Comparative toxicity studies on glucuronide-forming compounds in icteric and nonicteric newborn Gunn rats

PEDIATRIC

PHARMACOLOGY

AND

T H E R A P E U T I C S WilliamL. Nyhan, Editor; Harry C. Shirkey, Consultant

Comparative toxicity studies on glucuronideforming compounds in icteric and nonicteric ne vborn Gunn rats Comparative studies on the toxicity o[ glucuronide-forming compounds were conducted in icteric and nonicteric newborn Gunn rats. Toxicity was quantitated by determining the L.D.~o in two-day-old rats. Statistically significant differences in toxicity between icterie and nonieteric littermates were found in six of ten gIucuronide-forming compounds. This animal model appears to be a useful addition to preclinical toxicologic investigations with new drugs that may be given to premature and newborn infants.

Roger A. Yeary, D.V.M. COLUMBUS~

OHIO

T 14 E R E c A Z I V ~ inability of the premature and newborn infant to form glucuronides significantly complicates the use of drugs in the perinatal period. This p a p e r is a report of investigations on the utility of the Gunn rat as an animal model for examining the toxicologic significance of U D P glucuronyltransferase deficiency in the newborn infant. Gunn 1 described a mutation in the Wistar rat in which nonhemolytic icterus appeared as a homozygous recessive trait. Postpartum icterus is rarely observed in mammalian speFrom the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, The Ohio State University. Presented in part at the annual meeting of the Society of Toxicology, Williamsburg, Virginia, March 12, 1969. Supported by National Institutes of Health Grants Nos. 1 RO1 HD-O3867-O1A1 and FR 05463. Address: 1900 Coffey Road, Columbus, Ohio 43210,

cies other than primates. The genotype for icterus has been given the notation jj.2 One cannot readily distinguish nonicteric heterozygotes (Jj) from nonicteric homozygous (JJ) dominants. However, with experience there is little difficulty in distinguishing the heterozygous newborn from the icteric newborn by visual examination. Icterus occurs in the Gunn rat as a result of deficient U D P glucuronyltransferase activity, 3 and consequently plasma levels of unconjugated bilirubin are abnormally high. 2 Kernicterus is frequently observed in icteric Gunn rats. The characteristics of the Gunn rat are shown in Table I.

MATERIALS AND M E T H O D S A breeding colony of Gunn rats was established using a monogamous mating system pairing nonicteric (Jj) females with icteric (jj) males. T h e rats were housed in polyethylene cages with ground corn cobs as Vol. 77, No. 1, pp. 139-142

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The Journal oJ Pediatrics July 1970

Yeary

Table I. Characteristics of the Gunn rat

Genotype Plasma bilirubin Icterus Glucuronyltransferase activity for: Bilirubin p-Aminobenzoic acid p-Nitrophenol

# > 5.0 rag./100 ml. Present Deficient Deficient Normal~

I

E

< 0.2 rag./100 ml. Absent Subnormal Subnormal Normal

I

H

< 0.2 mg./100 ml. Absent Normal Normal NormM

*Normal in adults only; deficient in newborn animals.

bedding. Purina L a b Chow and water were offered ad libitum and a 12 hour day-night lighting cycle was controlled automatically. T h e animal room w a s air-conditioned, and the colony was maintained as a closed random-bred breeding colony. Matings which would allow reproduction of dominant homozygous (JJ) rats were not permitted in order to avoid the difficulty of distinguishing nonicteric dominants (JJ) from heterozygotes (Jj) and to obtain the most efficient system for production of icteric (j j) animals. Verification of genotype of any breeding pair could be made by plasma bilirubin determinations and examination of the recorded characteristics of two or more litters. T h e best method for quantitatively studying the toxicity of a compound in newborn animals is the estimation of the median lethal dose (L.D.s0). 4 Groups of newborn rats were arranged so that the L.D.s0 of a given glucuronide-forming compound in icteric UDP-glucuronyltransferasedeficient rats could be compared with that found in their nonicteric littermates. This was accomplished by foster nursing litters of two-day-old rats so that one female would be nursing a litter of icteric newborn rats and another female would be nursing a litter of nonicteric newborn rats. The size of each litter was restricted to ten animals. T h e newborn rats w e r e removed from the cage only as necessary for classification and administration of test compounds. Compounds were selected on ot~e or more of the following criteria: (a) its major metabolic pathway was by glucuronidation; (b) it was a member of a chemical class with a

glucuronide-forming linkage; and (c) it was implicated in iatrogenic disease of infants. The acute toxicity of each compound was determined by parallel assays using at least one litter of ten two-day-old newborn rats per dosage level; four or more dosage levels were employed. In some instances, twoday-old Sprague-Dawley rats were utilized for comparison with a more commonly used strain of rats that is not deficient in U D P glucuronyltransferase activity at birthJ T h e animals were observed daily and the L.D.50 was calculated for the cumulative deaths after fourteen days. The method of Litchfield and Wilcoxon ~ was used for calculating the L.D.50 and for statistical comparisons of the calculated values. RESULTS

AND DISCUSSION

The results of these studies are summarized in Table II. Phenolic compounds. The three phenolic compounds investigated were acetaminophen (N-acetyl-p-aminophenol), p-nitrophenol, and o-aminophenol. Aeetaminophen was the only compound of this group that was not significantly more toxic in the icteric newborn rat. The similar L.D.50 value for p-nitrophenol in the Sprague-Dawley and icteric newborn Gunn rats was not anticipated, p-Nitrophenol is metabolized by liver homogenates at the same rate in adult U D P glucuronyltransferase-deficient Gunn rats as in homozygous dominants. 7 However, after observing the differences in the L.D.~0 value between icteric (j j) and nonicteric newborn rats (Jj), we have found that there is indeed a significant difference in the in vitro rate

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Toxicity studies on glucuronide-forming compounds

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Table II. Results of acute toxicity studies in icteric and nonicteric newborn rats L.D.,o (mg./Kg.) Compound Phenolic /)-Nitrophenol p-AminophenoI Acetaminophen

Route

Gu~n #

[

Gunn #

[

Spr~gue-D~Iex

Oral Oral Oral

122 175 i45

111-134)* 148-206) 98-213)

185 ( 162-2ii)t 400 ( 360-444)t 110 ( 80-152)9

134 (118-I53) 390 ( 339-348)t 390 ( 368-413)9

Primary alcoholic Chloramphenicol Trichlorethanol

S.C. Oral

660 185

594-733) 138-248)

635 (567-711) 285 ( 228-356)9

525 ( 477-578)t

Aromatic carboxylic Sodium salicylate p-Aminobenzoic acid

Oral S.C.

355 303-416) 1900 1650-2185)

470 ( 423-522)9 2450 (2095-2865)9

470 ( 416-531)t 3300 (2973-3661)t

Aliphatic carboxylic Indomethacin

Oral

42

40-49)

N-glucuro.nides Sodium sulfathiazole Dapsone

Oral Oral

560 80

441-711) 53-121)

Aniline HCI

Oral

122

99-151)

47 ( 4 2 - 5 3 ) 880 ( 681-1135)t 63 ( 4 3 - 9 3 )

1620 (i530-I740)9

I10 ( 8 9 - 1 3 7 )

~Ninety-five per cent confidence limits in parentheses. ?Significantly different from Gunn jj, P < 0.05.

of glucuronidation of p-nitrophenol in Iiver homogenates from jj Gunn and heterozygous Jj littermates. Similar studies will be extended to the other substrates to determine if toxicity correlates with rate of biotransformation, since displacement of protein bound bilirubin may be a contributing factor to the greater toxicity in the icteric rat# Furthermore, there is evidence suggesting that there may be a multiplicity of glucuronyltransferase enzymes with differing substrate specificities# Primary alcoholic compounds. Trichlorethanol was significantly more toxic in the icteric newborn rat. However, chloramphenicol toxicity was identical for both Gunn rat genotypes but was more toxic in the Sprague-Dawley strain. There appears to be little, if any, correlation with bilirubin conjugation. Weiss and co-workers 9 suggested that chloramphenicol conjugation occurs by a mechanism unrelated to bilirubin conjugation. Our data would agree with a hypothesis that bilirubin and chloramphenicol conjugation are catalyzed by different enzymes. Aromatic carboxylic compounds. Both sodium salicytate and p-aminobenzoic acid were more toxic in the icteric newborn G u n n

rat when compared with nonicteric littermates and with Sprague-Dawley newborn rats. Schmid and associates r~ reported that icteric adult Gunn rats metabolized o-aminobenzoic acid only about one third as well as nonicteric littermates. One would also expect both compounds to be capable of displacing protein bound bilirubin?, 11 Aliphatic carboxylic functional groups. Indomethacin forms a glucuronide on the carboxylic side chain. There was no difference in the toxicity of this compound in the Gunn rat genotypes. Studies in progress indicate that the same is true for mefenamic acid. N-glucuronides. Sodium sulfathiazole forms an N-glucuronide, but this accounts for a relatively small percentage of the excreted product. Nevertheless, this drug presented a striking difference in toxicity in the three groups of newborn rats. T h e sulfonamides have been shown to be capable of displacing bilirubin from plasma proteins, s Dapsone toxicity was similar in icteric and nonicteric rats. Aniline. Aniline is largely metabolized by hydroxytatiox rather than N-glucuronidation and the L.D.s0 for icteric and nonicteric

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Yeary

newborn rats did not differ significantly. Additional studies should be conducted in the G u n n rat with compounds which neither bind significantly to plasma proteins nor are metabolized by glucuronidation to further d o c u m e n t the assumption that toxicity differences are found only with compounds interacting by the two mechanisms. O n e m a y use adult G u n n rats for comparing the toxic effects of glucuronide-forming compounds in icteric in contrast to nonicteric animals. However, there are certain disadvantages that limit the use of adult rats. A p r a g m a t i c consideration is that one can economically obtain large numbers of newborn animals for statistically significant studies, whereas it becomes costly to rear such numbers of animals to an adult age. M o r e i m p o r t a n t is that there a p p e a r to be several isozymes for gIucuronyltransferase and we have evidence that there are developmental differences for at least one substrate, p-nltrophenol, even in the jj G u n n rat. T h e newborn jj cannot metabolize p-nitrophenol as well as heterozygous (Jj) littermates, but there is no significant difference between rates of p-nitrophenol glucuronidation in jj and Jj adult G u n n rats. Although comparisons between jj and J J G u n n rats would be preferred to comparisons between jj and Jj, the problem of distinguishing JJ from Jj newborn rats makes such comparisons unfeasible. W e have utilized the Sprague-Dawley strain for comparisons between jj G u n n newborn rats and J J SpragueDawley newborn rats but are uncertain about the variability resulting f r o m interstrain differences in the toxic effects of chemical agents. SUMMARY Acute toxicity studies of glucuronide-forming compounds in icteric (j j) newborn G u n n rats were c o m p a r e d by parallel assay with nonicteric littermates (Jj). Six of ten compounds investigated were significantly more toxic in the icteric G u n n rats wlhen compared with their nonicteric littermates. Five of seven compounds were more toxic in the icteric newborn G u n n rat than in the

The Journal o[ Pediatrics July 1970

Sprague-Dawley newborn rat. T h e greater toxicity m a y be the result of decreased glucuronidation or displacement of bilirubin f r o m plasma proteins, or both. Further studies are indicated to determine which of these factors most clearly correlates with toxicity. This animal model appears to be of value for preclinical studies on new compounds that m a y be given to p r e m a t u r e and newborn infants. The technical assistance of Ronald Grothaus, Diane Gerkin, and Marcia Fleming is greatly appreciated. We are grateful to Dr. G. B. Odell, Johns Hopkins University, for the foundation stock for the Gunn rat colony. REFERENCES

1. Gunn, C. H.: Hereditary acholuric jaundice in a new mutant strain of rats, J. Hered. 29: 137, 1938. 2. Johnson, L., Sarmiento, F., Blanc, W. A., and Day, It.: Kernicterus in rats with an inherited deficiency in glucuronyl transferase, Amer. J. Dis. Child. 97: 591, 1959. 3. Schmid, R., Axelrod, J., Hammaker, L., and Rosenthal, I. M.: Congenital defects in bilirubin metabolism, J. Clin. Invest. 36: 927, 1957. 4. Yeary, R. A.: Drug toxicity in newborn animals, Appl. Ther. 9: 918, 1967. 5. Dutton, G. J.: Neonatal drug toxicity caused by defective glucuronide synthesis: Unsuitability of the rat as a test animal?, Proc. Europ. Soc. for the Study of Drug Toxicity 4: 121, 1964. 6. Litchfield, J. T., and Wilcoxon, F.: A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96: 99, 1949. 7. VanLeusden, H. A., Bakkern, J. A., Zilliken, J. M., and Stolte, L. A. M.: P-nitrophenol glucuronide formation by homozygous adult Gunn rats, Biochem. Biophys. Res. Comm. 7: 67, 1962. 8. Johnson, L., Garcia, M. L., Figueroa, E., and Sarmiento, F.: Kernicterus in rats lacking glucuronyl transferase, Amer. J. Dis. Child. 101: 322, 1961. 9. Weiss, C. F., Glazko, A. J., and Westofa, J. K.: Chloramphenicol in the newborn infant, a physiologic explanation of its toxicity when given in excessive doses, New Eng. J. MeG 262: 787, 1960. 10. Sehmid, R., Axelrod, J., Hammaker, L., and Swarm, R. L.: Congenital jaundice in rats due to a defect in glueuronide formation, J. Clin. Invest. 37: 1123, 1958. 11. Odell, G. B.: Studies in kernicterus. I. The protein binding of bilirubin, J. Clin. Invest. 38: 823, 1959.