Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.     

Acute sodium arsenite administration induces pulmonary CYP1A1 mRNA, protein and activity in the rat

Modulation of the cytochrome P450 (CYP) monooxygenase system (P450) by arsenite was investigated in male, adult Sprague-Dawley rats treated with a single dose (75 micromol/kg, sc) of sodium arsenite (As3+). Total CYP content and P450-dependent 7-pentoxyresorufin O-pentylation (PROD) and 7-ethoxyresorufin O-deethylation (EROD) activities of liver microsomes decreased maximally (33, 35, and 50% of control, respectively) 1 day after As3+ treatment. Maximum decreases of CYP content and P450 catalytic activities corresponded with maximum increases of microsomal heme oxygenase (HO) activity and with increased total plasma bilirubin concentrations. EROD activity increased maximally in lung (300%) 5 days after a single dose of As3+. Lung CYP1A1 mRNA and protein levels also increased maximally 5 days after treatment. A small but significant increase in EROD activity (65%) was observed in lung microsomes 24 h following a 1 h infusion of bilirubin (7.5 mg/kg) into rats. However, administration of bilirubin to the lung via intratracheal injection (0.25 and 2.5 mg/kg) did not increase CYP1A1 monooxygenase activity or mRNA. This study demonstrates that P450 is modulated in an isozyme (CYP1A1 vs CYP2B1/2) selective manner in rat lung after acute As3+ administration. Administration of bilirubin, a potential aryl hydrocarbon receptor (AHR) ligand, by infusion or intratracheal instillation did not upregulate pulmonary CYP1A1 at the mRNA level under our treatment conditions.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Modulation of drug-metabolizing enzymes by extracts of a herbal medicine Evodia rutaecarpa in C57BL/6J mice

Evodia rutaecarpa is a traditional Chinese medicine used for the treatment of gastrointestinal disorders and headache. To assess the possible drug interactions, effects of methanol and aqueous extracts of E. rutaecarpa on drug-metabolizing enzymes, cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. Treatment of mice with methanol extract by gastrogavage caused a dose-dependent increase of liver microsomal 7-ethoxyresorufin O-deethylation (EROD) activity. In liver, methanol extract at 2 g/kg caused 47%, 7-, 8-, 4-fold, 81% and 26% increases of benzo(a)pyrene hydroxylation (AHH), EROD, 7-methoxyresorufin O-demethylation (MROD), 7-ethoxycoumarin O-deethylation (ECOD), benzphetamine N-demethylation, and N-nitrosodimethylamine N-demethylation activities, respectively. Aqueous extract at 2 g/kg caused 68%, 2-fold, and 83% increases of EROD, MROD, and ECOD activities, respectively. For conjugation activities, methanol extract elevated UGT and GST activities. Aqueous extract elevated UGT activity without affecting GST activity. Immunoblot analyses showed that methanol extract increased the levels of CYP1A1, CYP1A2, CYP2B-, and GSTYb-immunoreactive proteins. Aqueous extract increased CYP1A2 protein level. In kidney, both extracts had no effects on AHH, ECOD, UGT, and GST activities. Three major bioactive alkaloids rutaecarpine, evodiamine, and dehydroevodiamine were present in both extracts. These alkaloids at 25 mg/kg increased hepatic EROD activity. These results demonstrated that E. rutaecarpa methanol and aqueous extracts could affect drug-metabolizing enzyme activities. Rutaecarpine, evodiamine, and dehydroevodiamine contributed at least in part to the increase of hepatic EROD activity by extracts of E. rutaecarpa. Thus, caution should be paid to the possible drug interactions of E. rutaecarpa and CYP substrates.     

The effect of xenobiotics on microRNA expression in the rat liver

The effect of xenobiotics on microRNA expression in the rat liver has been investigated. Based on results of bioinformatics analysis several microRNAs that can interact with 3'-untranslated regions of cytochrome P450 (CYP) mRNAs have been selected. These included three microRNAs (miR-21, miR-221, miR-222) for CYP1A1 mRNA as a putative target and two microRNAs (miR-143, miR-152) for CYP2B1 mRNA as a putative target. Using the RT-PCR method, expression levels of these microRNAs have been detected in the liver of rats treated with inducers of CYP1A and CYP2B, benzo(a)pyrene (BP), phenobarbital (PB), and DDT. In rats treated with both BP and DDT the hepatic content of miR-21, miR-221 and miR-222 was 2?3 times lower than in the control animals, while ethoxyresorufin-O-deethylase (EROD) activity of CYP1A1 demonstrated a 5.5?8.7-fold increase. In PB-treated rats miR-143 expression remained unchanged, the level of miR-152 increased 2-fold, while pentoxyresorufin-O-deetylase (PROD) activity of CYP2B increased 10.5-fold. In the liver of DDT-treated rats PROD activity demonstrated a 20.8-fold increase; expression of miR-143 increased 2-fold, and miR-152 expression remained unchanged. Bioinformatics analysis of putative miR-target interactions showed that the selected microRNAs can potentially bind such target as AhR, ESR1, GR, CCND1, PTEN mRNAs. Thus, the expression profile of miR-21, miR-221, miR-222, miR-143, miR-152 may vary in dependence on the CYP inducer used. Analysis in silico has shown that besides genes encoding CYP1A/2B other genes including those involved in hormonal carcinogenesis should be considered as potential targets of the investigated microRNAs.     

Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[a]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.     

Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx–DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx–DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx–DNA adduct formed was determined using ³²P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP–glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx–DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx–DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

Cytochrome P450 1A-dependent enzyme activities in the liver of dab (Limanda limanda): kinetics, seasonal changes and detection limits

Concentrations of total cytochrome P450 and cytochrome P450 1A (CYP 1A) and activities of ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) were measured in the liver of prespawning, spawning and postspawning dab (Limanda limanda) from the German Bight. Between all P450-dependent parameters measured significant correlations were found. Generally, during prespawning and spawning season higher values were measured in the liver of males compared to females, but the ratio between sexes changed during spawning time, when concentrations and activities in the liver of males decreased and increased in the liver of females. The activity and the signal-to-noise ratio decrease in the order EROD, ECOD and PROD. This decrease is accompanied by an increase in K_m. The findings indicate that the different activities can be attributed to the strongly overlapping substrate specificity and the different enzyme affinities of one enzyme, CYP 1A, towards the three substrates. A biphasic kinetic of ECOD indicates that in addition to CYP 1A a second isozyme catalyses the O-deethylation of ethoxycoumarin in the liver of dab. Interestingly, the ratio between EROD activity and CYP 1A concentration varied seasonally but did not differ significantly between sexes.     

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx-DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx-DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx-DNA adduct formed was determined using 32P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP-glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx-DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx-DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17β at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids - testosterone, 17β-estradiol, free estrone and androstenone - was not the primary cause of the reduced enzyme activities.     

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I(2)) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005 mg/kg Se, and I(2) deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T(4) (TT(4)), total T(3) (TT(3))], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I(2) deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I(2) and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I(2) status.     

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.     

Genetics of aryl hydrocarbon hydroxylase induction in mice: Response of the lung to cigarette smoke and 3-methylcholanthrene

When mice from different inbred strains are injected intraperitoneally with 3-methylcholanthrene (MC), the activity of aryl hydrocarbon hydroxylase (AHH) rapidly increases in livers of some strains but not others. AHH plays a role in the metabolism of polycyclic hydrocarbons. Alleles at a small number of loci account for most of the variation in inducibility of hepatic AHH among mice, when MC is used as the inducing agent. Cigarette smoke is a common source of carcinogenic polycyclic hydrocarbons in the environment. Since some of the hydrocarbons in cigarette smoke are metabolized by AHH, the activity of AHH in tissues may affect the carcinogenicity of smoke in those tissues. The purpose of these experiments was to see whether induction of AHH in lung in response to cigarette smoke is regulated by the same genes that regulate induction of AHH in liver in response to MC. Mouse strains AKR/J and C57L/J and six recombinant inbred (RI) lines derived from them were tested for the response of AHH in lung and liver to parenteral MC or inhalation of cigarette smoke. Inducibility (the ratio of MC-induced AHH activities to basal AHH activities) in liver from MC-treated RI lines is bimodal and compatible with Mendelian segregation of genes at a small number of loci. Increased activities of AHH in MC-treated liver are associated with increased ability to metabolize BP and whole smoke condensates to mutagens detected by Salmonella typhimurium TA1538. Inducibility of AHH in lung in response to MC is not bimodal, and no definite conclusion about the number of loci can be made. When actual levels of AHH activity are considered, following the administration of MC as inducing agent, there is a correlation (r=0.89, p<0.01) between AHH levels in liver and lung, suggesting that some genes affecting liver also affect lung. Basal and MC-induced AHH levels in lung are also correlated (r=0.86, p<0.01). Mice with high basal activities have two to threefold higher levels of AHH after MC treatment than do mice with low basal activities. Induction of AHH in pulmonary tissues occurs in all mice after either parenteral MC or smoke inhalation. In contrast to MC treatment, AHH activities in lungs following smoke inhalation are not correlated with AHH levels in liver after MC (r=0.49) and are only weakly correlated with basal (r=0.66, 0.05相似文献   

The anti-carcinogenic plant compound indole-3-carbinol differentially modulates P450-mediated steroid hydroxylase activities in mice.

Indole-3-carbinol (I3C), a component of cruciferous vegetables, exhibits anti-carcinogenic activity in a variety of model systems. This activity has been attributed in part to the induction of cytochrome P450 CYP1A subfamily members and the resulting increased metabolic inactivation of chemical carcinogens. The present study was undertaken to assess the effects of I3C on several constitutive P450 activities that contribute to both carcinogen and steroid hormone metabolism. Mice were administered I3C in their diet at estimated daily doses of 250, 500 and 750 mg/kg for 1 week. Liver microsomes from treated and untreated mice were subsequently assayed for CYP1A-mediated ethoxy-resorufin O-deethylase (EROD) activity, estradiol 2-hydroxylase activity and seven different testosterone hydroxylase activities. I3C elevated EROD, estradiol 2-hydroxylase and testosterone 6 alpha-hydroxylase activities in a dose-dependent manner. The other six testosterone hydroxylase activities were not significantly affected by in vivo treatment with I3C. In addition to its effects on steroid hydroxylase activities, I3C also elevated NADPH-cytochrome P450 reductase activity, a necessary component to the P450 monooxygenase system. We next examined the direct in vitro effects of I3C and its acid condensation products, as are generated in the stomach following ingestion, on the P450 catalytic activities. Testosterone 6 beta-hydroxylase, the major testosterone hydroxylase activity in untreated mice, was significantly inhibited (IC50 approximately 12 micrograms/ml) by the acid condensation products of I3C. In contrast, all other P450 activities were not appreciably affected by I3C or its acid condensation products. These results indicate that I3C can elicit both inductive and suppressive effects on the constitutive P450s that participate in carcinogen and steroid hormone metabolism. This pleiotropic effect on hepatic catalytic enzymes may contribute to the anti-carcinogenic properties of this compound.     

Comparison of the metabolism of benzo[alpha]pyrene and binding to DNA caused by rat liver nuclei and microsomes.

Administration of 3-methylcholanthrene (3MC) to rats greatly enhanced the aryl hydrocarbon hydroxylase (AHH) activity of liver nuclei. However, the binding in vitro [3H]benzo[alpha]pyrene (BP) to DNA within the nuclei which occurred at the same time as hydroxylation of BP was much less enhanced. Thin layer chromatography of the metabolites of BP produced by these nuclei revealed the same metabolites in similar relative amounts as were produced by rat liver microsomes prepared from rats which had received 3MC. The binding to DNA was further analysed by hydrolysis of the DNA and fractionation on a Sephadex column. This analysis revealed that the binding to DAN in nuclei was very similar in nature to that which occurred when calf-thymus DNA was added to microsomes metabolising BP.     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[alpha]pyrene in tissue homogenates from inbred Syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[alpha]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SSLak, LSH/SlLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagans. Dimethylnitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[alpha]pyrene as substrate. MC does not induced AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC. Activity of DMND and conversion of DMN to mutagen are correlated (r = 0.59) in hamster liver. Microsomes of hamster liver are more effective than those from mouse in converting DMN to mutagen, despite similar DMND activities in livers from the two species.     

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.