3-methylcholanthrene Pretreatment Research Articles

Hepatic cytochrome P450s metabolize aristolochic acid and reduce its kidney toxicity

Cytochrome P450s metabolize the naturally occurring nephrotoxin aristolochic acid. Using liver-specific cytochrome P450 reductase-null mice we found that a low but lethal dose of aristolochic acid I was ineffective in wild-type mice. Induction of hepatic CYP1A by 3-methylcholanthrene pretreatment markedly increased the survival rate of wild type mice given higher doses and these mice were protected from aristolochic acid I-induced renal injury. Clearance of aristolochic acid I in null mice was slower compared to control and the 3-methylcholanthrene-pretreated wild type mice. The levels of aristolochic acid I in the kidney and liver were much higher in null mice but much lower in 3-methylcholanthrene-treated compared to control wild type mice. Hepatic microsomes from 3-methylcholanthrene-treated wild type mice had greater activity compared to untreated mice. Finally, aristolochic acid I was more cytotoxic than its major metabolite aristolactam I and this cytotoxicity was decreased in human renal tubular epithelial HK2 cells in the presence of a reconstituted hepatic microsome-cytosol (S9) system. These results indicate that hepatic P450s play an important role in metabolizing aristolochic acid I into less toxic metabolites and thus have a detoxification role in aristolochic acid I-induced kidney injury.

Investigation of sanguinarine and chelerythrine effects on CYP1A1 expression and activity in human hepatoma cells

Quaternary benzo[ c]phenanthridine alkaloids (QBA) sanguinarine and chelerythrine exhibit a wide spectrum of biological activities whence they are used in dental care products. Recent studies indicated that cytochrome P450 CYP1A attenuates sanguinarine toxicity both in vivo [Williams, M.K., Dalvi, S., Dalvi, R.R., 2000. Influence of 3-methylcholanthrene pretreatment on sanguinarine toxicity in mice. Vet. Hum. Toxicol. 42, 196–198] and in vitro [Vrba, J., Kosina, P., Ulrichová, J., Modrianský, M., 2004. Involvement of cytochrome P450 1A in sanguinarine detoxication. Toxicol. Lett. 151, 375–387]. However, CYP1A converts sanguinarine to the products that form DNA adducts [Stiborová, M., Šimánek, V., Frei, E., Hobza, P., Ulrichová, J., 2002. DNA adduct formation from quaternary benzo[ c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique. Chem. Biol. Interact. 140, 231–242]. In our work we examined the effects of sanguinarine and chelerythrine on CYP1A1 expression and catalytic activity in human hepatoma cells—HepG2. Sanguinarine and chelerythrine did not affect basal and dioxin-inducible expression of CYP1A1 mRNA and protein in HepG2 cells. The enzymatic activity of CYP1A1 was assessed by the fluorescent measurement of 7-ethyxoresorufin- O-deethylase (EROD) activity. We observed a slight decrease of dioxin-induced EROD activity in HepG2 cells by sanguinarine and chelerythrine. This decrease was attributed to the inhibition of CYP1A1 catalytic activity, as revealed by enzyme kinetic studies on recombinant CYP1A1 protein. The IC50 values for the inhibition of CYP1A1 by sanguinarine and chelerythrine were 2.1 and 1.9 μM, respectively. In conclusion, albeit the CYP1A modulates QBA cytotoxicity and genotoxicity, the QBA themselves do not affect CYP1A1 expression. The data indicate that studied alkaloids do not have specific cellular target and their biological effects are rather pleiotropic.

Effect of hypoxia alone or combined with inflammation and 3-methylcholanthrene on hepatic cytochrome P450 in conscious rabbits.

1 To investigate the effect of moderate hypoxia alone or combined with an inflammatory reaction or after 3-methylcholanthrene (3MC) pre-treatment on cytochrome P450 (P450), conscious rabbits were exposed for 24 h to a fractional concentration of inspired O2 of 10% (mean PaO2 of 34 mmHg). Hypoxia decreased theophylline metabolic clearance (ClM) from 1.73+/-0.43 to 1.48+/-0.13 ml min-1 kg-1 (P<0. 05), and reduced (P<0.05) the formation clearance of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU) and 1,3-dimethyluric acid (1,3DMU). Hypoxia reduced the amount of CYP1A1 and 1A2 but increased CYP3A6 proteins. 2 Turpentine-induced inflammatory reaction reduced (P<0.05) the formation clearance of 3MX, 1MU, and 1,3DMU, and diminished the amount of CYP1A1, 1A2 and 3A6 proteins. However, when combined with hypoxia, inflammation partially prevented the decrease in ClM, especially by impeding the reduction of 1,3DMU. The amount of CYP1A1 and 1A2 remained reduced but the amount of CYP3A6 protein returned to normal values. 3 Pre-treatment with 3MC augmented the ClM by 114% (P<0.05) due to the increase in the formation clearance of 3MX, 1MU and 1,3DMU. 3MC treatment increased the amount of CYP1A1 and 1A2 proteins. Pre-treatment with 3MC prevented the hypoxia-induced decrease in amount and activity of the P450. 4 It is concluded that acute moderate hypoxia and an inflammatory reaction individually reduce the amount and activity of selected apoproteins of the P450. However, the combination of hypoxia and the inflammatory reaction restores P450 activity to near normal values. On the other hand, pre-treatment with 3MC prevents the hypoxia-induced depression of the P450.

Induction Time Course of Cytochromes P450 by Phenobarbital and 3-Methylcholanthrene Pretreatment in Liver Microsomes of Alligator mississippiensis

Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48–72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

Microsomal cytochrome P450-mediated liver and brain anandamide metabolism

Anandamide (AN) is an arachidonic acid congener, found in the brain, that binds to the cannabinoid receptor and elicits cannabinoid-like pharmacological activity. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to a wide variety of metabolites, yielding many physiologically potent compounds. To determine if AN could be similarly oxidized by P450s, its metabolism by mouse liver and brain microsomes was examined. Mouse hepatic microsomal incubation of AN with NADPH resulted in the generation of at least 20 metabolites, determined after HPLC separation by increased UV-absorbance at 205 nm. Pretreatment of mice with various P450 inducers resulted in increased hepatic microsomal formation of several AN metabolites, with dexamethasone being the most effective inducer. Phenobarbital pretreatment resulted in a metabolic profile similar to that observed after dexamethasone pretreatment, whereas 3-methylcholanthrene pretreatment selectively increased the formation of several other metabolites. Clofibrate pretreatment had no effect on hepatic AN metabolism. Polyclonal antibodies prepared against mouse hepatic P450 3A inhibited the formation of several AN metabolites by hepatic microsomes from untreated mice as well as the formation of those metabolites found to be increased after dexamethasone pretreatment. AN metabolism by brain microsomes resulted in the formation of two NADPH- and protein-dependent metabolites. Hepatic P450 3A antibody partially inhibited the formation of only one of these metabolites. Thus, P450 3A is a major contributor to AN metabolism in the liver but not in the brain. The physiological consequences of P450-mediated AN metabolism remain to be determined.

Purification and Characterization of a Newly Identified Isoform of Cytochrome-P450 Responsible for 3-Hydroxylation of 2,5,2′,5′-Tetrachlorobiphenyl in Hamster Liver

In the hamster liver, 2,5,2′,5′-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2′,5′-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the aminoterminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2′,5′-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16α- and 16β-hydroxylations of testosterone. In microsomal metabolism of 2,5,2′,5′-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3′,4′-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2′,5′-TCB, in hamster liver.

Glucuronidation and isomerization of all-trans- and 13-CIS-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats

Glucuronidation and isomerization of all- trans-am-retinoic acid (tr-RA) and 13- cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13- cis form to the all- trans form than vice versa. Also some 9- cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13- cis-retinoyl-β-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.

Involvement of P450 1A1 in Benzo(a)Pyrene but Not in Benzo(a)Pyrene‐7,8‐Dihydrodiol Activation by 3‐Methylcholanthrene‐Induced Mouse Liver Microsomes

Synchronous fluorescence spectrophotometry for benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts was used to study the activation pathway of benzo(a)pyrene in C57BL/6 mice. Benzo(a)pyrene but not benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced mouse liver microsomes was inhibited by a monoclonal antibody (Mab 1-7-1) against CYP1A1/2 suggesting that 1A1 probably takes part in the first P450 reaction. However, aryl hydrocarbon hydroxylase activity, a classical measure of benzo(a)pyrene metabolism, was not inhibited by the same concentration of Mab 1-7-1. None of the other antibodies used, detecting 2A, 2B, 2C or 2E subfamilies, inhibited the adduct formation. Troleandomycin and gestodene, chemical inhibitors of human 3A4, inhibited benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced microsomes to some extent only in high concentrations. Although liver microsomes from 3-methylcholanthrene-induced mice catalyzed the formation of BPDE-DNA in vitro clearly more than uninduced microsomes, 3-methylcholanthrene pretreatment in vivo decreased the adduct formation in benzo(a)pyrene-treated mice. These results emphasize the significance of detoxicating and DNA-repairing pathways in vivo. Finally, synchronous fluorescence spectrophotometry for BPDE-DNA measures the end-point of the three-step activation pathway while aryl hydrocarbon hydroxylase measures a one-step hydroxylation. Thus, these methods should be used rather as corroborative than mutually exclusive assays.

Effects of phenobarbital and 3-methylcholanthrene pretreatment on the pharmacokinetics of praziquantel in rats

The effects of phenobarbital (PB) and 3-methylcholanthrene (MC) pretreatment on the pharmacokinetics of praziquantel (PZQ), a schistosomicide were studied in Sprague-Dawley rats. Blood samples at different time intervals were obtained by severing the tail vein and were analyzed for unchanged PZQ by HPLC. The PB-pretreated rats showed a 6-fold decrease in AUC, a 5-fold decrease in Cmax and an 8-fold increase in CLtot compared to the saline treated controls. The MC-pretreated rats and their olive-oil treated controls did not show any statistically significant differences in the above parameters. These results suggest that PZQ is extensively metabolised by PB-inducible cytochrome P-450 isoforms and not by MC-inducible isoforms. These findings also suggest that the bioavailability of praziquantel could be altered to a significant extent in humans taking drugs that are phenobarbital type inducers.

Influence of agents affecting monooxygenase activity on taurolithocholic acid-induced cholestasis

In rats, pretreatment with certain ketones results in enhanced taurolithocholic acid (TLCA)-induced reduction in bile flow, whereas pretreatment with inhibitors of protein synthesis diminishes the effect on bile flow of cholestatic regimens. In the present study, the possible role of cytochrome P-450 in the ketone potentiation phenomenon was investigated. Male rats were pretreated with inducers or inhibitors of hepatic cytochrome P-450 and the impact of these pretreatments on TLCA-induced cholestasis assessed. Phenobarbital, 3-methylcholanthrene, chlordecone or mirex were used as inducers, and SKF 525-A, piperonyl butoxide, or cobaltous chloride as inhibitors of monooxygenase activity. Phenobarbital and 3-methylcholanthrene pretreatment enhanced TLCA-induced reduction of bile flow, while mirex and chlordecone were without effect. The three inhibitors of monooxygenase activity did not diminish TLCA-induced cholestasis. Instead, piperonyl butoxide and cobaltous chloride appeared to enhance the action of TLCA. Consequently, an increase in cytochrome P-450 (or specific isozymes) as a common denominator in the potentiation phenomenon appears unlikely. While hepatic proteins may play an important role in the potentiation of TLCA-induced cholestasis following pretreatment with ketones, the pattern of potentiation after pretreatment of rats with different inducers or inhibitors of cytochrome P-450 does not appear to implicate this family of proteins.

Effects of Phenobarbital and 3-Methylcholanthrene Pretreatment on the Pharmacokinetics and Pharmacodynamics of Furosemide in Rats

The effects of pretreatment with the enzyme inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) on the pharmacokinetic and pharmacodynamic parameters of furosemide were examined in rats. The nonrenal clearance (4.58 versus 6.18 mL/min/kg) increased significantly in PB-treated rats. This suggested that the nonrenal metabolism of furosemide increased by pretreatment with PB. This relationship was supported by the results of a tissue homogenate study; the amounts of furosemide remaining per gram of tissue after 30 min of incubation of 50 μg of furosemide with the 9000 × g supernatant fraction of liver, stomach, and kidney tissue homogenates decreased significantly in PB-treated rats. The contents of hepatic cytochrome P-450 (1.29 versus 2.15 nmol/mg protein) and the weights of liver and stomach increased significantly in PB-treated rats, suggesting that the metabolizing enzymes for furosemide are induced by pretreatment with PB. The 8-h urine output per 100 g of body weight increased significantly in PB-treated rats; however, the 8-h urinary excretion of furosemide per 100 g of body weight (797 versus 635 μg) decreased significantly in PB-treated rats. Alterations in the urine output might be due to the hormonal alterations in the concentration—effect relationship for furosemide in PB-treated rats. In 3-MC–treated rats, pharmacokinetic and pharmacodynamic parameters of furosemide were not significantly different, indicating that the metabolizing enzymes for furosemide were not induced by pretreatment with 3-MC. However, the contents of hepatic cytochrome P-450 and the weights of liver and stomach increased significantly.

Metronidazole and antipyrine metabolism in the rat: Clearance determination from one saliva sample

1. The applicability of a simple, non-invasive method for assessment of metronidazole and antipyrine metabolism in rats in vivo was investigated. 2. In 48 sample pairs of blood and pilocarpine-stimulated saliva from six rats the concentration of metronidazole was almost identical (r = 0.97). 3. In 26 rats the clearance could be determined from one sample without loss of precision and accuracy compared with conventional determinations (r = 0.99). If urine was collected for 24 h the fractional clearance representing each elimination pathway could be determined. 4. Pretreatment with phenobarbitone increased the fractional clearance of metronidazole by oxidation and glucuronidation 3.8-fold and 1.6-fold, respectively, whereas 3-methylcholanthrene pretreatment increased the rate of oxidation 10-fold and decreased the rate of glucuronidation 0.5-fold. 5. The clearance and fractional clearances of metronidazole and antipyrine administered in a mixture could be determined from the same saliva sample and urine collected for 24 h without drug-drug interactions. 6. Phenobarbitone pretreatment increased the formation rate of all metabolites of metronidazole and antipyrine administered in a mixture, whereas beta-naphthoflavone increased the formation rates of only the oxidative metronidazole metabolites, norantipyrine and 4-hydroxyantipyrine, but not metronidazole glucuronide or 3-hydroxymethylantipyrine. 7. A mixture of metronidazole and antipyrine and non-invasive sampling are recommendable for the study of the differential metabolism of foreign compounds in rats in vivo.

Cloning and characterization of two major 3-methylcholanthrene inducible hamster liver cytochrome P450s

We have studied the immunochemical properties of two major 3-methylcholanthrene inducible hamster liver cytochrome P450 isozymes, P450 MC1 and P450 MC4. Immunoblots using specific antibodies against P450 MC1 and P450 MC4 demonstrated that these two P450s were present in very low levels in control hamster livers and were greatly induced by 3-methylcholanthrene treatment. P450 MC1 was immunochemically different from P450 MC4, rat P450c and P450d, and rabbit LM 4. The immunorelated polypeptide to P450 MC1 was not present in the control or the 3-methylcholanthrene-treated rat liver microsomes, whereas it was present in two human liver microsomal preparations. On the other hand, P450 MC4 was immunochemically related to rat P450d and rabbit LM 4. The immunorelated polypeptide to P450 MC4 was present in the human and 3-methylcholanthrene-treated rat liver microsomes. We also isolated full-length cDNA clones encoding P450 MC1 and P450 MC4 mRNAs from a 3-methylcholanthrene-induced hamster liver cDNA library. The full-length cDNA clones of P450 MC1 and P450 MC4 contained 1771 and 1868 base pairs, which encoded polypeptides of 494 and 513 amino acids, respectively. RNA blot analysis revealed that the mRNAs for P450 MC1 and P450 MC4 were 2100 and 2600 bases in length, respectively. 3-Methylcholanthrene pretreatment increased the P450 MC1 mRNA level by 16-fold and the P450 MC4 mRNA level by 11-fold in the hamster livers. A comparison of the deduced amino acid sequences with other cytochrome P450s revealed that P450 MC1 was most similar to the mouse P450 15α with 75% sequence identity, whereas P450 MC4 shared 87% identity with the rat P450d or mouse P 3450. These results indicated that P450 MC1 was a unique member (CYP2A8) in the P450IIA subfamily, whereas P450 MC4 was the hamster P450IA2.

Acetaminophen structure-toxicity studies: In vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide

High doses of 3-hydroxyacetanilide (3HAA), a structural isomer of acetaminophen, do not produce hepatocellular necrosis in normal male hamsters or in those sensitized to acetaminophen-induced liver damage by pretreatment with a combination of 3-methylcholanthrene, borneol, and diethyl maleate. Although 3HAA was not hepatotoxic, the administration of acetyl-labeled [ 3H or 14C]3HAA (400 mg/kg, ip) produced levels of covalently bound radiolabel that were similar to those observed after an equimolar, hepatotoxic dose of [G- 3H]acetaminophen. The covalent nature of 3HAA binding was demonstrated by retention of the binding after repetitive organic solvent extraction following protease digestion. Hepatic and renal covalent binding after 3HAA was approximately linear with both dose and time. In addition, 3HAA produced only a modest depletion of hepatic glutathione, suggesting the lack of a glutathione threshold. 3-Methylcholanthrene pretreatment increased and pretreatment with cobalt chloride and piperonyl butoxide decreased the hepatic covalent binding of 3HAA, indicating the involvement of cytochrome P450 in the formation of the 3HAA reactive metabolite. The administration of multiple doses or a single dose of [ ring- 3H]3HAA to hamsters pretreated with a combination of 3-methylcholanthrene, borneol, and diethyl maleate produced hepatic levels of 3HAA covalent binding that were in excess of those observed after a single, hepatotoxic acetaminophen dose. These data suggest that the nature and/or the intracellular processing of the reactive metabolites of acetaminophen and 3HAA are different. These data also demonstrate that absolute levels of covalently bound xenobiotic metabolites cannot be utilized as absolute predictors of cytotoxic potential.

Biotransformation of lovastatin—III: Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes

The effects of the H 2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [ 14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6′β-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6′β-hydroxy-lovastatin but 63–66% less of the 3″-hydroxy and 6′-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3″-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6′β-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3″-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6′-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).

Effect of 3-Methylcholanthrene Pretreatment on Aflatoxin B1Metabolism in Hamster Liver

AbstractThe effects of 3-methylcholanthrene (MC) pretreatment of hamsters on the hepatic metabolism of aflatoxin B1 (AFB1) have been investigated in studies with subcellular, isolated hepatocytes and in vivo. Pretreatment of hamsters with MC (2.5 mg/100 g body wt.) 24 hr. before sacrifice not only increased microsomal cytochrome P-450 content by 70% but also increased the specific activity of cytochrome P-450 dependent metabolic activation of AFB1 as judged by AFB1-DNA binding by 65%. Even in isolated hepatocytes, AFB1-DNA binding was elevated by 65% in MC-treated hepatocytes. The ratios of AFB1-glutathione (AFB1-SG) to AFB1-DNA were about the same in both hepatocyte system. Hepatic AFB1-DNA binding was also increased by 85% in intact animals after MC treatment. It appears that the increased hepatic AFB1-DNA binding after MC treatment of hamsters is a result of higher specific activity and higher levels of cytochrome P-450 species involved in the activation of AFB1.

Pharmacokinetics and toxicity of mitomycin C in rodents, given alone, in combination, or after induction of microsomal drug metabolism.

The pharmacokinetics of mitomycin (MMC) was studied in Wistar rats. Up to five half-lives, the plasma concentration-time curve was biphasic. The AUC changed linearly with increasing doses between 0.5 and 7.5 mg/kg, which corresponds to 0.2 and 3 times the LD50 value in rats. Most of the drug was metabolized, and only 1%-2% and 10%-15% of the dose was eliminated unchanged by biliary and urinary excretion, respectively. The AUC of MMC at the LD50 is slightly less than that reported for the human MTD. Inoculation of MMC together with 5-fluorouracil and doxorubicin did not change the terminal half-life of MMC but decreased the total body clearance and the volume of distribution. The lack of significant influence of phenobarbital and 3-methylcholanthrene pretreatment on the terminal elimination half-life suggests that microsomal drug-metabolizing enzymes inducible by these compounds do not play a decisive role in the in vivo biotransformation of MMC.

Qualitative and quantitative differences in the induction and inhibition of hepatica benzo[a]pyrene metabolism in the rat and hamster

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [ 3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; 5μg/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., × 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., × 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., × 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10-and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.

Phenolsulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene

The effect of phenobarbital and 3-methylcholanthrene pretreatment on the biliary excretion of phenolsulphonphthalein (PSP) was investigated in male Wistar rats. The dye was injected at a single dose of 200 mumol/kg body wt. About 20% of the compound was excreted as a glucuronide in the controls, the liver UDP-glucuronyltransferase activity toward PSP being 0.064 +/- 0.005 nmol.min-1.mg protein-1. Treatment for two weeks with phenobarbital (354 mumol.kg body wt-1.day-1) caused a transient increase in conjugated and unconjugated PSP excretion, but glucuronyltransferase activity was not modified. 3-Methylcholanthrene pretreatment for 4 days (75 mumol.kg body wt-1.day-1) also enhanced biliary excretion of the dye, but the increase corresponded only to the glucuronide and glucuronyltransferase activity was significantly enhanced by 20%. Our data indicate that not only the rate of biotransformation but also other factors could be responsible for increased PSP biliary excretion following administration of microsomal enzyme inducers.

The effect of pyrazole, phenobarbital, ethanol and 3-methylcholanthrene pretreatment on the in vivo and in vitro genotoxicity of N-nitrosopyrrolidine.

The in vitro genotoxicity of N-nitrosopyrrolidine (NPy) has been studied in Salmonella typhimurium strain TA1535 in the presence of untreated and pyrazole-, phenobarbital (PB)-, 4-day ethanol (EtOH)-, 10-day EtOH- and 3-methylcholanthrene (3-MC)-pretreated male Sprague-Dawley rat liver S-9 fractions. Unless stated otherwise, the last pretreatment exposure was 24 h prior to sacrifice and isolation of hepatic enzymes. Pyrazole and EtOH (10-day exposure) both effectively induced the conversion of NPy into a mutagen at doses as low as 500 microM. PB and EtOH (4-day exposure) had a modest enhancing effect on the number of revertants scored, while 3-MC and uninduced S-9 fractions gave results not significantly different from background (no NPy). The same pretreatment protocols were used to determine the in vivo genotoxicity of NPy in rat liver using the technique of alkaline elution. The inducing agents had the exact opposite effect in vivo with control, 3-MC- and 4-day EtOH-treated animals showing the highest level of DNA damage. Pyrazole and 10-day EtOH pretreatments gave DNA elution rate constants comparable to animals not treated with NPy. However, in 10-day EtOH-pretreated animals which were administered NPy without a 24-h interval between EtOH and NPy exposure, DNA damage was observed at the same high levels as was seen in uninduced and 3-MC treated rats. The results are discussed in terms of a detoxification role for microsomal proteins and that the observed in vivo DNA damage may be induced by enzymes associated with the nuclear compartment.