3-Methylcholanthrene hydroxylation

Recently, the mechanism of 6-nitro-BaP ring hydroxylation has been elucidated by using 3-deutero-6-nitro-BaP (144). When incubated with 3-methylcholanthrene-induced rat liver microsomes, this deuterated analogue yielded the same metabolite profile previously observed with 6-nitro-BaP. Spectroscopic analysis of 3-hydroxy-6-nitro-BaP and 6-nitro-BaP-3,9-hydroquinone indicated that 30% of the deuterium label had migrated to carbon 2, presumably via an NIH shift. Therefore, it appears that 6-nitro-BaP-2,3-oxide is a common intermediate for these two metabolites. 

The pharmacological action of codeine is increased by induction as this increases demethylation to morphine. Induction by phenobarbital decreases the toxicity of organo-phosphates, but increases that of phosphorothionates. Studies with the drug warfarin have shown that induction by both phenobarbital and 3-methylcholanthrene will change the stereochemistry of the product, as can be seen in Table 5.24. Thus, hydroxylation in the 8-position in the R-isomer is increased 12 times compared with only 4 times with the S-isomer following 3-methylcholanthrene induction. 

N-hydroxylation is not the only or major route of metabolism in vivo, nor is it the only reaction catalyzed by the microsomal enzymes. Ring hydroxylation is the major route of metabolism, the products of which are not carcinogenic. These alternative routes of metabolism are inducible in vivo by pretreatment with agents such as phenobarbital and 3-methylcholanthrene (see chap. 5). 

Cytochromes P450 often convert drugs or other foreign compounds to forms that are more readily excreted.499 However, the result is not always beneficial. For example, 3-methylcholanthrene, a strong inducer of cytochrome P450, is converted to a powerful carcinogen by the hydroxylation reaction.500 See also Box 18-E. 

Treatment of immature male rats with phenobarbital for three days increased the 6 ft-, la-, and 16a-hydroxyl ati on of testosterone by liver microsomes to different degrees (36). The 16a-hydroxyl ati on reaction was stimulated several-fold, whereas the 6ft- and 7a-hydroxylations were stimulated to a smaller extent. In contrast to these results, the administration of 3-methylcholanthrene had little or no stimulatory effect on the 6ft- or 16a-hydroxylation of testosterone by liver microsomes but caused a significant increase in the 7 a-hydroxy 1 ati on reaction. 

Rats were treated with vehicle (control), phenobarbital (PB), or 3-methylcholanthrene (3-MC). Cytochrome P450, lipid, and reductase fractions were prepared and reconstituted. The reductase and lipid fractions were prepared from PB-treated rats. No hydroxylation activity was detected when hemoprotein was omitted from the reaction mixture. In Experiment 1, benzo[a]pyrene metabolism was measured by formation of fluorescent phenolic metabolites, and benzphetamine metabolism was measured by the rate of benzphetamine-dependent NADPH oxidation. In Experiment 2, the metabolism of pentobarbital, benzo[a]pyrene, and chlorcyclizine was measured by product formation. Experiment 1 was taken from Ref. (53) and Experiment 2 was taken from Ref. (55). 

Ebel, S. Liedtke, R. Missler, B. [Quantitative determination of nitrofurantoin in body fluids by direct injection HPLC]. Arch.Pharm.(Weinheim)., 1980, 313, 95-96 Jonen, H.G. Oesch, F. Platt, K.L. 4-Hydroxylation of nitrofurantoin in the rat. A 3-methylcholanthrene-inducible pathway of a relatively nontoxic compound. Drug Metab.Dispos., 1980, 8, 446-451... 

Nearly 50 years ago, it was noted that rats exposed to 3-methylcholanthrene (3-MC) displayed a marked increase in metabolic capacity toward that substrate and other polycyclic aromatic hydrocarbons (PAHs) . This enhanced metabolic activity was referred to as aryl hydrocarbon hydroxylase (AHH) based on the ability of these enzymes to efficiently hydroxylate aromatic hydrocarbons ". It is now known that AHH activity is the collective activities of the CYPlAl, CYPl A2, and CYPIBI enzymes. 

In the apparently major pathway for the conversion of cholesterol into 5 -cholestane-3a,7a,12a-triol, the step following the formation of 7a-hydroxy-4-cholesten-3-one is a 12a-hydroxylation yielding 7a,12a-dihydroxy-4-cholesten-3-one (Fig. 1). The reaction is catalyzed by the microsomal fraction fortified with NADPH (15,37). The conversion of 5-cholestene-3, 7a-diol into 5-cholestene-3/5,7a,12a-triol, which is a reaction in another pathway for the formation of 5/5-cholestane-3a,7a,12a-triol, is also catalyzed by the microsomal fraction fortified with NADPH (30,37), as is the 12a-hydroxylation of 5/5-cholestane-3a,7a-diol and 7a-hydroxy-5)5-cholestan-3-one (37). The rates of 12a-hydroxylation of these C27-steroids differ considerably the rate with 5-cholestene-3/5,7a-diol is about one-tenth and with 5 -cholestane-3a,7a-diol about half of that with 7a-hydroxy-4-cholesten-3-one (37). Einarsson (37) and Suzuki et al. (38) have studied some properties of the 12a-hydroxylase system with special reference to the possible participation of electron carriers such as NADPH-cytochrome c reductase and cytochrome P-450. The 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one was inhibited by cytochrome c, indicating that NADPH-cytochrome c reductase might be involved. However, no direct evidence for the participation of flavins was obtained. If NADPH-cytochrome c reductase participates, it is not rate-limiting, since the activity of this enzyme increases upon treatment with thyroxine whereas the activity of the 12a-hydroxylase decreases (39). Suzuki et al. (38) found no inhibition of 12a-hydroxylation by carbon monoxide, whereas Einarsson (37) obtained some inhibition. The 12a-hydroxylase activity was unaffected by methylcholanthrene treatment (40) and lowered by phenobarbital treatment (37,38). These observations indicate that the cytochrome(s) P-450 induced by methylcholanthrene and... 

The microsomal a-hydroxylation of NNN can be monitored by HPLC, with detection of the 2,4-dinitrophenyihydrazones of 12 and 13 (70). Using this assay, and the one described above for NPYR, the effects of modifiers on NNN and NPYR a-hydroxylation in rats and in Syrian golden hamsters have been determined. The results, which are summarized in Table XIV, suggest the presence of multiple enzymatic forms that catalyze a-hydroxylation, as observed with DMN. For example, 3-methylcholanthrene pretreatment of rats resulted in induction of 2 -hydroxylation of NNN but repression of 5 -hydroxylation. 2 -Hydroxylation of NNN was not induced by pretreatment of hamsters, in contrast to the rat, whereas 5 -hydroxylation of NNN in the hamster was induced by phenobarbital, but not affected by 3-methylcholanthrene. The induction in hamsters of a-hydroxylation of NPYR and 5 -hydroxylation of NNN by ethanol pretreatment could result in higher carcinogenicity of these compounds in... 

Puntarulo S, Cederbaum AI (1992) Effect of phenobarbital and 3-methylcholanthrene treatment on NADPH- and NADH-dependent production of reactive oxygen intermediates by rat liver nuclei. Biochim Biophys Acta 1116 17-23 Que BG, Downey KM, So AG (1980) Degradation of deoxyribonucleic acid by a 1,10-phenanthroline-copper complex the role of hydroxyl radicals. Biochemistry 19 5987-5991... 

Although 3-methylcholanthrene treatment stimulates the hydroxylation of 3.4-benzpyrene and zoxazolamine and the N-demethylation of 3-methyl-4-mono-methylaminoazobenzene (Table 8). it does not increase (in some cases decreases) the metabolism of ethylmorphine. hexobarbital, and aminopyrine. By contrast. 

In humans, administration of o,p -DDD [l,l-dichloro-2-(4-chlorophenyl)-2-(2-chlorophenyl) ethane], phenobarbital, diphenylhydantoin, or phenylbutazone causes a marked elevation in the urinary excretion of 6 )5-hydroxycortisol presumably by accelerating the metabolism of glucocorticoids. Accordingly, treatment of animals with phenobarbital stimulates the hydroxylation by hepatic microsomes of corticosterone, deoxycorticosterone, cortisone, and cortisol, whereas 3-methylcholanthrene has negligible effect on the hydroxylation of cortisone or cortisol. The physiological consequences of this induction, however, remain obscure. 

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