The exploration of N6-deoxyadenosine methylation in mammalian genomes.

N6-methyladenine (N6-mA, m6dA, or 6mA), a prevalent DNA modification in prokaryotes, has recently been identified in higher eukaryotes, including mammals. Although 6mA has been well-studied in prokaryotes, the function and regulatory mechanism of 6mA in eukaryotes are still poorly understood. Recent studies indicate that 6mA can serve as an epigenetic mark and play critical roles in various biological processes, from transposable-element suppression to environmental stress response. Here, we review the significant advances in methodology for 6mA detection and major progress in understanding the regulation and function of this non-canonical DNA methylation in eukaryotes, predominantly mammals.

Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver.

Enrofloxacin (EF) is a widely used fluoroquinolone, usually regarded as a safe and effective treatment for bacterial infections. Adverse effects of EF have previously been demonstrated in some species, but so far there have been no studies looking specifically at the impact of EF on pigs. In this study, three different doses of EF (5, 25 and 125 mg kg bw-1 ) were administrated to Bama pigs. The results showed that lipid peroxidation of pig liver tissue occurred with all EF doses. The 125 mg kg dose of EF induced catalase (CAT) and glutathione peroxidase (GSH-px) and increased CYP450 content in pig liver microsomes. The activity of microsomal NADPH-cytochrome C reductase (NCCR) was elevated at both the 25 and 125 mg kg doses of EF. Microsomal erythromycin N-demethylase (ERND) and aminopyrin N-demethylase (AND) were inhibited by high doses of EF, while aniline-4-hydroxylase (AH) was unaffected. None of the EF treatments affected superoxide dismutase (SOD) or cytochrome b5 content. Antioxidases and microsomal enzymes may work together to resist the adverse effects of EF. Proteomic analysis revealed increased protein expression of carboxylesterase (CES) and alpha-enolase (ENO1) in microsomes as a stress response to EF. These results provide new information about the adverse effect of fluoroquinolones and help guide their usage more effectively in the clinic or animal breeding.

Biotransformation and tissue distribution of protopine and allocryptopine and effects of Plume Poppy Total Alkaloid on liver drug-metabolizing enzymes.

In this study, the biotransformation in the plasma, urine and feces of rats following oral administration of protopine (PRO) and allocryptopine (ALL)were explored using HPLC-QqTOF MS. An HPLC-MS/MS method for the determination of tissues was developed and applied to the tissue distribution study in rats following intragastric administration of Plume Poppy Total Alkaloid for 3 weeks. A total of ten PRO metabolites and ten ALL metabolites were characterized in rats in vivo. Among these metabolites, six PRO metabolites and five ALL metabolites were reported for the first time. The predicated metabolic pathways including ring cleavage, demethylation following ring cleavage, and glucuronidation were proposed. The low-concentration residue of PRO and ALL in various tissues was detected at 24 h and 48 h after dosing, which indicated that both compounds could be widely distributed in tissues and exist as low levels of residue. The activities of erythromycin N-demethylase, aminopyrine N-demethylase and NAD (P)H quinone oxidoreductase in female rats can be induced post-dose, but these activities were inhibited in male rats. The proposed biotransformation and residues of PRO and ALL and their effects on enzymes may provide a basis for clarifying the metabolism and interpreting pharmacokinetics.

Toxicity study of ionic liquid, 1-butyl-3-methylimidazolium bromide on guppy fish, Poecilia reticulata and its biodegradation by soil bacterium Rhodococcus hoagii VRT1.

This study deals with the toxic effect of ionic liquid, 1-butyl-3-methylimidazolium bromide (BMImBr) on guppy fish, Poecilia reticulata. The fishes were exposed to various concentrations of ionic liquid for 96h. The activity of antioxidant enzymes viz. catalase, glutathione S-transferase and superoxide dismutase were found to be increased with increase in concentration. The BMImBr resistant bacterium were isolated from garden soil by enrichment method and identified as Rhodococcus hoagii VRT1 by 16S rDNA sequencing. An isolated bacterium was effective in biodegradation of compound in 8 days which was analyzed by changes in BOD and COD and later on confirmed by HRMS analysis. Higher concentrations of compound induced DNA damage in liver cells while degraded product did not show adverse impact on the DNA integrity.

Effect of atrazine and chlorpyrifos exposure on cytochrome P450 contents and enzyme activities in common carp gills.

Chlorpyrifos (CPF) and atrazine (ATR) are the most widely used organophosphate insecticides and triazine herbicides, respectively, worldwide. This study aimed at investigating the effects of ATR, CPF and mixture on common carp gills following 40-d exposure and 40-d recovery experiments. Cytochrome P450 content, activities of aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND) and the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) were determined. In total, 220 common carps were divided into eleven groups, and each group was treated with a specific concentration of ATR (4.28, 42.8 and 428 µg/L), CPF (1.16, 11.6 and 116 µg/L) or ATR-CPF mixture (1.13, 11.3 and 113 µg/L). The results showed that P450 content and activities of APND and ERND in fish exposed to ATR and mixture were significantly higher than those in the control group. After the 40-d recovery treatment (i.e., depuration), the P450 content and the activities of APND and ERND in fish decreased to the background levels. A similar tendency was also found in the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) in common carp gills. The CPF-treated fish showed no significant difference from the control groups, except for a significant CYP1C induction. These results indicated that CYP enzyme levels are induced by ATR but were only slightly affected by CPF in common carp gills. In addition, the ATR and CPF exposure showed an antagonistic effect on P450 enzymes in common carp gills.

The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers.

Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10µgL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver.

Mitragyna speciosa Korth leaves extracts induced the CYP450 catalyzed aminopyrine-N-demethylase (APND) and UDP-glucuronosyl transferase (UGT) activities in male Sprague-Dawley rat livers.

BACKGROUND: Mitragyna speciosa leaves have been abused by drug addicts as some of the alkaloids (mainly mitragynine) from the plant possess opiate and cocaine-like effects. These bring to its prohibition in Malaysia in 2004 as consumption of M. speciosa leaves has been perceived to lead to the abuse of other drugs such as cannabis and heroin. METHODS: In the current study, the in vitro and in vivo effects of M. speciosa methanolic, aqueous and total alkaloid leaves extracts on drug metabolizing enzymes, namely, cytochrome P450s (CYP450s) and UDP-glucuronosyl transferase (UGT) had been evaluated in rat liver cytosolic fraction and microsomes. Aminopyrine and p-nitrophenol (pNP) were employed as probe substrates in aminopyrine N-demethylase (APND) and UGT enzyme assays, respectively. Furthermore, mitragynine was also tested in vitro for its likelihood to inhibit APND and UGT activity. The assessment of the enzyme activity was conducted using spectrophotometric methods. RESULTS: In vitro, the IC50 value could only be obtained for the methanolic extract in APND study (595.30±30.78 µg/mL) and not in other studies due to the enzyme percentage inhibitions being <70%. In contrast to the in vitro study, the oral treatment of male Sprague-Dawley rats for 14 days with 50, 100 and 200 mg/kg of methanolic and aqueous extracts and with 5, 10 and 20 mg/kg of total alkaloid extract showed a profound increment on the APND and UGT activities. CONCLUSIONS: The current findings showed that possibilities exist for herb-drug interaction with increased clearance of drugs, which are primarily metabolized by CYP450s and UGT1A6 among M. speciosa leaves extract users.

Multi-biomarker responses in fishes from two typical marine aquaculture regions of South China.

The impact of typical pollutants upon the fish-farming was assessed by use of a battery of biomarkers in two typical marine aquaculture regions in South China. Biotransformation parameters including 7-ethoxyresorufin-O-deethylase (EROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (ERND), glutathione-S-transferase (GST) and Malondialdehyde (MDA) were measured in five cultured fish species. Pollutants such as polycyclic aromatic hydrocarbon (PAHs), organochlorinated compounds (OCs), heavy metals and antibiotics (quinolones and sulfonamides) in sediments were characterized. Higher pollutant residue levels were observed in Dapeng Cove. EROD, APND and ERND activities were lower in fish from Dapeng Cove compared with fish from Hailing Island, while it is just on the contrary for GST and MDA. ERND, APND and GST showed sensitivity corresponding to different pollutants. Small fish species seemed to exhibit more sensitive to pollutants. The study further supports usefulness of multi-biomarker approach considering multiple species to define the effects of anthropogenic inputs in marine aquaculture systems.

Reversal of experimental ethanol-induced liver steatosis by borage oil.

The aim of study was to evaluate the hepatoprotective effect of borage oil containing predominantly gamma-linolenic acid in rats with alcoholic steatohepatitis. Liver of ethanol-treated animals was characterized by fatty and hydropic dystrophies. Liver triglyceride contents and activitiies of serum marker enzymes were significantly increased. Ethanol increased nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-induced chemiluminescence and the contents of liver thiobarbituric acid reactive substances (TBARS). The reduced glutathione content in the liver was decreased. Ethanol enhanced liver microsomal cytochrome P-450 (CYP450) content, aniline p-hydroxylase and amydopyrine-N-demethylase activities. The treatment with borage oil improved the liver morphology, decreased triglyceride contents and normalized serum marker enzyme activities. Borage oil developed an antioxidant effect in ethanol-treated rats. The treatment with this compound decreased NADPH-induced chemiluminescence and the content of lipid peroxidation products. Borage oil normalized CYP450 content compared with the ethanol-treated group. CYPI450 2E1 isoform is a main source of free oxygen radicals in the liver of ethanol-treated rats and we propose that the antioxidant effect of borage oil is realized via the normalization of CYP450 content and activities of CYP450-related microsomal oxidases, as borage oil can improve the lipid surrounding of CYP450. In our opinion, the hepatoprotection by borage oil in alcoholic steatosis is connected with its antioxidant properties.

Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response.

Organic pollutants, heavy metals and pharmaceuticals are continuously dispersed into the environment and have become a relevant environmental emerging concern. In this study, a situ assay to assess ecotoxicity of mixed pollutants was carried out in three typical sites with different priority contaminations in Guangzhou, China. Chemical analysis of organic pollutants, metals and quinolones in three exposure sites were determined by GC-ECD/MS, ICP-AES and HPLC, as well as, a combination of biomarkers including: ethoxyresorufin O-deethylase (EROD); aminopyrine N-demethylase (APND); erythromycin N-demethylase (ERND); glutathione S-transferase (GST); malondialdehyde (MDA); CYP1A; and P-glycoprotein (P-gp) mRNA expressions were evaluated in Mugilogobius abei. Results of chemical analysis in sediment samples revealed that the dominant chemicals were organic pollutants and heavy metals in Huadi River while quinolones in the pond. Bioassays indicated that differences among sites were in relation to some specific biomarkers. EROD and GST activities significantly increased after 72 h in situ exposure, but no difference was observed among the exposure sites. APND, ERND and MDA exhibited dissimilar change patterns for different priority pollutants. CYP1A and P-gp mRNA expressions were significantly induced at all exposure sites, whilst P-gp activity was typical for S2 with the highest levels of quinolones. The molecular biomarkers seemed to be more susceptible than enzyme activities. These assays confirmed the usefulness of applying a large array of various combined biomarkers at different levels, in assessing the toxic effects of mixed pollutants in a natural aquatic environment.

Effects of atrazine on cytochrome P450 enzymes of zebrafish (Danio rerio).

In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in males and females of adult zebrafish (Danio rerio) were studied. The liver microsomal cytochrome P450 content, NADPH-P450 reductase, aminopyrine N-demethylase (APND), and erythromycin N-demethylase (ERND) activity were measured. Zebrafish were exposed to control and 3 treatments (0.01, 0.1, and 1 mg L(-1)) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, within the range of test atrazine concentrations, either P450 content or P450 isozyme activities could be induced by atrazine. Compared to controls, P450 content was significantly increased at all atrazine concentrations at days 10, 15, and 20; thereafter, at day 25, all concentrations decreased to approximately the control levels, both in males and females. In addition, the strongest induction of P450 content was observed on day 15 in males and day 10 in females at treatment concentrations of 1 mg L(-1). NADPH-P450 reductase activities showed mild increase in males; however, the females exhibited significant induction on days 15, 20, and 25; especially, at concentrations of 0.01 mg L(-1), the induction level was consistently increased during the experiment. The inducements of APND and ERND in males were mainly observed on the days 5, 10, and 15, which showed less distinct induction, while significant induction was observed in cases of treatments during all days in females. In conclusion, atrazine induces P450 enzymes in zebrafish, and the effects may function as significant toxicity mechanisms in zebrafish. Additionally, it also confirms the importance of using a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

Biodegradation of hazardous triphenylmethane dye methyl violet by Rhizobium radiobacter (MTCC 8161).

Rhizobium radiobacter MTCC 8161 completely decolorized methyl violet (10 mg l(-1)) within 8 h both at static and shaking conditions. The decolorization time increased with increasing dye concentration. The effect of different carbon and nitrogen sources on the decolorization of methyl violet was studied. The maximum decolorization was observed in the presence of sucrose (1%) and urea (1%). UV-Visible, HPLC and FTIR analysis of extracted products confirmed biodegradation of methyl violet. The significant increase in the activities of lignin peroxidase and aminopyrine N-demethylase in the cells obtained after decolorization indicated involvement of these enzymes in the decolorization process. In addition to methyl violet, this strain also shows an ability to decolorize various industrial dyes, (red HE7B, yellow 4G, blue 2B, navy blue HE22, red M5B and red HE3B).

[Effects of ethyl acetate extract of Semen Hoveniae on liver microsomal cytochrome P450 isoenzyme in rat].

OBJECTIVE: To investigate the effects of the ethyl acetate extract of Semen Hoveniae (ESH) on liver microsomal cytochrome P450 isoenzyme in rats. METHOD: The rats were given orally the ESH in the doses of 0.14, 0.17, 0.2 g x kg (equivalent to the crude herb) for 10 days respectively. Rat liver microsomal cytochrome P450, NADPH-Cyt C reductase, erythromycin N-demethylase (ERD), Aniline hydroxylase (ANH), aminopyrine N-demethylase (ADM) activities were quantitated by UV chromatography. The levels of mRNA expression of CYP1A1, CYP2C11, CYP2E1 and CYP3A1 were detected by semi-quantitative reverse transcripatase-polymerase chain reaction (RT-PCR). RESULT: The cytochrome P450 content, NADPH-Cyt C reductase activities and erythromycin N-demethylase (ERD) activities were not affected. Aniline hydroxylase (ANH) activities in liver were decreased by up to35.1%; aminopyrine N-demethylase (ADM) activitiesin liver were increased by up to 42.4%. The mRNA expression of CYP1A1, CYP2C11 and CYP3A1 were found to be increased markedly. CONCLUSION: A specific effect of ESH on liver microsomal cytochrome P450 isoenzyme in rats was observed in this investigation. ESH had various effects on liver microsomal cytochrome P450 isoenzyme.

Hepatic injury induces contrasting response in liver and kidney to chemicals that are metabolically activated: role of male sex hormone.

Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase activities significantly only in male mice. Increases in Cyp2e1 and Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.

Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride.

Induction of CYP 2E1 by carbon tetrachloride (CCl(4)) is one of the central pathways by which CCl(4) generates oxidative stress in hepatocytes. Experimental liver injury was induced in rats by CCl(4) to determine toxicological actions on CYP 2E1 by microsomal drug metabolizing enzymes. In this report, ethanolic extract of propolis at a dose of 200 mg/kg (po) was used after 24 h of toxicant administration to validate its protective potential. Intraperitoneal injection of CCl(4) (1.5 ml/kg) induced hepatotoxicity after 24 h of its administration that was associated with elevated malonyldialdehyde (index of lipid peroxidation), lactate dehydrogenase and gamma-glutamyl transpeptidase release (index of a cytotoxic effect). Hepatic microsomal drug metabolizing enzymes of CYP 2E1 showed sharp depletion as assessed by estimating aniline hydroxylase and amidopyrine N-demethylase activity after CCl(4) exposure. Toxic effect of CCl(4) was evident on CYP 2E1 activity by increased hexobarbitone induced sleep time and bromosulphalein retention. Propolis extract showed significant improvement in the activity of both enzymes and suppressed toxicant induced increase in sleep time and bromosulphalein retention. Choleretic activity of liver did not show any sign of toxicity after propolis treatment at a dose of 200 mg/kg (id). Histopathological evaluation of the liver revealed that propolis reduced the incidence of liver lesions including hepatocyte swelling and lymphocytic infiltrations induced by CCl(4). Electron microscopic observations also showed improvement in ultrastructure of liver and substantiated recovery in biochemical parameters. Protective activity of propolis at 200 mg/kg dose was statistically compared with positive control silymarin (50 mg/kg, po), a known hepatoprotective drug seems to be better in preventing hepatic CYP 2E1 activity deviated by CCl(4). These results lead us to speculate that propolis may play hepatoprotective role via improved CYP 2E1 activity and reduced oxidative stress in living system.

Decolourization of azo dye methyl red by Saccharomyces cerevisiae MTCC 463.

Saccharomyces cerevisiae MTCC 463 decolourizes toxic azo dye, methyl red by degradation process. Methyl red (100mgl(-1)) is degraded completely within 16min in plain distilled water under static anoxic condition, at the room temperature. Effect of physicochemical parameters (pH of medium, composition of medium, concentration of cells, concentration of dye, temperature and agitation) on methyl red decolourization focused the optimal condition required for decolourization. Biodegradation (fate of metabolism) of methyl red in plain distilled water was found to be pH dependent. Cells of Saccharomyces cerevisiae could degrade methyl red efficiently up to 10 cycles in plain distilled water. Analysis of samples extracted with ethyl acetate from decolourized culture flasks in plain distilled water (pH 6.5) and at pH 9 using UV-VIS, TLC, HPLC and FTIR confirm biodegradation of methyl red into several different metabolites. A study of the enzymes responsible for the biodegradation of methyl red in the control and cells obtained after decolourization in plain distilled water (pH 6.5) and at pH 9 showed different levels of the activities of laccase, lignin peroxidase, NADH-DCIP reductase, azoreductase, tyrosinase and aminopyrine N-demethylase. A significant increase in the activities of lignin peroxidase and NADH-DCIP reductase was observed in the cells obtained after decolourization in plain distilled water (pH 6.5), however cells obtained at pH 9 shows increased activities of azoreductase, tyrosinase, lignin peroxidase and NADH-DCIP reductase. High efficiency to decolourize methyl red in plain distilled water and low requirement of environmental conditions enables this yeast to be used in biological treatment of industrial effluent containing azo dye, methyl red.

Zinc mediates normalization of hepatic drug metabolizing enzymes in chlorpyrifos-induced toxicity.

The present study investigated the protective effects of zinc in attenuating the altered activities of drug metabolizing enzymes in the livers of rats intoxicated with chlorpyrifos. Male Sprague-Dawley rats received oral chlorpyrifos treatment (at a dose level of 13.5 mg/kg body weight in corn oil every alternate day), zinc supplementation alone (at a dose level of 227 mg/l in drinking water), or combined chlorpyrifos plus zinc treatments for a total duration of 8 weeks. The effects of different treatments were studied on the specific activities of various drug metabolizing enzymes including cytochrome P(450), cytochrome b(5), NADPH cytochrome-c-reductase, NADH cytochrome-c-reductase, aminopyrene-N-demethylase (APD) and aromatic hydrocarbon hydroxylase (AHH). Additionally, serum zinc levels were also determined in each of the treatment groups at the end of the study. Chlorpyrifos treatment resulted in a significant decrease in the serum zinc concentrations. Analogous to these changes, we observed significant depression in the activities of majority of the drug metabolizing enzymes investigated in the present study, except for AHH, where the decrease in enzyme activity was not statistically significant. However, zinc treatment to chlorpyrifos treated animals effectively restored the depressed serum zinc levels to within normal limits. Similarly, co-administration of zinc to chlorpyrifos intoxicated animals normalized the enzymatic activities of cytochrome P(450), NADPH cytochrome-c-reductase and NADH cytochrome-c-reductase within normal range. Collectively, these findings suggest that zinc plays an important role in regulating the hepatic activities of drug metabolizing enzymes in chlorpyrifos intoxicated animals, although it remains to be determined whether such protective effects of zinc are regulated directly, or through some indirect mechanism.

Biotransformation enzymes in Cunninghamella blakesleeana (NCIM-687).

Presence of higher enzyme levels of aminopyrine N-demethylase, aniline hydroxylase and 11-beta hydroxylase activities were observed in Cunninghamella blakesleeana grown in potato-dextrose medium for 96 h. The enzyme activity preferred NADPH as a cofactor and showed inhibition with CO, indicating cytochrome P450 mediated reactions. A significant increase in aniline hydroxylase enzyme activity was observed when mycelia incubated in incubation medium containing different inducers (viz. camphor, cholesterol, naphthalene, veratrole, phenobarbital, n -hexadecane and ethyl alcohol) when compared with mycelia incubated in same way but in absence of inducers. Cunninghamella blakesleeana (NCIM 687) have shown the ability to degrade cholesterol, camphor and naphthalene when 96 h grown mycelia incubated in incubation medium containing these organic compounds.

Influences of 3-methylcholanthrene, phenobarbital and dexamethasone on xenobiotic metabolizing-related cytochrome P450 enzymes and steroidogenesis in human fetal adrenal cortical cells.

AIM: To explore the influence and possible mechanism of xenobiotics on adrenal steroidogenesis during fetal development. METHODS: Primary human fetal adrenal cortical cells were prepared, cultured and treated with 3-methylcholanthrene, phenobarbital and dexamethasone. The activities of 7-ethoxyresorufin O-dealkylase, benzphetamine, aminopyrine and erythromycin N-demethylases were measured by enzyme assays. At the same time, quantitative analysis of steroid hormones cortisol, aldosterone, testosterone and progesterone were carried out in cultural medium by radioimmunoassays. RESULTS: The activities of benzphetamine and aminopyrine N-demethylase were increased in the cultural fetal adrenal cells treated with phenobarbital (0.25-1 mmol/L) for 24 h. Dexamethasone (25-100 micromol/L) also increased the activity of erythromycin N-demethylase. The activity of 7-ethoxyresorufin O-dealkylase was undetected in the cells treated without and with 3-methylcholanthrene (0.5-2 micromol/L). Meanwhile, the contents of medium cortisol, aldosterone and progesterone were decreased after treatment with 3-methylcholanthrene. Cortisol, aldosterone and progesterone concentrations were also slightly decreased with phenobarbital. Dexamethasone enhanced the productions of cortisol and progesterone remarkably. The trend of testosterone concentration was uncertain after 3-methylcholanthrene, phenobarbital or dexamethasone treatment. CONCLUSION: 3-Methylcholanthrene, phenobarbital or dexamethasone could interfere with the synthesis of cortisol, aldosterone and progesterone in primary human fetal adrenal cortical cells, which likely act through xenobiotic metabolizing-related cytochrome P450 isoform activation.

Inhibition of drug metabolizing enzymes (cytochrome P450) in vitro as well as in vivo by Phyllanthus amarus SCHUM & THONN.

An alcoholic extract of Phyllanthus amarus (P. amarus) was found to inhibit cytochrome P450 (P450) enzymes both in vivo as well as in vitro. This was studied using specific resorufin derivatives, as substrate for isoenzymes in the P450 super family. Concentration needed for 50% inhibition of 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 was 4.6 microg/ml while concentration needed for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2 was 7.725 microg/ml and 7-pentoxyresorufin-O-depentylase (PROD), CYP2B1/2 was found to be 4.18 microg/ml indicating that the extract inhibited the P450 enzymes at very low concentration. Extract also inhibited the activity of aniline hydroxylase (an indicator of CYP 2E1 activity, IC(50) 50 microg/ml) and aminopyrine demethylase (an indicator of CYP 1A, 2A 2B, 2D and 3A activity, IC(50) >1000 microg/ml). Oral administration of the extract was also found to reduce the elevated P450 enzyme activities produced by phenobarbitone by 50% at 250 mg/kg body weight. The implication of these results on the inhibition of carcinogenesis produced by the extract is discussed.