Phenobarbital-treated

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

We have examined whether the sulfur that was bound to the proteins of a reconstituted system from the liver of phenobarbital-treated rats was bound to both the reductase and cytochrome P-450. I this experiment, the reconstitued system was incubated with [ s] parathion. The reaction mixture was dialyzed and applied to a Sephadex G-25 column to remove the last traces of unreacted parathion and its noncovalently bound metabolites. The protein fraction from the Sephadex column was reduced in volume and subjected to SDS-polyacrylamide gel electrophoresis in the absence of either dithiothreitol or mercaptoethanol. The results are shown in Figure 5. There was considerable protein and radioactivity at the origin. This material at the origin represents an aggregate of reductase... 

LED, lowest effective dose ElID, highest ineffective dose in-vitro test, p.g/mL in-vivo test, mg/kg bw/day ip, intraperitoneal po, oral Active only with 30% hamster liver S9 not with rat liver S9 S9 from phenobarbital-treated rats Activity detected in YEPD medium Acute feeding... 

Chloroprene induced a small increase in sex-linked recessive lethal mutations in Drosophila melanogaster without a dose-response relationship in one study, but had no effect in another study. Chloroprene was not mutagenic to V79 Chinese hamster lung cells in the presence of S15 liver supernatants from phenobarbital-treated mice and rats. 

In isolated rat hepatocytes obtained from acetone- or phenobarbital-treated rats, the metabolism of toluene at low (below 100 pM) or high (100-500 pM) concentration was increased, in particular after phenobarbital treatment. Ethanol (7 and 60 mM) inhibited the overall metabolism of toluene (sum of benzyl alcohol, benzaldehyde, benzoic acid and hippuric acid), leading to accumulation of benzyl alcohol (Smith-Kielland Ripel, 1993). 

In rats, the conversion of vinyl bromide to reactive metabolites occurs primarily in hepatocytes. Irreversible binding of such metabolites to proteins and RNA has been established both with rat-liver microsomes in vitro and in rats in vivo. They can also alkylate the cytochrome P450 prosthetic group of phenobarbital-treated rat-liver microsomes. Exposure of rats to vinyl bromide causes a decrease in hepatic cytochrome P450 (lARC, 1986). 

Rifampicin and pregnenolone-16a-carbonitrile (PCN) induce members of the CYP3A family and represent a third type of inducer, in that the substrate specificity of the microsomes from treated animals differs from that of the microsomes from either phenobarbital-treated or TCDD-treated animals. Inducing substrates of this class include endogenous and synthetic glucocorticoids (e.g., dexamethasone), pregnane... 

COMPARISON OF EPOXIDE HYDROLASE ACTIVITY IN LIVER MICRO-SOMES FROM PHENOBARBITAL-TREATED RATS WITH THAT OF THE PURIFIED ENZYME IN THE PRESENCE OF LIPID FOR A SERIES OF ARENE OXIDES ... 

Jorgensen, A. J. and Majumdar, A. P, Influence of tryptophan on the level of hepatic microsomal cytochrome P-450 in well-fed normal, adrenalectomized, and phenobarbital-treated rats, Biochim. Biophys. Acta, 444[2], 453, 1976. 

The metabolic formation of carbonyl sulfide from carbon disulfide was confirmed in an in vivo study (Dalvi and Neal 1978). After intraperitoneal injection of 14C-carbon disulfide in nonpretreated rats, carbonyl sulfide was excreted by the lung in greater quantities than carbon dioxide. Pretreatment with phenobarbital, however, resulted in a greater amount of excretion of carbon dioxide than carbonyl sulfide. In both experiments, excretion of 14C-carbonyl sulfide and carbon dioxide accounted for 14-43% of the total administered radioactivity, with about twice as much carbon dioxide. These results indicate that phenobarbital treatment caused induction of cytochrome P-450 which catalyzed the conversion of carbon disulfide to carbonyl sulfide faster in pretreated rats than in rats not pretreated with phenobarbital. The role of the cytochrome P-450 monooxygenase system in catalyzing carbonyl sulfide formation was also confirmed by in vitro studies (Dalvi et al. 1974, 1975). The rate of carbonyl sulfide formation was NADPH-dependent and increased with microsomes obtained from phenobarbital-treated rats. 

Recently Hansson and Wikvall studied 12 -hydroxylation in a reconstituted system consisting of highly purified cytochrome P-450 LM4 from rabbits [101]. The cytochrome P-450 fractions used were electrophoretically homogenous, but the cytochrome P-450 from starved rabbits had up to 4 times higher capacity to catalyse 12a-hydroxylation than had cytochrome P-450 from untreated, phenobarbital-treated, or -naphthoflavone-treated rabbits. It should be mentioned that treatment with /8-naphthoflavone increases the amount of cytochrome P-450 LM4 in the liver. Amino acid analyses, peptide-mapping experiments as well as absorption spectral and circular dichroism spectral analyses revealed physical differences between cytochrome P-450 LM4 preparations from starved and phenobarbital-treated animals. It was concluded that the cytochrome P-450 LM4 fraction was heterogenous and contained a species of cytochrome P-450 specific for 12a-hydroxylation. 

Conclusive evidence that a species of cytochrome P-450 was involved in the hydroxylation was presented by Okuda et al., who showed that the photochemical action spectrum for reversal of the carbon monoxide inhibition of 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol in rat liver exhibited a maximum at 450 nm [134]. Pedersen et al. [135] and Sato et al. [136] reported simultaneously that small amounts of cytochrome P-450 could be solubilized from the inner membranes of rat liver mitochondria that was active towards cholesterol as well as 5)8-cholestane-3a,7a,12a-triol in the presence of ferredoxin, ferredoxin reductase and NADPH. The mechanism of hydroxylation is thus the same as that operative in the biosynthesis of steroid hormones in the adrenals and in the la-hydroxylation of 25-hydroxyvitamin D in the kidney (Fig. 8). The liver mitochondrial cytochrome P-450 was not active in the presence of microsomal NADPH-cytochrome P-450 reductase [135,136]. Ferredoxin reductase as well as ferredoxin were active regardless of whether they were isolated from rat liver mitochondria or bovine adrenal mitochondria [133]. The partially purified cytochrome P-450 had a carbon monoxide difference spectrum similar to that of microsomal cytochrome P-450 from liver microsomes and adrenal mitochondria. In the work by Pedersen et al. [133], the concentration of mitochondrial cytochrome P-450 in rat liver mitochondria from untreated rats was calculated to be only about 0.1 nmole/mg protein. Treatment of rats with phenobarbital increased the specific content of cytochrome P-450 in the mitochondria more than 2-fold, without significant increase in the 26-hydroxylase activity. The carbon monoxide spectrum of the reduced cytochrome P-450 solubilized from liver mitochondria of phenobarbital-treated rats exhibited a spectral shift of about 2 nm as compared to the corresponding spectrum obtained in analysis of preparations from untreated rats. This was taken as evidence that more than one species of cytochrome P-450 was present in the preparation. It was later shown by Pedersen et al. [137] and Bjbrkhem et al. [138] that the preparation was also able to catalyse 25-hydroxylation of vitamin D3 and that different enzymes are involved in... 

In vitro studies showed that rat liver microsomes activated with NADPH and molecular oxygen metabolized MMT (Hanzlik et al. 1980b). Preliminary studies with pooled liver microsomes from 5-6 normal or pheno-barbital-induced rats showed that reaction rates of metabolism were linear for the first 20 minutes. MMT and aminopyrine, a positive control compound that is metabolized exclusively by cytochrome P450, showed parallel responses to changes in incubation conditions (i.e. NADPH dependence, inhibition by carbon monoxide, induction by phenobarbital). Liver microsomes metabolized MMT with an estimated of 78 pM and a Vni of 3.12 nmol/mg protein/min. When the studies were done with liver microsomes from phenobarbital-treated rats, the remained the same, but the doubled (Hanzlik et al. 1980b). Lung microsomes were equally capable of metabolizing MMT, but phenobarbital induction did not enhance the response. 

Brown, C. A., 8c Black, S. D. (1989). Membrane topology of mammalian cytochrome P-450 from liver endoplasmic reticulum determination by trypsinolysis of Phenobarbital-treated microsomes. The Journal of Biological Chemistry, 264, 4442-4449. 

Ethynylestradiol was implicated as hepatocarcinogen in the rat and cocarcinogen for DMBA-induced mammary tumors in hamsters ". Benrekassa and Decloitre showed that this synthetic estrogen at concentrations of 0.1-1 mM inhibited benzo(a)pyrene metabolism in hepatocytes from naive or phenobarbital-treated rats. Pretreatment of rats with 3-methylcholantrene, an inducer of benzo(a)pyrene metabolism, abolished the inhibitory activity of the synthetic estrogen on benzo(a)pyrene metabolism. This observation suggests that the inhibitory activity of ethynylestradiol is probably not related to the major isozyme that metabolizes benzo(a)pyrene, namely cytochrome P450 1A, but rather to a different isozyme. 

Data on the inhibition of demethylation of aminopyrine in microsomes from Phenobarbital-treated rat liver CYP by 4-X-phenoxyanilines, resulted in the delineation of QSAR 7.20. 

JeSery EH, Manneiing GJ (1983) Interaction of constitutive and phenobarbital-induced cytochrome P-450 isozymes during the sequential oxidation of benzphetamine. Explanation for the difference in benzphetamine-induced hydrogen peroxide production and 455-nm complex formation in microsomes from untreated and phenobarbital-treated rats. Mol Pharmacol 23 748-757... 

One should also mention the cytological changes that appeared in the liver of phenobarbital-treated rats. Phenobarbital is normally a weak inducer of ALA-synthetase. It induced a fourfold increase in microsomal NADPH cjTochrome reductase by 6 to 8 hours, although electron microscope pictures did not reveal an increase in the endoplasmic reticulum [Kuriyama et ah, 64] until several days later, (see page 104, Section C). 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

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