Hepatic Microsomal Enzyme Induction Research Articles

Effects of PCBs and related compounds on hepatocarcinogenesis in rats and mice.

Commercial mixtures of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) can cause hepatocellular carcinoma in rats and mice. Present evidence indicates that these chemicals act as promoters and not initiators of hepatocarcinogenesis. Our results show that Firemaster BP-6 (FM) and its nontoxic major congener, 2,2', 4,4', 5,5'-hexabromobiphenyl (HBB), act as promoters in the two-stage model for hepatocarcinogenesis devised by Pitot and associates. A toxic congener, 3,3', 4,4', 5,5'-HBB, also was assessed for tumor-promoting activity. This congener, though not in FM, is similar to TCDD, in that both cause 3-methylcholanthrene (MC)-type induction of hepatic microsomal enzymes and produce similar toxic responses. FM contains several congeners which are mixed-type inducers in that they induce MC-type and phenobarbital (PB)-type enzymes. The toxicity of FM is most likely associated with its congeners which are mixed-type inducers and not to relatively nontoxic congeners such as 2,2', 4,4', 5,5'-HBB which are strictly PB-type inducers. Congener 3,3', 4,4', 5,5'-HBB acted as a tumor promoter only at a dose that was hepatotoxic. A synergistic effect on tumor promoting ability was produced by combining a nontoxic and nonpromoting dose of 3,3', 4,4', 5,5'-HBB with a promoting dose of 2,2', 4,4', 5,5'-HBB. Our results suggest that synergism between toxic and nontoxic congeners in FM may explain why mixtures such as FM have greater promoting ability than individual congeners. Our results also indicate that with PBB, toxicity and carcinogenicity are not necessarily related.

Effects of PCBs and Related Compounds on Hepatocarcinogenesis in Rats and Mice

Commercial mixtures of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) can cause hepatocellular carcinoma in rats and mice. Present evidence indicates that these chemicals act as promoters and not initiators of hepatocarcinogenesis. Our results show that Firemaster BP-6 (FM) and its nontoxic major congener, 2,2', 4,4', 5,5'-hexabromobiphenyl (HBB), act as promoters in the two-stage model for hepatocarcinogenesis devised by Pitot and associates. A toxic congener, 3,3', 4,4', 5,5'-HBB, also was assessed for tumor-promoting activity. This congener, though not in FM, is similar to TCDD, in that both cause 3-methylcholanthrene (MC)-type induction of hepatic microsomal enzymes and produce similar toxic responses. FM contains several congeners which are mixed-type inducers in that they induce MC-type and phenobarbital (PB)-type enzymes. The toxicity of FM is most likely associated with its congeners which are mixed-type inducers and not to relatively nontoxic congeners such as 2,2', 4,4', 5,5'-HBB which are strictly PB-type inducers. Congener 3,3', 4,4', 5,5'-HBB acted as a tumor promoter only at a dose that was hepatotoxic. A synergistic effect on tumor promoting ability was produced by combining a nontoxic and nonpromoting dose of 3,3', 4,4', 5,5'-HBB with a promoting dose of 2,2', 4,4', 5,5'-HBB. Our results suggest that synergism between toxic and nontoxic congeners in FM may explain why mixtures such as FM have greater promoting ability than individual congeners. Our results also indicate that with PBB, toxicity and carcinogenicity are not necessarily related.

Role of 3,5-dichlorophenyl methyl sulfone, a metabolite of m-dichlorobenzene, in the induction of hepatic microsomal drug-metabolizing enzymes by m-dichlorobenzene in rats

The increases in the hepatic microsomal aminopyrine N-demethylase activity and in the content of cytochrome P-450 produced by m-dichlorobenzene ( m-DCB) occurred after increases in the hepatic concentration of 3,5-dichlorophenyl methyl sulfone, a minor metabolite. The extent of increases in aminopyrine N-demethylase activity and in the content of cytochrome P-450 at 48 hr after po administration of 200 mg/kg (1.36 mmol/kg) of m-DCB was almost equal to that 72 hr after the ip administration of 25 μmol/kg of the sulfone (Kimura et al., 1983). m-DCB in liver was not detectable at that time, and the concentration of sulfone was 63 to 70% of that 48 to 72 hr after the ip administration of 50 μmol/kg of sulfone. Administration of m-DCB (200 mg/kg) produced a significant reduction in hexobarbital sleeping time, but this reduction was less than that produced by administration of the sulfone (50 μmol/kg). The protein band patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes from rats treated with the sulfone and m-DCB were similar to those of phenobarbital-treated rats but were different from those of 3-methylcholanthrene-treated rats. The sulfone showed type I interaction with the cytochrome P-450 ( K s, 0.17 m m). The sulfone was formed from the sulfide but reduction of the sulfone was not observed when it was incubated in a hepatic microsomal preparation. The pattern of induction by the sulfone and m-DCB was similar to that by phenobarbital and differed from that by 3-methylcholanthrene. From these results, 3,5-dichlorophenyl methyl sulfone is considered to be a major contributing factor of the inducing activity of m-DCB and to be a potent phenobarbital-like inducer.

Serum low-density lipoprotein and high-density lipoprotein cholesterol, and liver size in subjects on drugs inducing hepatic microsomal enzymes.

Serum low-density lipoprotein cholesterol and high-density lipoprotein cholesterol concentration and the ratio between them, major risk factors of coronary heart disease, and liver size were investigated in 18 subjects who were on enzyme-inducing anticonvulsants, phenytoin alone or in combination with phenobarbital and/or carbamazepine. The subjects with a high liver cytochrome P-450, indicating hepatic microsomal enzyme induction, who showed an increase in liver size, had an elevated high-density lipoprotein concentration and high-density lipoprotein cholesterol/total cholesterol ratio, and a reduced low/high-density lipoprotein cholesterol ratio. The high-density lipoprotein cholesterol concentration and its ratio to total cholesterol were directly and related to the ratio between low and high-density lipoprotein cholesterol were inversely related to the extent of liver enlargement. The serum cholesterol distribution profile associated with an increase in liver size was typical of subjects with a low risk of coronary heart disease. The results suggest that enzyme-inducers, such as phenytoin and phenobarbital, induce structural and functional changes in hepatocellular membranes associated with liver enlargement and cholesterol distribution characteristic of low susceptibility to atherosclerotic vascular disease.

Effects of isotretinoin on serum lipids and lipoproteins, liver and thyroid function

Seven patients with severe rosacea were treated with 1 mg/kg per day isotretinoin for 12 wk. There were significant increases in serum triglyceride ( p < 0.001) and cholesterol ( p < 0.001). Triglyceride associated with very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) increased ( p < 0.01), cholesterol in VLDL and LDL increased ( p < 0.01), and levels of HDL cholesterol decreased ( p < 0.01). There were changes in indices of liver function, with increased levels of γ-glutamyltransferase (GGT) ( p < 0.01), alkaline phosphatase (ALP) ( p < 0.01) and aspartate aminotransferase (AST) ( p < 0.01), and decreased bilirubin levels ( p < 0.05). Although levels of thyroxine and triiodothyronine were lower after treatment ( p < 0.05), there were no changes in basal levels of thyroid-stimulating hormone (TSH), luteinizing hormone (LH) or follicle-stimulating hormone (FSH), and responses to thyrotrophin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) were unchanged. These changes may partially be explained by induction of hepatic microsomal enzymes by isotretinoin.

Toxicologic studies associated with the agricultural use of municipal sewage sludge and health effects among sewage treatment plant workers

The proposed use of municipal sewage sludges as soil amendments on agricultural land involves potential health risks. Sludges may contain toxic heavy metals, synthetic organics, and pathogens. Studies of animals inhabiting sludge-amended soils, or foraging on crops grown on such soils, have shown an increased deposition of cadmium and polychlorinated biphenyls in animal tissues, hepatic microsomal enzyme induction, altered blood profiles, appearance of mutagens in blood and excreta, and lowered feed intake, feed efficiency, and milk production. Epidemiologic evidence of possible adverse health effects among sewage plant workers exposed to aerosolized pathogens or synthetic organic vapors during wastewater treatment or land spreading of sludge is still inconclusive. Other possible deleterious environmental effects include pollution of groundwater and toxicity to birds consuming worms or insects inhabiting sludgetreated soils.

Octachlorostyrene: A 90-day toxicity study in the rat

This study was designed to provide information on the subchronic toxicity of octachlorostyrene (OCS), a demonstrated environmental pollutant in fish from the Great Lakes of North America and the Norwegian coast in Europe. Groups of 15 male and 15 female rats were administered OCS mixed in the diet at 0.05, 0.5, 5.0, 50, or 500 ppm for 13 weeks. Increased liver weights were observed in male and female rats fed 50 ppm OCS and higher, while enlarged kidney and spleen were noted in the highest dose groups. Hepatic microsomal enzyme induction occurred at 5.0 ppm OCS and higher for the males and 50 ppm and higher for the females. The chemical produced serum biochemical changes at concentrations as low as 5.0 ppm. OCS treatment resulted in hematological disturbances starting with the 0.5-ppm dose group. Dose-dependent histological changes were observed in the thyroid, kidneys, and liver of the treated animals. OCS residues accumulated in a dose-related fashion in the liver and fat of treated animals. These results indicate that OCS produced toxic effects at low levels of exposure and accumulated in the tissues of rats.

Enzyme induction by phenobarbitone and vitamin K1 disposition in man.

The plasma disposition of an intravenous pharmacological dose of vitamin K1 was determined in seven healthy volunteers before and after chronic administration of phenobarbitone (100 mg nocte). Phenobarbitone produced a significant increase in antipyrine clearance and 6 beta-hydroxycortisol excretion in all volunteers, indicating induction of hepatic microsomal mixed-function oxidase enzymes. However, phenobarbitone administration had no effect on the plasma disposition of vitamin K1. We conclude therefore, that vitamin K1 is not metabolized to any significant extent, by phenobarbitone-inducible mixed-function oxidase enzymes.

Toxicologic studies with Japanese quail fed winter wheat grown on municipal sludge-amended soil.

Winter wheat was grown on soil amended with 100 dry tons per acre (224 metric tons/ha) of municipal sewage sludge from Syracuse, New York. The grain contained 1.43 and 0.98 ppm (mg/kg) dry weight, respectively, of cadmium and nickel. This wheat was incorporated as 60% of a semi-synthetic diet and fed to male and female Japanese quail for two generations. Male quail from the F1 generation fed sludge-grown wheat showed induction of hepatic microsomal enzymes,i.e., aminopyrene-N-demethylase and aniline hydroxylase, that indicated foreign compounds present in the wheat. Cadmium was significantly higher (p 0.05) concentrations of cadmium in kidney, liver, or eggs between the two dietary treatments. There were no observable changes in the tissue ultrastructure of liver and kidney as examined by electron microscopy in any of the treatment groups.

Urinary D-glucaric acid excretion during rifampicin/isoniazid and anticonvulsant enzyme induction.

As measured by urinary D-glucaric acid excretion, an index of hepatic enzyme induction, glutethimide was the most powerful of six such inducers tested. In patients with tuberculosis, rifampicin, 450 mg daily, induced excretion rates of the lower dose range of anticonvulsants in epileptics. The effect was detectable in the first few days but the degree and rate of rise to maximum excretion were variable. This may be due either to disposition of rifampicin or to genetic susceptibility to enzyme induction. Plasma beta-glucuronidase, an essential enzyme of the glucuronic acid pathway, could be induced independently of an increase in D-glucaric acid excretion. Plasma gamma-glutamyltranspeptidase-levels, an index of hepatic microsomal enzyme induction, were elevated in only 20 of 83 subjects receiving rifampicin and isoniazid, and in all of them urinary D-glucaric acid excretion was normal. Neither of these indices, therefore, showed hepatic enzyme induction during combined therapy when other pathways such as oxidative metabolism continued to be induced. Different active sites of rifampicin and isoniazid on glucuronic acid and other biochemical pathways emphasize the complexity of final metabolic effects in patients on long-term therapy.

Effect of Renal Failure and Bis(2-ethylhexyl) Phthalate Pretreatment on the Disposition and Metabolism of Antipyrine in the Rat

□ Renal failure patients undergoing hemodialysis are regularly exposed to phthalate plasticizers leached from dialysis tubings. Previous studies have shown that antipyrine is eliminated more rapidly in chronic renal failure patients compared with normal individuals. Therefore, the effect of bis(2-ethylhexyl) phthalate on the metabolism of antipyrine was investigated in normal and renal failure rats. In normal animals, the elimination kinetics of an intravenous dose of antipyrine (20mg/kg) was determined before and after 14days of peroral treatment with 2mL/kg/d of bis(2-ethylhexyl) phthalate. The plasma clearance of antipyrine increased markedly after bis(2-ethylhexyl) phthalate treatment. There was a corresponding decrease in the elimination half-life of antipyrine, whereas the apparent volume of distribution was not affected. Both liver weight and hepatic cytochrome P450 content increased following exposure to bis(2-ethylhexyl) phthalate, indicating the induction of hepatic microsomal enzymes. The fractional urinary recovery of the N-demethyl, 4-hydroxy, and 3-hydroxymethyl metabolites of antipyrine was not altered, suggesting that all three oxidative pathways were induced to the same extent. Renal failure alone did not affect the elimination kinetics of antipyrine. However, antipyrine clearance was induced to a greater extent by bis(2-ethylhexyl) phthalate treatment in the renal failure rats as compared with the control animals. The potential for phthalate plasticizers to alter hepatic drug metabolism in hemodialysis patients should be considered.

Drug induction of hepatic enzymes in the elderly.

Previous workers have suggested that hepatic enzyme induction by drugs does not occur in the elderly. We have studied the effect of a potent enzyme inducer--glutethimide--on a group of nine patients over the age of 70 years, using antipyrine clearance as a measure of hepatic drug metabolism. After six days treatment with glutethimide, antipyrine clearance rose from 22.8 +/- 4.5 ml/min to 38.3 +/- 7.6 ml/min (P less than 0.001). The results suggest that the elderly are at risk for the full range of drug interactions due to hepatic microsomal enzyme induction.

Octachlorostyrene: A 90-Day Toxicity Study in the Rat

Octachlorostyrerie: A 90-Day Toxicity Study in the RaL CHU, L, VILLENEUVE, D. C, SECOURS, V. E., YAGMINAS, A., REED, B., AND VALU, V. E. (1984). Fundam. Appl. Toxicol. 4, 547–557. This study was designed to provide information on the subchronic toxicity of octachlorostyrene (OCS), a demonstrated environmental pollutant in fish from the Great Lakes of North America and the Norwegian coast in Europe. Groups of 15 male and 15 female rats were administered OCS mixed in the diet at 0.05, 0.5, 5.0, 50, or 500 ppm for 13 weeks. Increased liver weights were observed in male and female rats fed 50 ppm OCS and higher, while enlarged kidney and spleen were noted in the highest dose groups. Hepatic microsomal enzyme induction occurred at 5.0 ppm OCS and higher for the males and 50 ppm and higher for the females. The chemical produced serum biochemical changes at concentrations as low as 5.0 ppm. OCS treatment resulted in hematological disturbances starting with the 0.5-ppm dose group. Dose-dependent histologjcal changes were observed in the thyroid, kidneys, and liver of the treated animals. OCS residues accumulated in a dose-related fashion in the liver and fat of treated animals. These results indicate that OCS produced toxic effects at low levels of exposure and accumulated in the tissues of rats.

The role of microsomal enzyme inducers in the reduction of misonidazole neurotoxicity.

In a series of studies, we have shown that phenytoin, 300 mg daily for one week, produces consistent hepatic microsomal enzyme induction, resulting in a decrease of 25% in misonidazole half-life, without causing any toxicity per se. A longer period of administration gives only a slightly greater induction. Phenobarbitone in a daily dose of 90 mg causes a reduction of 18% and 23% in misonidazole half-life after 1 and 2 weeks' pre-treatment respectively, but is less suitable clinically because of its sedative effect. A further series of studies using phenytoin as the inducing agent has shown that, despite adequate enzyme induction and increased misonidazole metabolism, it is impossible to increase the total dose of misonidazole beyond the usually accepted value of 12 g/m2 because of unacceptable neuropathy (a rate of 50% at a dose of 14 g/m2 over three weeks). In single doses of above 3.0-4.0 g of misonidazole, severe nausea and vomiting are prominent, so that this side effect is a determining factor in the treatment fractionation. Audiometric studies show no correlation between the incidence of peripheral neuropathy and abnormal audiograms, and have no value in the early prediction of neurotoxicity. It seems that despite causing increased metabolism, enzyme induction does not protect against neurotoxicity and thus will not permit the use of higher doses of misonidazole for increased radiosensitisation.

Maternal hepatic effects of 1,2,4,5-tetrachlorobenzene in the rat

1,2,4,5-Tetrachlorobenzene (TCB) is an industrial intermediate used in the production of 2,4,5-trichlorophenoxyacetic acid. This herbicide contains trace quantities of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Because of possible maternal hepatic or reproductive effects of this uncharged, low-molecular weight, lipophilic chlorinated benzene 0, 30, 100, 300, and 1000 mg/kg/day of TCB was orally administered to rats on Days 9, 10, 11, 12, and 13 of gestation and the animals were sacrified on Day 14 of pregnancy. No maternal deaths were recorded and body weight gain was significantly decreased only in the 1000 mg/kg/day group. Maternal liver weight, liver to body weight ratio, and hepatic microsomal protein content were unaffected by TCB treatment. Although Day 14 NADPH-cytochrome c reductase activity was not affected, the maternal hepatic microsomal cytochrome P-450 content was significantly increased by administration of 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was slightly increased from 2.4 to 3.4 and 3.5 nmole/mg protein/min at doses of 300 and 1000 mg/kg TCB. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was greatly increased from 14 to 30, 40, 50, and 49 pmole/mg protein/min in pregnant rats administered 0, 30, 100, 300, and 1000 mg/kg/day TCB. The microsomal rates of p-nitrophenol and phenolphthalein glucuronidation in vitro were not increased by TCB administration. The maternal hepatic microsomal enzyme induction observed after TCB administration to pregnant rats suggests the presence both cytochrome P-450 and P-448 inducers in the sample of 1,2,4,5-tetrachlorobenzene used.

Unidirectional cross-tolerance between the carbamate insecticide propoxur and the organophosphate disulfoton in mice

Previous studies have shown that subchronic treatment of mice with the organophosphate insecticide, disulfoton, or the carbamate insecticide, propoxur, leads to the development of tolerance to their toxicity. Tolerance to disulfoton was due to a decrease in the number of muscarinic cholinergic receptors, while tolerance to propoxur appeared to be due to an induction of hepatic microsomal enzymes. In the present study we investigated if cross-tolerance between disulfoton and propoxur would occur. Cross-tolerance was evaluated by measuring acute toxicities, cholinesterase and carboxylesterase inhibition and hypothermic and antinociceptive effects. Mice tolerant to propoxur were cross-tolerant to the hypothermic and anticholinesterase effects of disulfoton. Similarly, when mice were pretreated with the microsomal enzyme inducer, phenobarbital, the toxicity of disulfoton was decreased. Mice made tolerant to disulfoton were cross-tolerant to the organophosphate chlorpyrifos, but were more sensitive than controls to the toxicity of propoxur. The acute toxicity of the organophosphate malathion was also increased in disulfoton-tolerant mice. Propoxur is metabolized by mixed function oxidases and possibly by a carboxylesterase. While hepatic microsomal enzymes appeared to be unchanged in disulfoton-tolerant mice, brain and liver carboxylesterase activities were significantly inhibited. Pretreatment of mice with the specific carboxylesterase inhibitor triorthotolylphosphate is known to greatly potentiate the toxicity of malathion and also potentiated, to a lesser extent, the toxicity of propoxur. The results indicate that chronic exposure to an anticholinesterase insecticide may lead to tolerance to the toxicity of the inducing compound by more than one mechanism and that the toxicity of other insecticides may be either increased or decreased.

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat.

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.

Characterization of delayed neurotoxicity and induction of hepatic microsomal enzymes in the mouse following chronic oral administration of Tri- [formula omitted]-Cresyl phosphate (TOCP)

Characterization of delayed neurotoxicity and induction of hepatic microsomal enzymes in the mouse following chronic oral administration of Tri- [formula omitted]-Cresyl phosphate (TOCP)

Effect of low-dose phenobarbital on hepatic microsomal udp-glucuronyl transferase activity

To determine whether hepatic microsomal enzyme induction occurs in rats following administration of phenobarbital at doses similar to those used in humans (0.5 to 7.5 mg/kg), UDP-glucuronyl transferase (UDPGT) and cytochrome P-450 activities were measured in liver homogenate and microsomal preparations from control rats and rats treated for 6 days with phenobarbital at 1 and 3 mg per kg per day. While no significant increases in liver weight and protein content of homogenate and microsomal preparations were observed with either dose of the drug, both UDPGT and P-450 activities were enhanced significantly following administration of phenobarbital at 3 mg per kg per day. The activity of P-450 was increased by approximately 30% and that of UDPGT by 15–24 and 45–66%, respectively, employing bilirubin and p-nitrophenol as the acceptor substrate. The extent of induction of bilirubin or p-nitrophenol UDPGT was similar when measured with “native” enzyme or with enzyme activated by UDP- N-acetyl glucosamine, digitonin or deoxycholate. These data suggest that the discordant effects of phenobarbital on UDPGT and cytochrome P-450 previously reported in humans and rats may not be attributable solely to differences in the drug doses employed.

Protracted Hypervitaminosis A— Method of Treatment?

To the Editor.— We were interested to read of the case of vitamin A poisoning recently described by William A. Farris, MD, and John W. Erdman, MD (1982;247:1317). We note that the symptoms and signs of raised intracranial pressure persisted for over a month after cessation of supplements, and the serum total vitamin A and retinol binding protein levels remained elevated for almost two months. Unfortunately, while the problem of vitamin A poisoning continues to cause concern, no specific form of therapy has been described that might shorten the duration of toxic reaction after heavy overdosage. We have recently described the treatment of a case of vitamin D 2 poisoning by hepatic microsomal enzyme induction using glutethimide. 1 Rapid metabolism of vitamin D to more readily excretable and biologically less active forms has been suggested to occur after hepatic enzyme induction. 2 From what is known of vitamin A metabolism,