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Effect of alteration of rat hepatic mixed-function oxidase (MFO) activity on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
45,5 13-519 (1978)
Effect of Alteration of Rat Hepatic Mixed-Function Oxidase (MFO) Activity on the Toxicity of 2,3,7,8-Tetrachlorodibenzop-Dioxin (TCDD) PATRICKW. BEATTY,~ILLIAM Center
in Environmental School Received
K. VAUGHN,ANDROBERT A. NEAL
Toxicology, Department of Medicine, Nashville,
August
l&1977;
accepted
of Biochemistry, Tennessee December
Vanderbilt
University
37232 19,1977
Effect of Alteration of Rat Hepatic Mixed-Function Oxidase (MFO) Activity on the Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). BEAT-N, P. W., VAUGHN, W. K., AND NEAL, R. A. (1978). Toxicol. Appl. Pharmacol. 45,5 13-5 19. The effect of alteration of the activity of the hepatic mixed-function oxidase (MFO) enzyme systems on the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in rats has been examined. These experiments have indicated the naturally occurring age and sex-related differences in hepatic MFO activity in rats are inversely correlated with the 20-day LD50 of TCDD in these animals. The effect of administering inducers and inhibitors of the hepatic MFO enzyme systems on the 20-day LD50 of TCDD in rats has also been examined. In all cases there was an inverse relationship between the activity of the hepatic MFO system at the time of TCDD administration and the toxicity of TCDD as determined by the 20-day LD50.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant in the manufacture of 2,4,5trichlorophenol (Crummet and Stehl, 1973). TCDD is one of the most toxic synthetic chemicals known. It has an oral LD50 of approximately lpg/kg in the adult male guinea pig (Schwetz et al., 1973). It is not known at this time whether the toxicity of TCDD is the result of the action of the parent compound or some metabolite(s). In one study of the metabolism of TCDD in mammals (Vinopal and Casida, 1973), the authors were unable to detect metabolism of TCDD by microsomal preparations from mouse, rat, and rabbit liver. In addition, these authors were not able to demonstrate the in vivo metabolism of TCDD in the mouse. However, in two in vivo studies carried out in rats using l14ClTCDD it has been reported that small amounts (3-18%) of the administered radioactivity are excreted in the urine (Allen et al., 1975; Rose et al., 1976). These data suggest the excretion of TCDD metabolites in the urine. However, attempts at isolation and identification of metabolites of TCDD from urine, feces, or in vitro incubations have not been successful. If TCDD is metabolized by mammals, the most logical enzyme catalyzing this metabolism would be the hepatic MFO enzyme systems. The most probable metabolite formed by the action of these enzyme systems would be one in which the 1, 4, 6, or 9 position (all equivalent) is hydroxylated to form a phenol derivative of TCDD. The MFO-catalyzed metabolism of TCDD to a phenol derivative would most likely proceed 513
~l-o08X/78/0452-0521%02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
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by a carbon-hydrogen insertion of an oxygen atom (Selander et al., 1975a,b; Tomaszewski et al., 1975). The MFO-catalyzed metabolism of TCDD could increase or decrease the effect or have no effect on the toxicity of TCDD. If an increase in the toxicity of TCDD were observed on increasing the activity of the hepatic MFO enzyme systems, it would suggest that a metabolite(s) of TCDD was largely responsible for the toxicity. On the other hand, if a decrease in toxicity were seen on increasing the activity of the hepatic MFO system, it would suggest that TCDD itself was responsible for the toxicity. In these studies we have increased and decreased the activity of the hepatic MFO enzyme systems in adult male, adult female, and weanling male rats and examined the effect of these changes in MFO activity on the toxicity of TCDD. METHODS
Adult male, castrated adult male, adult female, and weanling male Sprague-Dawley rats were used in these experiments. TCDD was a gift of the Dow Chemical Co. Its purity was judged to be greater than 98%, as determined by gas chromatography (Rose et al., 1976). Hemin,’ 3-methylcholanthrene,’ testosterone propionate,’ 3,4-benzo[alpyrene,’ piperonyl butoxide, glucose 6-phosphate dehydrogenase,4 cobaltous chloride,3 glucose 6-phosphate,4 NADP,4 NADPH,4 aniline-HCJ5 p-aminophenol,5 and aminopyrine5 were obtained commercially. Rat liver microsomes were prepared, as described by Neal (1967). Aniline hydroxylase was assayed by the method of Brodie and Axelrod (1948) as modified by Kato and Gillette (1965). Aminopyrine-ZV-demethylase was assayed by the method of Cochin and Axelrod (1959). The formaldehyde formed was measured by the method of Nash (1953). Benzpyrene hydroxylase (aryl hydrocarbon hydroxylase) was assayed according to Gielen et al, (1972) as modified by Poland and Glover (1973). Fluorescence because of hydroxylated metabolites of benzpyrene was expressed as quinine sulfate units, according to Hook et al. (1972). Microsomal protein was measured by the biuret reaction (Layne, 1957) modified to contain 0.1 ml of 1% deoxycholate. LD50 values were determined by a standard bioassay probit analysis (Finney. 197 I). In these experiments, groups of six rats were either untreated or pretreated with inducers or inhibitors of hepatic MFO activity. For each LD50 determination, TCDD was administered ip in olive oil at four dosages chosen to include the LD50 value. Dosages of TCDD in experiments with adult male, adult female, and weanling male rats ranged from 20 to 80 ,ug/kg, 10 to 60 ,ug/kg, and 5 to 50 pug/kg, respectively. Unless otherwise specified, statistical significance was determined by analysis of variance and Duncan’s multiple range test (Duncan, 1970). RESULTS
In the initial experiments examining the relation between hepatic MFO activity and TCDD toxicity, we utilized the naturally occurring age and sex-dependent differences in ’ Sigma Chemical Co., St. Louis, Missouri. ’ ICN Life Sciences Group, Cleveland, Ohio. ’ Fisher Scientific Co., Pittsburg, Pennsylvania. 4 Boehringer-Mannheim, Indianapolis, Indiana. 5 J. T. Baker Chemical Co., Glen Ellyn. Illinois.
515
HEPATXC MFO ACTIVITY AND TCDD TOXICITY
the activity of these enzyme systems in rats. The hepatic MFO activity in adult male Sprague-Dawley rats is two to three fold greater than that of adult female rats (Quinn et al., 1958). The hepatic MFO activity in weanling male rats is quantitatively similar to that of adult females. During maturation (30-40 days of age), however, the activity quickly rises to adult male levels. Table 1 presents the 20-day LD50 of TCDD in adult TABLE 1 TWENTY-DAY LD50 OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN ADULT MALE, ADULT FEMALE, AND MALE WEANLING RATS“ Animals
(&kg,
LD50b mean + SE)
60.2 k 7.8 24.6 + 2.0c 25.2 * 1.4c
Adulte male Adult female Weanling male
a TCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. The volume of olive oil injected was kept constant at 3 ml/kg. The weanling male rats were 25 days old at the start of these experiments. b LD50 values were determined by probit analysis based on the number of survivors at each dose 20 days after TCDD injection. c Significantly different (p < 0.05) compared to adult males, Duncan’s multiple range test. TABLE
2
OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 OF CASTRATION OF MALE RATS OR TESTOSTERONE TREATMENT OF FEMALE RATS
EFFECT ON HEPATIC MIXED-FUNCTION TETRACHLORODIBENZO-P-DIOXIN (TCDD)
Sex Male Female
Aminopyrine demethylase”vb
Aniline hydroxylase”*’
LD50 (pug/kg, mean + SE)d.”
None Castration
1.641 + 0.209 1.207 + 0.04Y
0.0143 2 0.0007 0.0098 f 0.0007f
60.2 f 7.8 39.1 * 2.18
None Testosterone
1.089 k 0.148 1.298 + 0.189
0.0084 + 0.0007 0.0124 &- 0.0015’
24.6 + 2.0 44.5 k 1.5’
Treatment
’ Hepatic microsomes were prepared, as described under Methods, from male rats 2 weeks after castration and in females 24 hr after the last dose of testosterone propionate (100 mg/kg/day for 3 days). Values shown are the mean t SE of values from three rats. b Nanomoles of HCHO formed per minute per milligram of protein. c Micrograms ofp-aminophenol formed per minute per milligram of protein. dTCDD was administered ip in olive oil at four dosages chosen to include the LD50. Six rats were injected at each dose. TCDD was administered 2 weeks after castration and 24 hr after the last injection of testosterone propionate (100 mg/kg/day for 3 days). e LD50 values were determined by probit analysis. based on the number of survivors at each dose 20 days after TCDD injection. ‘Significantly different (p < 0.05) compared to nontreated rats of the same sex. Student’s I test. g Significantly different (p < 0.05) compared to nontreated rats of the same sex. Duncan’s multiple range test.
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male, adult female, and weanling male Sprague-Dawley rats. TCDD exhibits nearly equal toxicity in adult females and weanling males, whereas the LD50 of TCDD in the adult males is more than twice that of the females and weanling males. The hepatic MFO activity of adult male rats can be decreased to values approximating those of adult females by castration (Siami ef al., 1972). Similarly, the hepatic MFO activity of female rats can be increased by administering testosterone (Siami et al., 1972). Table 2 shows the effect of prior castration of male rats or administering testosterone propionate at 100 mg/kg/day for 3 days to female rats on MFO activities. As can be seen, castration decreased and testosterone increased the TABLE 3 HEPATIC MICROSOMAL MIXED-FUNCTION OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) TO MALE WEANLING RATS PRETREATED WITH PHENOBARBITAL (PB), 3-METH~LCHOLANTHRENE (3-MC), OR TCDD” Treatment None PB 3-MC TCDD
Aniline hydroxylaseb
Benzpyrene hydroxylaseC
LD50 (,&kg, mean+ SE)dse
0.012 * 0.001 0.015 f- 0.001’ 0.014 * 0.000 0.028 + 0.001’
10.3+ 3.68 55.4 + 8.35f 213 + 25.4’ 226 rf: 0.001’
25.2 + 1.4 40.9 -L 1.39 44.1 & 1.29 36.8 5 1.8g
0 Hepatic microsomes were prepared as described under Methods. PB was injected ip at 50 mg/kg/day for 3 days, and microsomes were prepared 24 hr after the last injection. 3-MC (40 mg/kg) and TCDD (5 pug/kg) were administered ip 72 hr prior to the preparation of microsomes. Values shown are the mean + SE of values from three rats. b Micrograms ofp-aminophenol formed per minute per milligram of protein. c Quinine-SO,, units per minute per milligram of protein. dTCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. Phenobarbital was administered (50 mg/kg/day) by ip injection for 3 days. The various doses of TCDD were administered 24 hr after the last dose of PB. 3-MC (40 mg/kg) and TCDD (5 pg/kg) were administered ip 72 hr prior to administration of the various doses of TCDD. e LD50 values were determined by probit analysis, based on the number of survivors at each dose 20 days after TCDD injection. JSignificantly different (p < 0.05) compared to nontreated weanlings, Student’s t test. g Significantly different (p < 0.05) compared to untreated weanlings. Duncan’s multiple range test.
MFO activities in these rats, compared to the respective controls of the same sex. Table 2 also presents the effects of these same treatments on the LD50 of TCDD in male and female rats. Castration decreased the 20-day LD50 of TCDD in adult male rats, and testosterone administration nearly doubled the amount of TCDD required for an LD50 dose in adult female rats. These studies of the relationship of hepatic MFO activity and TCDD-mediated lethality were extended to include the effects of inducers of the hepatic monooxygenase enzymes on TCDD toxicity. Both phenobarbital (PB) and 3-methylcholanthrene (3MC) are classic inducers of the hepatic MFO enzyme systems (Conney, 1967). In addition, TCDD itself is a potent inducer of aryl hydrocarbon hydroxylase (Poland and Glover, 1974). Table 3 shows the resulting MFO activities in hepatic microsomes of male weanling rats pretreated with PB, 3-MC, or TCDD. PB pretreatment caused a
significant increase in both microsomal MFO activities assayed, as did pretreatment with TCDD. 3-MC caused a significant increase in benzpyrene hydroxylation but had
HEPATIC
MFO
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517
TOXICITY
no effect on aniline hydroxylation. Table 3 also shows the effect of pretreatment with PB, 3-MC, or TCDD on the toxicity of TCDD. All three pretreatments exerted a significant protective effect against TCDD-related lethality. The effect of decreases in the hepatic MFO activity in ~iuo on the LD50 of TCDD in rats was also examined. The administration of CoCl, to rats has been shown to reduce the activity of hepatic MFO systems in the liver (Tephly and Hibbeln, 1971). The administration of hemin to rats also causes a reduction in hepatic MFO activity (Bock et al., 1973). TABLE 4 EFFECT OF COBALTOLJS CHLORIDE AND HEMIN ON HEPATIC MIXED-FUNCTION OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN WEANLING RATS’
LD50
Benzpyrene Treatment
hydroxylaseb
None COCI, Hemin Piperonyl butoxide
24.2 f 5.1 10.2 * 8.8’ 12.8 + 7.0’
(,&kg,
mean & SEpd 33.8 20.6 27.5 19.7
k 13.8 k 3.9 + 3.8 Ifr 4.3
(1Hepatic microsomes were prepared 24 hr after the last injection of CoCl, (60 mg/kg/day SCfor 2 days) or hemin (20 mg/kg/day ip for 4 days). The values shown are the mean & SE of values from three rats. * Quinine-SO, units per minute per milligram of protein. c TCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. CoCl, was administered SC at 20 mg/kg/day for 2 days, and the various doses of TCDD were injected 24 hr after the last dose of CoCI,. Hemin was injected ip at 20 mg/kg/day for 4 days, and the various doses of TCDD were injected 24 hr after the last dose of hemin. Piperonyl butoxide was administered ip at 400 mg/kg 2 hr prior to the injection of TCDD and once per day for 2 days thereafter. d LD50 values were determined by probit analysis, based on the number of survivors at each dose 20 days after TCDD injection. e Significantly different (p < 0.05) compared to untreated weanlings. Student’s t test.
Table 4 presents the effects of CoCI, and hemin pretreatment on benzpyrene hydroxylase activity in weanling rats. Both CoCl, and hemin pretreatment resulted in an approximate 50% decrease in benzpyrene hydroxylase activity, as compared to untreated controls. The effect of CoCl,, hemin, and piperonyl butoxide on the toxicity of TCDD in weanling male rats is also shown in Table 4. Piperonyl butoxide is a wellknown competitive inhibitor of hepatic MFO enzyme systems. The effect of piperonyl butoxide on benzpyrene hydroxylase activity was not included because of the competitive nature of its inhibition of this enzyme system. In each case, treatment with the inhibitor resulted in the lowered LD50 values for TCDD in these rats. These lowered LD50 values were not, however, statistically significant. This is most likely because of the large variance in the LD50 value for TCDD in the control rats. The LD50 of TCDD in untreated weanling male rats in Table 4 is larger than that seen in
518
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AND
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untreated weanling male rats in Table 3. This difference is perhaps due to the somewhat greater age of the rats used in the experiment shown in Table 4 (30 days) than those used in the experiment shown in Table 3 (25 days). At 30 days of age the hepatic MFO activity of weanling male rats is just beginning to rise to adult values. This may account for the increased LD50 of TCDD in the controls of the experiment in Table 4 as compared to Table 3. This transition to adult enzyme activities may also account for the large variability in the LD50 values of the control animals in Table 4. DISCUSSION
The results of the experiments described herein indicate that an inverse relationship exists between the activity of the hepatic MFO enzyme systems and the toxicity, as measured by the ip LD50 of TCDD in the rat. Examination of adult male, adult female, and immature male rats reveals that the adult males with naturally high MFO activity are less susceptible to the lethal effects of TCDD. Induction of the hepatic MFO enzyme system in females by administration of testosterone or in weanling males by various chemical inducers such as PB, 3-MC, or a small dose of TCDD itself is correlated with a decrease in TCDD toxicity in these animals. Decreasing the hepatic MFO activity by castration of adult male rats or by administration of inhibitors of this enzyme system such as CoCl,, hemin, or piperonyl butoxide to weanling male rats is associated &ith an increase in the toxicity of TCDD with respect to noncastrated or untreated controls. Although other explanations such as altered steroid concentrations, changes in fatty acid metabolism, changes in cellular ultrastructure, etc. may account for this effect, a more likely explanation is that TCDD is metabolized by the hepatic MFO enzyme systems in rats and that this metabolism results in the formation of a chemical species which is less toxic than the parent compound. Verification of this hypothesis awaits the demonstration of the excretion of a metabolite(s) of TCDD in vim, examination of the toxicity of this metabolite(s), and the demonstration of increased excretion of this metabolite(s) in response to elevated hepatic MFO activity.
ACKNOWLEDGMENTS
This work was supported by USPHS Grants ES00267 and ES01552. Patrick Beatty also gratefully acknowledges the training support provided by Grant ES07028. REFERENCES ALLEN, J. R., VAN MILLER, J. P., AND NORBACK, D. H. (1975). Tissue distribution, excretion and biological effects of 114Cltetrachlorodibenzo-p-dioxin in rats. Food Cosmet. Toxicol. 13, 501-505. BOCK, K. W., FROHLING, W., AND REMMER, H. (1973). Influence of fasting and hemin on microsomal cytochromes and enzymes. Biochem. Pharmacol. 22,1557-1564. BRODIE, B. B., AND AXELROD, J. (1948). The fate of acetanilide in man. J. Pharmacol. Exp. Ther. 94,29-38. COCHIN, J., AND AXELROD, J. (1959). Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine, and normorphine. J. Pharmacol. Exp. Ther. 125, 105-l 10.
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ACTIVITY
AND
TCDD TOXICITY
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CONNEY, A. H. (1967). Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev. 19,3 17-366. CRUMMETT,W. B., ANDSTEHL,R. H. (1973). Determinationof chlorinateddibenzop-dioxins anddibenzofuransin variousmaterials.Environ. Health Perspect. 5, 15-25. DUNCAN,D. B. (1970). Multiple comparisons.Methods for comparingregressioncoefficients. Biometrics 26, 141-142. FINNEY,D. J. (1971).Probit Analyses, pp. 66-70. CambridgeUniv. Press,London/NewYork. GIELEN,J. E., GOUJON,F. M., AND NEBERT,D. W. (1972), Genetic regulation of aryl hydrocarbonhydroxylaseinduction.J. Biol. Chem. 247, 1125-l 137. HOOK,G. E. R., BEND,J. R., AND FOUTS,J. R. (1972). Mixed-function oxidasesand the alveolarmacrophage.Biochem. Pharmacol. 21,3267-3277. KATO, R., ANDGILLETTE,J. R. (1965). Effect of starvationof NADPH-dependentenzymesin liver microsomes of maleand femalerats.J. Pharmacol. Exp. Ther. 150,279-284. LAY-NE,E. (1957). Spectrophotometricand turbidimetric methodsfor measuringproteins. Methods Enzymol. 3,447-454.
NASH, T. (1953). The calorimetric estimationof formaldehydeby meansof the Hantzsch Reaction.Biochem. J. 55,4 16-421. NEAL, R. A. (1967). Studieson the metabolismof diethyl 4-nitrophenyl phosphorothionate (parathion)in vitro. Biochem. J. 103, 183-191. POLAND,A., AND GLOVER,E. (1973). Chlorinated dibenzo-p-dioxins:Potent inducersof 6aminolevulinicacid synthetaseandaryl hydrocarbonhydroxylase.II. A study of the structure activity relationship.Mol. Pharmacol. 9, 736-747. POLAND,A., AND GLOVER,E. (1974). Comparisonof 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene.Mol. Pharmacol. 10,349-359.
QUINN,G. P., AXELROD,J., AND BRODIE,B. B. (1958). Species,strain and sex differencesin metabolismof hexobarbitone,amidopyrine,antipyrine and aniline.Biochem. Pharmacol. 1, 152-159. ROSE,J. Q., RANSEY,J. C., WENTZLER, T. H., HUMMELL,R. A., ANDGEHRING,P. J. (1976). The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following singleand repeatedoral dosesto the rat. Toxicol. Appl. Pharmacol. 36,209-226. SCHWETZ, B. A., NORRIS, J. M., SPARSCHU, G. L., ROWE,V. K., GEHRING,P. J., EMERSON, J. L., ANDGERBIG,C. G. (1973).Toxicology of chlorinateddibenzop-dioxins.Environ. Health Perspect. 5,87-99.
SELANDER, H. G., JERINA,D. M., ANDDALY, J. W. (1975a).Metabolismof chlorobenzene with hepaticmicrosomesandsolubilizedcytochromeP-450 systems.Arch. Biochem. Biophys. 168, 309-321. SELANDER, H. G., JERINA,D. M., PICCOLO, D. E., ANDBERCHTOLD, G. A. (1975b).Synthesis of 3- and 4chlorobenzene oxides. Unexpected trapping results during metabolismof [i4C]chlorobenzeneby hepaticmicrosomes. J. Amer. Chem. Sot. 97,4428-4430. SIAMI, G., SCHULERT, A. R., ANDNEAL, R. A. (1972). A possiblerole for the mixed function oxidaseenzymesystemin the requirementfor seleniumin the rat. J. Nutr. 102,857-86 1. TEPHLY,T. R., AND HIBBELN,P. (1971). The effect of cobalt chloride administrationon the synthesisof hepatic microsomalcytochromeP-450. Biochem. Biophys. Res. Common. 42, 589-595.
TOMASZEWSKI, J. E., JERINA,D. M., ANDDALY, J. W. (1975).Deuteriumisotopeeffectsduring formation of phenolsby hepatic monoxygenases.Evidencefor an alternative to the arene oxide pathway. Biochemistry 14, 2024-2031. VINOPAL,J. H., ANDCASIDA,J. E. (1973). Metabolic stability of 2,3,7,8-tetrachlorodibenzo-pdioxin in mammalianliver microsomalsystemsand in living mice.Arch. Environ. Contam. Toxicol. 1, 122-132.
AND
APPLIED
PHARMACOLOGY
45,5 13-519 (1978)
Effect of Alteration of Rat Hepatic Mixed-Function Oxidase (MFO) Activity on the Toxicity of 2,3,7,8-Tetrachlorodibenzop-Dioxin (TCDD) PATRICKW. BEATTY,~ILLIAM Center
in Environmental School Received
K. VAUGHN,ANDROBERT A. NEAL
Toxicology, Department of Medicine, Nashville,
August
l&1977;
accepted
of Biochemistry, Tennessee December
Vanderbilt
University
37232 19,1977
Effect of Alteration of Rat Hepatic Mixed-Function Oxidase (MFO) Activity on the Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). BEAT-N, P. W., VAUGHN, W. K., AND NEAL, R. A. (1978). Toxicol. Appl. Pharmacol. 45,5 13-5 19. The effect of alteration of the activity of the hepatic mixed-function oxidase (MFO) enzyme systems on the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in rats has been examined. These experiments have indicated the naturally occurring age and sex-related differences in hepatic MFO activity in rats are inversely correlated with the 20-day LD50 of TCDD in these animals. The effect of administering inducers and inhibitors of the hepatic MFO enzyme systems on the 20-day LD50 of TCDD in rats has also been examined. In all cases there was an inverse relationship between the activity of the hepatic MFO system at the time of TCDD administration and the toxicity of TCDD as determined by the 20-day LD50.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant in the manufacture of 2,4,5trichlorophenol (Crummet and Stehl, 1973). TCDD is one of the most toxic synthetic chemicals known. It has an oral LD50 of approximately lpg/kg in the adult male guinea pig (Schwetz et al., 1973). It is not known at this time whether the toxicity of TCDD is the result of the action of the parent compound or some metabolite(s). In one study of the metabolism of TCDD in mammals (Vinopal and Casida, 1973), the authors were unable to detect metabolism of TCDD by microsomal preparations from mouse, rat, and rabbit liver. In addition, these authors were not able to demonstrate the in vivo metabolism of TCDD in the mouse. However, in two in vivo studies carried out in rats using l14ClTCDD it has been reported that small amounts (3-18%) of the administered radioactivity are excreted in the urine (Allen et al., 1975; Rose et al., 1976). These data suggest the excretion of TCDD metabolites in the urine. However, attempts at isolation and identification of metabolites of TCDD from urine, feces, or in vitro incubations have not been successful. If TCDD is metabolized by mammals, the most logical enzyme catalyzing this metabolism would be the hepatic MFO enzyme systems. The most probable metabolite formed by the action of these enzyme systems would be one in which the 1, 4, 6, or 9 position (all equivalent) is hydroxylated to form a phenol derivative of TCDD. The MFO-catalyzed metabolism of TCDD to a phenol derivative would most likely proceed 513
~l-o08X/78/0452-0521%02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
514
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by a carbon-hydrogen insertion of an oxygen atom (Selander et al., 1975a,b; Tomaszewski et al., 1975). The MFO-catalyzed metabolism of TCDD could increase or decrease the effect or have no effect on the toxicity of TCDD. If an increase in the toxicity of TCDD were observed on increasing the activity of the hepatic MFO enzyme systems, it would suggest that a metabolite(s) of TCDD was largely responsible for the toxicity. On the other hand, if a decrease in toxicity were seen on increasing the activity of the hepatic MFO system, it would suggest that TCDD itself was responsible for the toxicity. In these studies we have increased and decreased the activity of the hepatic MFO enzyme systems in adult male, adult female, and weanling male rats and examined the effect of these changes in MFO activity on the toxicity of TCDD. METHODS
Adult male, castrated adult male, adult female, and weanling male Sprague-Dawley rats were used in these experiments. TCDD was a gift of the Dow Chemical Co. Its purity was judged to be greater than 98%, as determined by gas chromatography (Rose et al., 1976). Hemin,’ 3-methylcholanthrene,’ testosterone propionate,’ 3,4-benzo[alpyrene,’ piperonyl butoxide, glucose 6-phosphate dehydrogenase,4 cobaltous chloride,3 glucose 6-phosphate,4 NADP,4 NADPH,4 aniline-HCJ5 p-aminophenol,5 and aminopyrine5 were obtained commercially. Rat liver microsomes were prepared, as described by Neal (1967). Aniline hydroxylase was assayed by the method of Brodie and Axelrod (1948) as modified by Kato and Gillette (1965). Aminopyrine-ZV-demethylase was assayed by the method of Cochin and Axelrod (1959). The formaldehyde formed was measured by the method of Nash (1953). Benzpyrene hydroxylase (aryl hydrocarbon hydroxylase) was assayed according to Gielen et al, (1972) as modified by Poland and Glover (1973). Fluorescence because of hydroxylated metabolites of benzpyrene was expressed as quinine sulfate units, according to Hook et al. (1972). Microsomal protein was measured by the biuret reaction (Layne, 1957) modified to contain 0.1 ml of 1% deoxycholate. LD50 values were determined by a standard bioassay probit analysis (Finney. 197 I). In these experiments, groups of six rats were either untreated or pretreated with inducers or inhibitors of hepatic MFO activity. For each LD50 determination, TCDD was administered ip in olive oil at four dosages chosen to include the LD50 value. Dosages of TCDD in experiments with adult male, adult female, and weanling male rats ranged from 20 to 80 ,ug/kg, 10 to 60 ,ug/kg, and 5 to 50 pug/kg, respectively. Unless otherwise specified, statistical significance was determined by analysis of variance and Duncan’s multiple range test (Duncan, 1970). RESULTS
In the initial experiments examining the relation between hepatic MFO activity and TCDD toxicity, we utilized the naturally occurring age and sex-dependent differences in ’ Sigma Chemical Co., St. Louis, Missouri. ’ ICN Life Sciences Group, Cleveland, Ohio. ’ Fisher Scientific Co., Pittsburg, Pennsylvania. 4 Boehringer-Mannheim, Indianapolis, Indiana. 5 J. T. Baker Chemical Co., Glen Ellyn. Illinois.
515
HEPATXC MFO ACTIVITY AND TCDD TOXICITY
the activity of these enzyme systems in rats. The hepatic MFO activity in adult male Sprague-Dawley rats is two to three fold greater than that of adult female rats (Quinn et al., 1958). The hepatic MFO activity in weanling male rats is quantitatively similar to that of adult females. During maturation (30-40 days of age), however, the activity quickly rises to adult male levels. Table 1 presents the 20-day LD50 of TCDD in adult TABLE 1 TWENTY-DAY LD50 OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN ADULT MALE, ADULT FEMALE, AND MALE WEANLING RATS“ Animals
(&kg,
LD50b mean + SE)
60.2 k 7.8 24.6 + 2.0c 25.2 * 1.4c
Adulte male Adult female Weanling male
a TCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. The volume of olive oil injected was kept constant at 3 ml/kg. The weanling male rats were 25 days old at the start of these experiments. b LD50 values were determined by probit analysis based on the number of survivors at each dose 20 days after TCDD injection. c Significantly different (p < 0.05) compared to adult males, Duncan’s multiple range test. TABLE
2
OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 OF CASTRATION OF MALE RATS OR TESTOSTERONE TREATMENT OF FEMALE RATS
EFFECT ON HEPATIC MIXED-FUNCTION TETRACHLORODIBENZO-P-DIOXIN (TCDD)
Sex Male Female
Aminopyrine demethylase”vb
Aniline hydroxylase”*’
LD50 (pug/kg, mean + SE)d.”
None Castration
1.641 + 0.209 1.207 + 0.04Y
0.0143 2 0.0007 0.0098 f 0.0007f
60.2 f 7.8 39.1 * 2.18
None Testosterone
1.089 k 0.148 1.298 + 0.189
0.0084 + 0.0007 0.0124 &- 0.0015’
24.6 + 2.0 44.5 k 1.5’
Treatment
’ Hepatic microsomes were prepared, as described under Methods, from male rats 2 weeks after castration and in females 24 hr after the last dose of testosterone propionate (100 mg/kg/day for 3 days). Values shown are the mean t SE of values from three rats. b Nanomoles of HCHO formed per minute per milligram of protein. c Micrograms ofp-aminophenol formed per minute per milligram of protein. dTCDD was administered ip in olive oil at four dosages chosen to include the LD50. Six rats were injected at each dose. TCDD was administered 2 weeks after castration and 24 hr after the last injection of testosterone propionate (100 mg/kg/day for 3 days). e LD50 values were determined by probit analysis. based on the number of survivors at each dose 20 days after TCDD injection. ‘Significantly different (p < 0.05) compared to nontreated rats of the same sex. Student’s I test. g Significantly different (p < 0.05) compared to nontreated rats of the same sex. Duncan’s multiple range test.
516
BEATTY, VAUGHN, AND NEAL
male, adult female, and weanling male Sprague-Dawley rats. TCDD exhibits nearly equal toxicity in adult females and weanling males, whereas the LD50 of TCDD in the adult males is more than twice that of the females and weanling males. The hepatic MFO activity of adult male rats can be decreased to values approximating those of adult females by castration (Siami ef al., 1972). Similarly, the hepatic MFO activity of female rats can be increased by administering testosterone (Siami et al., 1972). Table 2 shows the effect of prior castration of male rats or administering testosterone propionate at 100 mg/kg/day for 3 days to female rats on MFO activities. As can be seen, castration decreased and testosterone increased the TABLE 3 HEPATIC MICROSOMAL MIXED-FUNCTION OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) TO MALE WEANLING RATS PRETREATED WITH PHENOBARBITAL (PB), 3-METH~LCHOLANTHRENE (3-MC), OR TCDD” Treatment None PB 3-MC TCDD
Aniline hydroxylaseb
Benzpyrene hydroxylaseC
LD50 (,&kg, mean+ SE)dse
0.012 * 0.001 0.015 f- 0.001’ 0.014 * 0.000 0.028 + 0.001’
10.3+ 3.68 55.4 + 8.35f 213 + 25.4’ 226 rf: 0.001’
25.2 + 1.4 40.9 -L 1.39 44.1 & 1.29 36.8 5 1.8g
0 Hepatic microsomes were prepared as described under Methods. PB was injected ip at 50 mg/kg/day for 3 days, and microsomes were prepared 24 hr after the last injection. 3-MC (40 mg/kg) and TCDD (5 pug/kg) were administered ip 72 hr prior to the preparation of microsomes. Values shown are the mean + SE of values from three rats. b Micrograms ofp-aminophenol formed per minute per milligram of protein. c Quinine-SO,, units per minute per milligram of protein. dTCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. Phenobarbital was administered (50 mg/kg/day) by ip injection for 3 days. The various doses of TCDD were administered 24 hr after the last dose of PB. 3-MC (40 mg/kg) and TCDD (5 pg/kg) were administered ip 72 hr prior to administration of the various doses of TCDD. e LD50 values were determined by probit analysis, based on the number of survivors at each dose 20 days after TCDD injection. JSignificantly different (p < 0.05) compared to nontreated weanlings, Student’s t test. g Significantly different (p < 0.05) compared to untreated weanlings. Duncan’s multiple range test.
MFO activities in these rats, compared to the respective controls of the same sex. Table 2 also presents the effects of these same treatments on the LD50 of TCDD in male and female rats. Castration decreased the 20-day LD50 of TCDD in adult male rats, and testosterone administration nearly doubled the amount of TCDD required for an LD50 dose in adult female rats. These studies of the relationship of hepatic MFO activity and TCDD-mediated lethality were extended to include the effects of inducers of the hepatic monooxygenase enzymes on TCDD toxicity. Both phenobarbital (PB) and 3-methylcholanthrene (3MC) are classic inducers of the hepatic MFO enzyme systems (Conney, 1967). In addition, TCDD itself is a potent inducer of aryl hydrocarbon hydroxylase (Poland and Glover, 1974). Table 3 shows the resulting MFO activities in hepatic microsomes of male weanling rats pretreated with PB, 3-MC, or TCDD. PB pretreatment caused a
significant increase in both microsomal MFO activities assayed, as did pretreatment with TCDD. 3-MC caused a significant increase in benzpyrene hydroxylation but had
HEPATIC
MFO
ACTIVITY
AND
TCDD
517
TOXICITY
no effect on aniline hydroxylation. Table 3 also shows the effect of pretreatment with PB, 3-MC, or TCDD on the toxicity of TCDD. All three pretreatments exerted a significant protective effect against TCDD-related lethality. The effect of decreases in the hepatic MFO activity in ~iuo on the LD50 of TCDD in rats was also examined. The administration of CoCl, to rats has been shown to reduce the activity of hepatic MFO systems in the liver (Tephly and Hibbeln, 1971). The administration of hemin to rats also causes a reduction in hepatic MFO activity (Bock et al., 1973). TABLE 4 EFFECT OF COBALTOLJS CHLORIDE AND HEMIN ON HEPATIC MIXED-FUNCTION OXIDASE ACTIVITY AND TOXICITY OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN WEANLING RATS’
LD50
Benzpyrene Treatment
hydroxylaseb
None COCI, Hemin Piperonyl butoxide
24.2 f 5.1 10.2 * 8.8’ 12.8 + 7.0’
(,&kg,
mean & SEpd 33.8 20.6 27.5 19.7
k 13.8 k 3.9 + 3.8 Ifr 4.3
(1Hepatic microsomes were prepared 24 hr after the last injection of CoCl, (60 mg/kg/day SCfor 2 days) or hemin (20 mg/kg/day ip for 4 days). The values shown are the mean & SE of values from three rats. * Quinine-SO, units per minute per milligram of protein. c TCDD was administered ip in olive oil at four dosages chosen to include the expected LD50. Six rats were injected at each dose. CoCl, was administered SC at 20 mg/kg/day for 2 days, and the various doses of TCDD were injected 24 hr after the last dose of CoCI,. Hemin was injected ip at 20 mg/kg/day for 4 days, and the various doses of TCDD were injected 24 hr after the last dose of hemin. Piperonyl butoxide was administered ip at 400 mg/kg 2 hr prior to the injection of TCDD and once per day for 2 days thereafter. d LD50 values were determined by probit analysis, based on the number of survivors at each dose 20 days after TCDD injection. e Significantly different (p < 0.05) compared to untreated weanlings. Student’s t test.
Table 4 presents the effects of CoCI, and hemin pretreatment on benzpyrene hydroxylase activity in weanling rats. Both CoCl, and hemin pretreatment resulted in an approximate 50% decrease in benzpyrene hydroxylase activity, as compared to untreated controls. The effect of CoCl,, hemin, and piperonyl butoxide on the toxicity of TCDD in weanling male rats is also shown in Table 4. Piperonyl butoxide is a wellknown competitive inhibitor of hepatic MFO enzyme systems. The effect of piperonyl butoxide on benzpyrene hydroxylase activity was not included because of the competitive nature of its inhibition of this enzyme system. In each case, treatment with the inhibitor resulted in the lowered LD50 values for TCDD in these rats. These lowered LD50 values were not, however, statistically significant. This is most likely because of the large variance in the LD50 value for TCDD in the control rats. The LD50 of TCDD in untreated weanling male rats in Table 4 is larger than that seen in
518
BEATTY,
VAUGHN,
AND
NEAL
untreated weanling male rats in Table 3. This difference is perhaps due to the somewhat greater age of the rats used in the experiment shown in Table 4 (30 days) than those used in the experiment shown in Table 3 (25 days). At 30 days of age the hepatic MFO activity of weanling male rats is just beginning to rise to adult values. This may account for the increased LD50 of TCDD in the controls of the experiment in Table 4 as compared to Table 3. This transition to adult enzyme activities may also account for the large variability in the LD50 values of the control animals in Table 4. DISCUSSION
The results of the experiments described herein indicate that an inverse relationship exists between the activity of the hepatic MFO enzyme systems and the toxicity, as measured by the ip LD50 of TCDD in the rat. Examination of adult male, adult female, and immature male rats reveals that the adult males with naturally high MFO activity are less susceptible to the lethal effects of TCDD. Induction of the hepatic MFO enzyme system in females by administration of testosterone or in weanling males by various chemical inducers such as PB, 3-MC, or a small dose of TCDD itself is correlated with a decrease in TCDD toxicity in these animals. Decreasing the hepatic MFO activity by castration of adult male rats or by administration of inhibitors of this enzyme system such as CoCl,, hemin, or piperonyl butoxide to weanling male rats is associated &ith an increase in the toxicity of TCDD with respect to noncastrated or untreated controls. Although other explanations such as altered steroid concentrations, changes in fatty acid metabolism, changes in cellular ultrastructure, etc. may account for this effect, a more likely explanation is that TCDD is metabolized by the hepatic MFO enzyme systems in rats and that this metabolism results in the formation of a chemical species which is less toxic than the parent compound. Verification of this hypothesis awaits the demonstration of the excretion of a metabolite(s) of TCDD in vim, examination of the toxicity of this metabolite(s), and the demonstration of increased excretion of this metabolite(s) in response to elevated hepatic MFO activity.
ACKNOWLEDGMENTS
This work was supported by USPHS Grants ES00267 and ES01552. Patrick Beatty also gratefully acknowledges the training support provided by Grant ES07028. REFERENCES ALLEN, J. R., VAN MILLER, J. P., AND NORBACK, D. H. (1975). Tissue distribution, excretion and biological effects of 114Cltetrachlorodibenzo-p-dioxin in rats. Food Cosmet. Toxicol. 13, 501-505. BOCK, K. W., FROHLING, W., AND REMMER, H. (1973). Influence of fasting and hemin on microsomal cytochromes and enzymes. Biochem. Pharmacol. 22,1557-1564. BRODIE, B. B., AND AXELROD, J. (1948). The fate of acetanilide in man. J. Pharmacol. Exp. Ther. 94,29-38. COCHIN, J., AND AXELROD, J. (1959). Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine, and normorphine. J. Pharmacol. Exp. Ther. 125, 105-l 10.
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TOMASZEWSKI, J. E., JERINA,D. M., ANDDALY, J. W. (1975).Deuteriumisotopeeffectsduring formation of phenolsby hepatic monoxygenases.Evidencefor an alternative to the arene oxide pathway. Biochemistry 14, 2024-2031. VINOPAL,J. H., ANDCASIDA,J. E. (1973). Metabolic stability of 2,3,7,8-tetrachlorodibenzo-pdioxin in mammalianliver microsomalsystemsand in living mice.Arch. Environ. Contam. Toxicol. 1, 122-132.