism and excretion rtf drugs. Conipuruiiie I'harmocologv. Vol 2. p851. Pergamon Piess. New York. NY Michclson MJ. (lid) 1977. 6. Dutton GJ. Glucuronidc conjugation. Troe 1st Int Thurmnew Meeting. Vol (i. p79. Pergamon Press. London DM iirodtc and EG Eidas (lids) i%l. 7. Dutton GJ. The mechanism of glucuronidc formation. Ilioclierr Pliarm. 6, b5. 1961 8. Gillette JR. Individually different responses to drugs according tu age, sex and functional or pathological states. Drug Responses in Man. p24. Churchill, London. G Wolstcnholme and R Porter, (lids) 1967 9. Clarke LGC, Clarke ML. Garner's Veterinary Toxicology 7rd Edition. pp!56 158. Williams and Wilkins Co. Haltimore. 1967. 10. Jones LM. Veterinary Parmacolopy and Therapeutics. 7rd Edition, pp442-447. Iowa State University Press. Ames. 1905. I t. Steelier PG. (Ed) The Merck Index. Rth Edition, pKIO. Merck and Co, Rahveav, NJ, 1968. 12. Wilkenson TT. A review of drug toxicity in the cat. J Small Amaral Pract. 9, 21, I96R 13. Ochinc EM A comparative study of the biotransformaliini and excretion of phenol. PhD Dissertation. U or Missouri, 1969 14. Nyhan WL. Toxicology of drugs m the neonatal period. Jour Pediatrics. 59 (I), I. 1961. 1 5. Smith RL. Williams RT. Implication of the conjugation of drugs and cither exogenous compounds Glucuronic Acid. p4 57. Academic Press, NY. GJ Dutton (fed) 1966. 16. Bcrglund F. Levels of polychlorinated biphenyl in lood in Sweden. Environ lllth Pcrspcc. 1, 67, 1967 17. Vos Jll. Toxicity of PCR (in non human mammals and on birds Environ lllth Pcrspcc. I, 120. 1972 18. Garllicr LM, Arias IM. Studies of prolonged neonatal jaundice m the breast fed infant. Jour Pediatrics. 68 (I). 54. 1966 19. Sutherland JM. Keller WII. Novobicin and neonatal hypcrlnlt ruhinemta. Am J Dis Child. 101, 4-17. 190 1 20. Lokeitz II. Dowhen RM. Ifsics DY. Studies tin the effect of novobiocin and glucuronyl transferase. Pediatries 72, 47. 1967 21. Lcslcr R. Schmid R. Bilirubin metabolism. New I ng J Med. 270 (15). 779 1964 22. Allen JR. Cartons LA, Abrahamson IT. Marlar RJ Responses ol rats and non human primates to 2,5,2 '.5' tctrachlurohiphcnsl. Environ Research 9, 265, 1975 27. Billing till. Bilirubin metabolism and jaundice with special refer cnee to unconjugalcd hyperbilirubinemia. Annals of Clin lliocltcm. 7, 69. 1970 24. Kornberg A. Latent liver disease in persons recovered from catarrhal jaundice and in otherwise normal medical students as revealed by 1 be bilirubin excretion lest. Jour Clin Invest 21, 299. 1942 ' 25. Owens [7, loans J. Population studies mi gilbert's syndrome. Jour Med Genetics. 12, 152, 1976 26. Yaflc SJ. Levy G. Matsuz.awa T, llaltah T. Enhancement of glu cumnidc-conpigating capacity in a hyperbiliriibinenue inlani due to apparent enzyme induction by phcnobarbital. New bug J Med. 275, 1461. 1966 27. Smith PM. Middleton JE. Williams R. Studies on the iiiimlial incidence ami clinical history of patients with chronic tmcmijugated hyperbilirubinemia Gm. 8. 469. 1968 28. Arias IM. Gartner EM. Cohen M. Issci JIL Levi AL l In on., non hemolytic unconjugalcd hyperbilirubinemia with glucuron)l transferase deficiency. Amcr J Med. 47, 795, 1969 29. Krcck MJ. Sleisenger MIL Reduction of serum unconjugated bilirubin with hyperbilirubinaemia. Lancet I. 7 7. 1968 70. Thompson RPG. Slathers GM. Pilcher CWT, McLean At:M. Robinson J. Williams R. Treatment of unconjugalcd jaundicr witli dicoptiaoc Lancet 1. 4. 1969 . , DSW 0 3 1 5 6 2 STLCOPCB4015524 32. Whclton NM. Kruslev LP. Hilling HM. Reduction in scrum bilirubin by phcnobnrbiinl in adult unconjugalcd hyperbilirubinemia. Is niryme induction ro/wriMWc? Arncr J Med, 45* J00, 1968 33. Mnrver MS. Schmid R. Hinlransformntion in the liver: implications fen human disease. Ciastroenierolopy 55, ?82. IQ6H DSW 031563 165 STLCOPCB4015525