Microsomal Xenobiotic Metabolism Research Articles

Hepatoprotective properties of aqueous extract from Pentaphylloides fruticosa during chronic toxic hepatitis.

Hepatoprotective properties of Pentaphylloides fruticosa L. aqueous extract were studied using rat model of chronic toxic hepatitis. The preparation normalized alanine aminotransferase activity and bilirubin concentration in the plasma. Aqueous extract of Pentaphylloides fruticosa produced a protective effect on microsomal metabolism of xenobiotics and lipid peroxidation activity in the plasma and microsomal liver fraction.

Glutathione depletion in a liver microsomal assay as an in vitro biomarker for reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleophile trap for reactive species derived from xenobiotics. This has led to the development of an assay for the detection of reactive species generated by liver microsomal metabolism of xenobiotics. This assay has been used extensively to study reactive metabolites which initiate toxicity through a direct (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen formation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displayed GSH reactivity that was either dependent or independent (amodiaquine) on metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquinone, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoin and diquat, two other redox cycling xenobiotics. Induction of the mixed function oxidase system with Aroclor afforded little advantage when using rat liver microsomes, whilst there was considerable inter-individual variation in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived reactive species.

Strain differences in adrenal CYP2D16 expression in guinea pigs: Relationship to xenobiotic metabolism

Experiments were done to determine the mechanisms responsible for differences in adrenal microsomal xenobiotic metabolism between Strain 13 and English Short-Hair (ESH) guinea pigs. The rates of adrenal xenobiotic metabolism (bufuralol 1′-hydroxylase, benzo[a]pyrene hydroxylase, benzphetamine N-demethylase) were 2–3 times greater in microsomes from the Strain 13 animals. In both strains, xenobiotic-metabolizing activities were far greater in the inner zone (zona reticularis) than in the outer zones (zona fasciculata and zona glomerulosa) of the adrenal cortex. Northern blot analyses of total adrenal RNA with a CYP2D16 cDNA as the probe revealed significantly greater amounts of CYP2D16 mRNA in the Strain 13 guinea pigs. In addition, SDS-PAGE and Western blotting of adrenal microsomes demonstrated higher concentrations of CYP2D16 protein in Strain 13 than in ESH animals. Expression of CYP2D16 was predominantly in the inner zone of the adrenal, coinciding with the major site of xenobiotic metabolism. The results demonstrated higher levels of expression of CYP2D16 in adrenal glands from Strain 13 than from ESH guinea pigs, which may account for the strain differences in adrenal xenobiotic metabolism. Strain 13 guinea pigs should serve as a good experimental model for further studies on the regulation of adrenal CYP2D16.

Changes in Alveolar Lavage Materials and Lung Microsomal Xenobiotic Metabolism Following Exposures to HCl-Washed or Unwashed Crystalline Silica

Intratracheal exposures of rats to crystalline silica washed with HCl to remove iron contaminants have previously been shown to increase lung surfactant phospholipids (PL) and proteins and to alter the pulmonary microsomal cytochrome P450 system. We compared these effects of HCl-washed silica with those produced by exposures to unwashed silica and alumina. Both silica preparations produce increases in lung weights and alveolar lavage PL and proteins, but to different degrees. The increases produced by HCl-washed vs unwashed silica are lung weights, 2.2- vs 1.3-fold; lavage FL, 25.9- vs 3.7-fold; and lavage proteins, 11.1- vs 3.2-fold, respectively. Although the two silica particles increase lung microsomal protein concentrations (expressed per gram lung) by 50-60%, their effects on cytochrome P-450-mediated xenobiotic metabolism are quite different. Exposure to HCl-washed silica leads to a 2.3-fold increase in 7-ethoxyresorufin O-deethylation, a reaction catalyzed by cytochrome P4501A1, and a 0.5- to 0.6-fold reduction in 7-ethoxycoumarin O-deethylation, a reaction which may be catalyzed by cytochrome P-4502B1. Unwashed silica does not alter the metabolism of either xenobiotic when results are expressed per milligram microsomal protein. Administration of alumina produces only minor increases in lung weight and lavage PL and no effect on microsomal xenobiotic metabolism. These results show that the increases in alveolar lavage PL and proteins induced by administration of unwashed silica are exaggerated by 3- to 7-fold if the silica is treated with HCl. Furthermore, exposure to HCl-washed silica results in significant alterations of the lung microsomal cytochrome P450 system, but the unwashed silica has little effect. Although the reason(s) for these different effects is not known, measurements of iron levels and formation of hydroxyl radicals using ESR demonstrate that there is more iron associated with the unwashed than with the HCl-washed silica.

Major differences between lung, skin and liver in the microsomal metabolism of homologous series of resorufin and coumarin ethers

Phenoxazone and a homologous series of its ethers (methoxy to octoxy plus benzyloxy), and coumarin and a series of its ethers (methoxy to propoxy), were metabolized by liver, lung and skin microsomes of normal adult female BALB/c mice. For each series of substrates, and with each tissue, clear structure-activity relationships were seen, relating metabolic activity to the length of the ether side-chain. With the coumarin series of substrates the structure-activity relationships were almost identical in the three tissues, with liver more active than lung and lung more active than skin. Liver, lung and skin microsomes each showed very different structure-activity relationships, however, for metabolism of the phenoxazone series of substrates. Benzyloxyphenoxazone was metabolized almost twice as fast in lung as in liver, but for the other phenoxazone substrates the activities were much greater in liver than in lung or skin. Liver, lung and skin microsomal propoxy- and benzyloxyphenoxazone dealkylase activities differed in their sensitivities to inhibition by metyrapone and α-napthoflavone. The structure-activity relationship and inhibitor data for the phenoxazone substrates are consistent with a view that mouse lung and sking cyt. P-450 are predominantly similar to phenobarbitone-induced and 3-methylcholanthrene-induced forms of hepatic cyt. P-450 respectively. The results also show that the pattern of microsomal metabolism of xenobiotics in lung and skin cannot be reliably predicted from that in liver.

In vivo effect of ozone inhalation on xenobiotic metabolism of lung and liver of rats

Male Jcl:Wistar rats were exposed to 0.4 and 0.8 ppm O3 daily for 7 h for 14 d to examine the effect of O3 on xenobiotic metabolism of lung and liver microsomes. An exposure to 0.4 ppm O3 did not affect the microsomal xenobiotic metabolism of either lung or liver. On the other hand, 0.8 ppm O3 increased significantly the NADPH-cytochrome P-450 reductase activity and the cytochrome P-450 content of lung microsomes. The activities of lung benzo[a]pyrene hydroxylase and 7-ethoxycoumarin O-deethylase also increased significantly on d 7, and remained at a higher level by the d 14. These results show that exposures to 0.8 ppm O3 induce the xenobiotic metabolizing systems in the lung. In the liver, after the first day of exposure to 0.8 ppm O3, a significant reduction occurred in all components of the electron-transport systems examined as well as in the microsomal protein. A significant decrease was also observed in benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and aniline hydroxylation. The decreased activities recovered in the following period of exposure. In contrast, the p-nitroanisole N-demethylase activity was not altered during the 14-d exposures. These results suggest that some isozymes of the hepatic cytochrome P-450 are sensitive to O3 inhalation but other(s) are resistant.

Cytosolic factors that alter the metabolism of N, N-dimethyl-4-aminoazobenzene by rat liver microsomes

N, N-Dimethyl-4-aminoazobenzene (DAB), an azo dye carcinogen, is N-demethylated and 4′-hydroxylated by rat liver microsomes. Addition of hepatic cytosol to the microsomal system stimulated both pathways. This occurred in the presence of added NADPH or an NADPH-generating system. Cytosol was effective only when present prior to addition of substrate; no stimulation was seen when added after the reaction had begun. This suggested a direct effect on the microsomes rather than a chemical interaction with one or more metabolic intermediates of DAB. The degree of stimulation was somewhat different when using microsomes from phenobarbital- or β-naphthoflavone-treated animals, implying a selectivity of the cytosolic effect for various isozymes of cytochrome P-450. Some loss of stimulatory activity occurred with dialysis. Activity was restored by adding back glutathione (GSH) which can stimulate DAB metabolism even in the absence of cytosol. DAB metabolism is also stimulated by EDTA. Although both EDTA and cytosol inhibit lipid peroxidation, cytosol stimulated DAB metabolism even in the presence of EDTA. Therefore, suppression of lipid peroxidation does not explain satisfactorily the cytosolic effect. Separation of cytosolic proteins by gel filtration revealed a factor which inhibits N-demethylation but not 4′-hydroxylation of DAB. Heating at 100° partially inactivated the stimulatory activity. However, inhibitory activity was less susceptible to heat inactivation than was stimulatory activity. These results indicate that, in the whole cell, microsomal metabolism of xenobiotics is regulated to an appreciable extent by macromolecular cytosolic substances.

Selective induction and inhibition of renal mixed function oxidases in the rat and rabbit

Renal mixed function oxidases vary in response to inducers depending on the species. Pretreatment of rabbits with β-naphthoflavone (100 mg/kg × 4 days) or phenobarbital (60 mg/kg × 4 days) resulted in a highly selective induction of renal microsomal ethoxyresorufin-O-deethylase and benzphetamine-N-demethylase, respectively. Pretreatment of rats with β-naphthoflavone (100 mg/kg) induced renal microsomal ethoxyresorufin and ethoxycoumarindeethylation whereas phenobarbital (80 mg/kg) was without effect. Furthermore, benzphetamine-N-demethylase could not be detected in rat renal microsomes. These data suggest that the composition of renal cytochromes P-450 may vary between species. α-Naphthoflavone inhibited both ethoxyresorufin and ethoxycoumarin metabolism by rat and rabbit renal microsomes; inhibition was highly selective for renal microsomes from β-naphthoflavone-treated animals. Metyrapone did not inhibit rat renal microsomal mixed function oxidases from any treatment group. In contrast, rabbit renal microsomal xenobiotic metabolism from all treatment groups were strongly inhibited by metyrapone. Furthermore, polyacrylamide gel electrophoresis of solubilized renal microsomes from rabbits treated with phenobarbital revealed a new coomassie blue staining band in the range of 50 000–60 000 daltons; no such pattern was observed in rats. Electrophoresis of solubilized renal microsomes from both rats and rabbits treated with β-naphthoflavone revealed increased Coomassie blue staining. It is apparent from these results that the rat kidney lacks any phenobarbital inducible form of cytochrome P-450 and that rat renal microsomes contain at least two forms of cytochrome P-450 whereas rabbit renal microsomes contain at least three forms.

The induction of rat hepatic microsomal xenobiotic metabolism by n-octadecyl β-(3′,5′-di- tert-butyl-4′-hydroxyphenyl)-propionate

The hepatic effects of oral administration of n-octadecyl β-(3′,5′-di- tert-butyl-4′-hydroxyphenyl)propionate (OBPP) has been studied in male and female rats. OBPP administration produced both marked liver enlargement and the induction of a number of parameters of hepatic microsomal xenobiotic metabolism, including cytochrome P-450, mixed-function oxidase enzymes and UDPglucuronosyltransferase. Histological examination of liver sections from OBPP-treated rats revealed hypertrophy of the centrilobular cells of the liver lobule, and ultrastructural studies indicated a marked proliferation of the smooth endoplasmic reticulum. Reversibility studies demonstrated that the increase in liver size and in the activities of xenobiotic-metabolizing enzymes had substantially reverted to control levels 14 days after the cessation of OBPP treatment. It is concluded that OBPP is a potent inducer of hepatic xenobiotic metabolism in the rat, with properties similar to those of sodium phenobarbitone.

Constitutive and induced hepatic microsomal cytochrome P-450 monooxygenase activities in male Fisher-344 and CD rats. A comparative study

The hepatic microsomal mixed function monooxygenase system (MFO) is the major enzyme system responsible for the activation and deactivation of xenobiotics. This study was designed to compare the hepatic MFO system in Fischer-344 and CD (Sprague-Dawley) rats. Hepatic microsomes were prepared from control, phenobarbital (3 × 80 mg/kg)-, and 3-methylcholanthrene (3 × 20 mg/kg)-pretreated male F-344 and CD rats (49 days old). Both control and phenobarbital-treated F-344 rats had significantly lower microsomal epoxide hydratase activity than corresponding preparations from CD rats. In addition, the pattern of benzo( a)pyrene metabolism was significantly different between the strains. Microsomes from F-344 rats produced less dihydrodiols and quinones than the corresponding preparations from CD rats. The spectral characteristics of cytochrome P-450 in hepatic microsomes from both control and phenobarbital-treated F-344 rats were significantly different from those observed in CD rat hepatic microsomes. Specifically, the λ max for the reduced cytochrome P-450 CO complex occurred at a slightly longer wavelength and the reduced ETNC 430 455 nm peak ratios were larger by about 70%. No significant strain differences were detected in control rats or rats pretreated with either phenobarbital or 3-methylcholanthrene with regard to microsomal protein, benzphetamine- N-demethylase, NADPH-cytochrome c reductase, biphenyl-4-hydroxylase, biphenyl-2-hydroxylase, arylhydrocarbon hydroxylase, ethoxycoumarin- or ethoxyresorufin- O-deethylase activities. These results suggest that differences do exist in the in vitro hepatic microsomal metabolism of xenobiotics in these two strains of rats. These differences may be of importance with respect to the susceptibility of the two strains of rats to various toxic agents.

The circadian variation of hepatic microsomal drug and steroid metabolism in the golden hamster

Rhythmic variations in a number of parameters of hepatic microsomal drug and steroid metabolism and binding were observed in the adult male golden hamster. With an illumination schedule of a light phase between 06.30 and 18.30 h, maximal activities were encountered between 04.00 and 08.00 h with corresponding minima between 16.00 and 18.00 h. In addition, kinetic changes were observed in both the metabolism and spectral interaction of aniline and biphenyl at times of maximal and minimal activities. Possible mechanisms responsible for the circadian variation in hamster hepatic microsomal xenobiotic metabolism are discussed.