Hepatic Microsomal Cytochrome P450s Research Articles

Extraction of Ethyl-p-methoxycinnamate from Kaempferia galanga L.

In this study, ethyl-p-methoxycinnamate (EPMC) was extracted from Kaempferia galanga L. by different methods. The study results show that vacuum drying process led to a significant decrease in EPMC content in the herb (about 7%); EPMC obtained by water distillation not only had a high purity and yield but could also be applied for both fresh and dried herb; whereas, Soxhlet extraction with n-hexane solvent brought the highest extraction yield (1.99% on dried herb weight) despite lower purity when compared to that by water distillation method.
 Keywords
 Ethyl-p-methoxycinnamate, EPMC, Kaempferia galanga L., extraction.
 References
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Effects of Dendrobium huoshanense on expressions and activities of hepatic microsomal cytochrome P450s in mice

This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⁻¹), D. huoshanense high dose group (crude drug 7.5 g·kg⁻¹), and phenobarbital positive control group (0.08 g·kg⁻¹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.

The immature rat as a potential model for chemical risks to children: Ontogeny of selected hepatic P450s

Concern about potential susceptibilities of infants and children to chemicals has led to the consideration of immature rodents as potential test surrogates. Maturation of some hepatic microsomal cytochrome P450s (CYPs), that participate in metabolic activation of organic solvents and polycyclic aromatic hydrocarbons (PAHs), may differ significantly between humans and rodents. The present investigation was undertaken to delineate the ontogeny of selected hepatic CYPs in male and female Sprague-Dawley (S-D) rats, and to contrast them with developmental profiles in humans. Microsomes were prepared from the liver of sexed and unsexed postnatal day (PND) 1–90 rats, and total CYP450 levels, as well as CYP1A1/2, CYP2E1 and CYP2B1/2 activities and protein, were quantified. CYP1A1/2 and CYP2E1 activity and expression rose rapidly after birth, peaked from PND 21–40/50, and declined substantially to adult values by PND 90. The same ontogenic profiles were manifested when the enzyme activities were expressed per entire liver or liver normalized to body weight. CYP1A1/2 and CYP2E1 activity and protein expression were well correlated. CYP2B1/2 activity peaked abruptly on PND 21 and declined irregularly to adult values. These patterns are in contrast to human CYP1A2 and CYP2E1, which are reported to progressively increase in liver during the first few months to years of life. The three CYP protein developmental profiles were largely gender independent in rats. The immature rat does not appear to be a suitable model for assessing risks posed to infants and children by chemicals metabolically activated by CYP2E1, based on the findings of greater carbon tetrachloride hepatotoxicity in preweanlings and weanlings than in adult animals. Additional studies are required to determine whether immature S-D rats may be used as an animal model for substrates of other CYPs, as total CYP450 levels in the liver progressively rose during maturation, similarly to humans.

Pharmacokinetic changes of drugs in a rat model of liver cirrhosis induced by dimethylnitrosamine, alone and in combination with diabetes mellitus induced by streptozotocin.

Rats with liver cirrhosis induced by N-dimethylnitrosamine (LC) and rats with LC with diabetes mellitus induced by streptozotocin (LCD) have been developed as animal models for human liver cirrhosis and liver cirrhosis with diabetes mellitus, respectively. Changes in the pharmacokinetics of drugs (mainly non-renal clearance, CLNR) in LC and LCD rats reported in the literature compared with respective control rats were reviewed. This review mainly explains the changes in the CLNRs of drugs (which are mainly metabolized via hepatic microsomal cytochrome P450s, CYPs) in LC and LCD rats, in terms of the changes in in vitro hepatic intrinsic clearance (CLint; mainly due to the changes in CYPs in the disease state), free (unbound) fraction of a drug in the plasma (fp) and hepatic blood flow rate (QH) depending on the hepatic excretion ratio of the drug. Generally, changes in the CLNRs of drugs in LC and LCD rats could be well explained by the above-mentioned three factors. The mechanism of urinary excretion of drugs (such as glomerular filtration or renal active secretion or reabsorption) in LC and LCD rats is also discussed. The pharmacokinetics of the drugs reported in the LC and LCD rats were scarce in humans. Thus, the present rat data should be extrapolated carefully to humans.

Effects of bicyclol on the activity and expression of cytochrome P450 after hepatic warm ischemia/reperfusion in rats

Bicyclol is a synthetic antihepatitis drug with antioxidative property. This study was designed to investigate the effects of bicyclol on the activity, gene and protein expressions of hepatic microsomal cytochrome P450s (CYPs) during hepatic ischemia and reperfusion (I/R) in rats. The rats were subjected to 90 min of hepatic ischemia followed by reperfusion for 3 and 24 h. Bicyclol (300 mg/kg) was orally administered three times before hepatic ischemia in rats. Liver injury was evaluated by biochemical examinations. Hepatic microsomal malondialdehyde (MDA) was measured spectrophotometrically. Total hepatic CYP content and activities of four CYP isozymes were evaluated with a differential spectrophotometer and liquid chromatography–mass spectrometry analysis. The gene and protein expressions of four CYP isozymes were determined by reverse transcriptional-polymerase chain reaction and Western blotting assay. As a result, bicyclol significantly inhibited the elevation of serum alanine aminotransferase and hepatic microsomal MDA and prevented the decrease of total hepatic CYP content in I/R rats. In addition, bicyclol markedly attenuated the decrease of 2C6, 2C11, 3A1/2 activity and reduction of mRNA or protein expression of 2C6 and 3A in I/R rats. As for CYP2E1, bicyclol further aggravated the decrease of protein expression in I/R rats. These results suggested that bicyclol may ameliorate the abnormalities in the activity and expression of certain CYP isoforms during I/R, and this protective effect was likely due to its antioxidative and hepatoprotective properties.

Effects of dietary butylated hydroxytoluene on aflatoxin B 1-relevant metabolic enzymes in turkeys

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B 1 (AFB 1) is due to a combination of efficient AFB 1 activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB 1-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB 1 to the putative toxic metabolite, the exo-AFB 1-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB 1-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB 1 detoxification by GST.

Hepatic microsomal cytochrome P450S and chlorinated hydrocarbons in largha and ribbon seals from Hokkaido, Japan: Differential response of seal species to ah receptor agonist exposure

AbstractFrom 16 largha seals (Phoca larghá) and 15 ribbon seals (Phoca fasciata) in the coastal waters of Hokkaido, Japan, blubber chlorinated hydrocarbon (CHC) levels and hepatic cytochrome P450 (CYP) catalytic activities and their immunochemically detected protein content levels were measured. Concentrations of DDTs (2,2‐bis(4‐chlorophenyl)‐1,1‐dichloroethylene, p,p′‐DDE; 2,2‐bis(4‐chlorophenyl)‐1,1‐dichloroethane, p,p′‐DDD; dichlorodiphenyltrichloroethane, p,p′‐DDT), polychlorinated biphenyl congeners (PCBs), and chlordane compounds (oxychlordane, chlordanes, and nonachlors) in both species were in the range of 290 to 5,300, 420 to 4,000, and 130 to 1,500 ng/g lipid weight, respectively. Aryl hydrocarbon receptor (AhR) agonists, non‐ortho (IUPAC 77 and 126) and mono‐ortho (IUPAC 105, 118, and 156) coplanar PCB congeners, were also detected, and the 2,3,7,8‐tetrachlordibenzo‐p‐dioxin (TCDD) toxic equivalents (TEQs) were 4.9 to 120 pg TEQ/g lipid weight. Cross‐reactive proteins with polyclonal antibodies against rat CYP1A1 and CYP3A2 were notably detected in seal liver microsomes. Interestingly, a polyclonal antibody against rat CYP2B1 recognized proteins only at trace levels. In largha seals, both levels of alkoxyresorufin‐ (methoxy‐, ethoxy‐, pentoxy‐, and benzyloxyresorufin) O‐dealkylase (AROD) activities and proteins detected by polyclonal antibodies against rat CYP1A1 were significantly correlated with the concentrations of individual coplanar PCB congeners, total TEQs, and total PCBs. Threshold concentrations for TEQs in blubber of the largha seal to induce hepatic CYP1A protein and EROD activity were estimated to be 8.5 and 19 pg TEQ/g fat weight, respectively. In ribbon seals, similar correlations were not detected, although the TEQ levels were not significantly lower than those in largha seals. These results suggest that AROD activity and CYP1A1 protein in the liver of the largha seal could be a biomarker for the exposure to AhR agonists such as coplanar PCB congeners. This study also indicates differences in AhR‐mediated responses to the CHC exposures between largha and ribbon seals.

Hepatic CYP1A Induction in 3-Methylcholanthrene-Treated Transgenic Rats with Insufficient Blood Growth Hormone.

We have shown that the Mini rat, a transgenic rat strain carrying the antisense transgene for rat growth hormone, is a useful model to investigate the role of growth hormone in the expression of hepatic microsomal cytochrome P450s (CYPs). However, the induction of CYP isoforms by hepatic enzyme inducers in Mini rats has not been investigated. We selected two enzyme inducers, 3-methylcholantherene (2 mg/kg/day, i.p.) and phenobarbital (5 mg/kg/day, p.o.), and administered them to male Mini rats and their parental strain, Wistar rats, for 3 days. Enzyme activity assays and immunohistochemistry showed no differences in the expressions of CYP1A and CYP2B between untreated Wistar and Mini rats. 3-Methylcholanthrene administration induced CYP1A in both strains, but the induction was significantly higher in the Mini rat liver than in the Wistar rat liver. Phenobarbital administration increased CYP2B activity in both strains by more than 20-fold over their respective controls. Although there was no difference in the CYP2B immunohistochemistry between the two strains given phenobarbital, the CYP2B activity of phenobarbital-treated Mini rats was significantly higher than that of phenobarbital-treated Wistar rats. These results suggest that growth hormone may play a role in the hepatic enzyme induction.

Metabolism of ipecac alkaloids cephaeline and emetine by human hepatic microsomal cytochrome P450s, and their inhibitory effects on P450 enzyme activities.

In this study, we identified the metabolites and the CYP forms that are specifically involved in emetine O-demethylation in human liver microsomes, and cleared the inhibitory potential of cephaeline and emetine on the activity of the major drug-metabolizing CYP enzymes. Incubation of emetine with human liver microsomes yielded three metabolites identified by using HPLC by comparison of the retention time with the authentic sample of cephaeline, 9-O-demethylemetine and 10-O-demethylemetine. CYP3A4 and CYP2D6 were able to metabolize emetine to cephaeline and 9-O-demethylemetine, and CYP3A4 also participated in metabolizing emetine to 10-O-demethylemetine. Cephaeline and emetine inhibited probe substrates metabolism. IC50 for cephaeline against CYP2D6 and CYP3A4 were 121 and 1000 microM, respectively. For the emetine, CYP2D6 and CYP3A4 were 80 and 480 microM, respectively. Inhibition constants (Ki) for both compounds on the CYP2D6 and CYP3A4 activities were determined by graphic analysis of Dixon plots at various concentrations. The obtained Ki values of cephaeline for CYP2D6 and CYP3A4 were 54 and 355 microM, respectively, and the values of emetine were 43 and 232 microM, respectively. We concluded that these in vitro inhibitions of cephaeline and emetine would hardly increase plasma concentrations of co-administered drugs in clinical therapy.

Changes in amounts of cytochrome P450 isozymes and levels of catalytic activities in hepatic and renal microsomes of rats with streptozocin-induced diabetes

Hepatic microsomal cytochrome P450s, which are involved in the metabolism of drugs, hormones, prostaglandins and fatty acids, change when animals develop diabetes. We studied changes in cytochrome P450 isozymes in both hepatic and renal microsomes of rats with diabetes caused by streptozocin, and compared the results with changes in catalytic activities in the microsomes. In hepatic microsomes of diabetic rats, the amount of cytochrome P450 2E1, an acetone-inducible isozyme, was two and a half times that of control rats, and that of P450 4A2, a major renal isozyme, was three times that in the controls. The amounts of cytochrome P450s 2A1, 2C6, 2C7, 3A2, and 4A3 increased in hepatic microsomes of diabetic rats, and P450 2C11 decreased. Treatment with insulin restored these to the levels in the controls. The catalytic activities of aniline hydroxylation, 7-ethoxycoumarin O-dealkylation, testosterone 2β, 6β, 7α, and 16β-hydroxylation, and ω-, (ω − 1)-hydroxylation of lauric acid were low. In renal microsomes of diabetic rats, and testosterone 2α and 16α-hydroxylation activities were low. In renal microsomes of diabetic rats, cytochrome P450s 2E1, 4A2 and K-4 were induced, and ω- and (ω − 1)-hydroxylation activities were high. These changes were reversed by insulin treatment. The induction and suppression of cytochrome P450 isozymes in diabetic rats were consistent with changes in the catalytic activities. In both hepatic and renal microsomes, P450s 2E1 and 4A2 were induced, altered metabolism of ketones and fatty acids in diabetes may contribute to these changes.

Effects of aging and long-term caloric restriction on hepatic microsomal monooxygenases in female fischer 344 rats: Alterations in basal cytochrome P-450 catalytic activities

The effects of aging and caloric restriction on uninduced levels of hepatic microsomal cytochrome P-450s and specific P-450-related catalytic activities were evaluated in female Fischer 344 rats. Microsomes were isolated from livers of ad libitum (AL) fed rats 1, 3, 6, 16 and 26 months of age, and from calorie-restricted (CR) rats 6, 16 and 26 months of age. The recovery of microsomal protein was higher in CR than AL rats at 6, 16 and 26 months of age. Between ages 3 and 26 months there was a gradual decline in recovered microsomal P-450, but the differences between successive age categories were not significant. A different trend was observed for total cytochrome P-450 per liver. AL rats showed a 2-fold decline in P-450/liver between 6 and 26 months of age, while P-450/liver of corresponding CR rats increased 1.7-fold. Neither cytochrome b5 nor NADPH cytochrome C reductase varied consistently as a function of increased age, with all age groups showing greater reductase activity for CR rats than for AL rats. Phenobarbital (PB) and methylcholenthrene (MC) inducible cytochrome P-450 catalytic activities (pentoxy [PROD] and ethoxy [EROD] resorufin O-deethylases) exhibited similar age-related alterations that were distinctly different from the two other enzymes examined. Both PROD and EROD showed dramatic declines in activity between 1 and 3 months of age, with a slight increase in activity at 6 months and maintenance of the activity levels through 26 months. Benzphetamine N-demethylase (BND) and aniline p-hydroxylase (APH) activities were also altered as a function of increased age, with CR rats exhibiting increased isozyme activity above that seen for aging AL rats. Calorie restriction appears to have a modulating effect, compensating for an age-related decline of hepatic microsomal monooxygenase activity in female Fischer 344 rats.

Expression of hepatic microsomal cytochrome P450s as altered by uremia

The proportions of different hepatic microsomal cytochrome P450s expressed in uremic rats were studied with specific antibodies and with a steroid hydroxylase assay. In male uremic rats, the hepatic levels of P450 2C11, a male-specific form, and 3A2, a male-dominant form, were decreased to about 30% at 5 weeks after the induction of uremia. These changes were paralleled by decreases in the activities of testosterone 2α-, 16α-, and 6β-hydroxylation. The level of P450 2A1, abundant in immature rats, was increased 2-fold by uremia and accompanied by an increase in testosterone 7α-hydroxylation activity. The levels of P450 2C6 and 2E1 were not changed by uremia. The levels of male-specific and male-dominant forms such as P450 2C11 and 3A2 are affected by the serum level of testosterone, which was decreased in the male rats with uremia. Therefore, castrated rats were prepared to compare the effects of testosterone on hepatic cytochrome P450s in uremic rats with those in castrated rats. When testosterone was administered to the castrated rats, the decreased levels of both P450 2C11 and 3A2 returned to normal. However, the administration of testosterone to the uremic rats did not prevent the decrease in the levels of these P450s. In female rats, changes in the levels of cytochrome P450s were not as great during uremia as those in male uremic rats. The level of P450 2C12, a female-specific form, was not changed; the level of P450 2A1 was increased by 50%, that of 3A2 which is barely detected in female rats was increased by 60%, and that of 2E1 was increased by 25%. These results suggested that the changes in the hepatic levels of cytochrome P450s were affected by factors other than testosterone in uremic rats.

3-Methoxy-4-aminoazobenzene, a selective inducer for a high spin form of cytochrome P-448 in rat liver microsomes

Treatment of Splague Dawley rats with 3-methoxy-4-aminoazobenzene (3-MeO-AAB) resulted in striking increase of the activity of hepatic microsomal cytochrome P-450s which could efficiently catalyze the mutagenic activation of hepatocarcinogenic aromatic amines such as a tryptophan-pyrolysate component, Trp P-2, and a glutamic acid-pyrolysate component, Glu P-1. The 3-MeO-AAB-induced cytochrome P-450 (3-MeO-AAB-P-450) was examined for the molecular charactor by immuno-Western blotting using monoclonal antibody to 3-methylcholanthrene-induced cytochrome P-448 and was characterized as a high spin form of cytochrome P-448 (P-448H; m.w. 54,000).

Purification and partial characterization of hepatic microsomal cytochrome P-450s from phenobarbital- and 3-methylcholanthrene-treated rats.

Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.