Total Cytochrome P450 Content Research Articles

N-nitrosodimethylamine induced changes in the activities of carcinogen-metabolizing enzymes in the liver of male mice: role of glutathione and gossypol as antioxidants

The importance of nutrition in protecting living organisms from the toxic effects of environmental carcinogens has attracted the attention of many researchers. In order to exert their toxic and/or carcinogenic effects, most carcinogens need to be activated primarily by phase I drug-metabolizing enzymes including; cytochrome P450, cytochrome b5, arylhydrocarbon hydroxylase (AHH), N-nitroso-dimethylamine N-demethylase I (NDMA-N-dI), NADPH-cytochrome c reductase and the expression of cytochrome P450 2E1. Changes in the activity of these enzymes were determined in livers of male mice pretreated with either glutathione (GSH) or gossypol as antioxidants, for seven consecutive days before administration of NDMNA as single dose for two hours before decapitation of mice. The total hepatic content of cytochrome P450 and AHH activity were induced, whereas the activity of NDMA-N-dI and expression of cytochrome P450 2E1 were reduced after treatment of mice with NDMA as a single dose. On the other hand, preteatments of mice with either gossypol or GSH for seven consecutive days decreased the total cytochrome P450 content as well as AHH activity whereas the activity of NDMA-N-dI and the expression of cytochrome P450 2E1 increased. Treatment of mice with NDMA only induced the total hepatic content of cytochrome P450 and AHH activity. Interestingly pretreatment of mice with GSH for seven consecutive days before injection of NDMA was found to restore the induced cytochrome P450 and AHH activity caused by NDMA to their normal levels. However, pretreatment of mice with gossypol prior to administration of NDMA did not restore such activities to their normal levels. Treatment of mice with either GSH or gossypol induced the expression of cytochrome P450 2E1. Interestingly, gossypol/NDMA treated mice restored the induced cytochrome P450 2E1 expression, caused by gossypol, to its normal level. However, GSH/NDMA-treated mice did not restore the induced level of cytochrome P450 2E1 caused by GSH, to its normal level. It is concluded that, pretreatment of mice with GSH and gossypol prior to administration of NDMA induced the expression of cytochrome P450 2E1. Therefore, GSH and gossypol, could induce the toxicity and/or carcinogenicity of N-nitrosamines whereas they might protect the liver and probably other organs from the toxic and carcinogenic effects of other carcinogens such as polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene, through inhibition of total cytochrome P450 content and AHH activity. Key words: Aryl hydrocarbon hydroxylase, Cytochrome P450 2E1, N-nitrosodimethylamine N- demethylase, glutathione, antioxidants.

Predominant Role of Peripheral Catecholamines in the Stress‐Induced Modulation of CYP1A2 Inducibility by Benzo(α)pyrene

The potential involvement of catecholamines and in particular of alpha(2)-adrenoceptor-related signalling pathways, in the regulation of drug-metabolizing enzymes by stress was investigated in Wistar rats after exposure to the environmental pollutant benzo(alpha)pyrene. For this purpose, total cytochrome P450 content, the CYP1A2 mRNA levels, 7-methoxyresorufin-O-dealkylase (MROD), 7-pentoxyresorufin-O-dealkylase (PROD) and p-nitrophenol hydroxylase activity levels were determined in the livers of rats exposed to repeated restraint stress after treatment with benzo(alpha)pyrene coupled with pharmacological manipulations of peripheral and/or central catecholamines and alpha(2)-adrenoceptors. The data show that stress is a significant factor in the regulation of CYP1A2 induction and that catecholamines play a central role in the stress-mediated modulation of hepatic CYP1A2 inducibility by benzo(alpha)pyrene. The up-regulating effect of stress on benzo(alpha)pyrene-induced CYP1A2 gene expression was eliminated after a generalized catecholamine depletion with reserpine. Similarly, in a state where only peripheral catecholamines were depleted and central catecholamines remained intact after guanethidine administration, the up-regulating effect of stress was eliminated. It is apparent that stress up-regulates the induction of CYP1A2 by benzo(alpha)pyrene mainly via peripheral catecholamines, while central catecholamines hold a minor role in the regulation. Pharmacological manipulations of alpha(2)-adrenoceptors appear to interfere with the effect of stress on the regulation of CYP1A2 inducibility. Either blockade or stimulation of alpha(2)-adrenoceptors with atipamezole and dexmedetomidine respectively, eliminated the up-regulating effect of stress on CYP1A2 benzo(alpha)pyrene-induced expression, while it enhanced MROD activity. In contrast, stress and pharmacological manipulations of catecholamines and alpha(2)-adrenoceptors did not affect total P450 content, the CYP2B1/2-dependent PROD and the CYP2E1-dependent p-nitrophenol hydroxylase activities. In conclusion, stress is a significant factor in the regulation of the CYP1A2 inducibility by benzo(alpha)pyrene, which in turn is involved in the metabolism of a large spectrum of toxicants, drugs and carcinogenic agents. Although the mechanism underlying the stress effect on CYP1A2 induction has not been clearly elucidated, it appears that peripheral catecholamines hold a predominant role, while central catecholamines and in particular, central noradrenergic pathways hold a minor role.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100%) were reduced in SW (EROD: male (M) 36%, female (F) 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28%) and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73%) while PNPH (CYP2E1) was decreased in SW (M 31%, F 38%) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW) and DBA/2 mice of either sex (N = 12 per sex per group) were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP) concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD), p-nitrophenol-hydroxylase (PNPH), coumarin-7-hydroxylase (COH), and UDP-glucuronosyltransferase (UGT) activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100%) were reduced in SW (EROD: male (M) 36%, female (F) 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28%) and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73%) while PNPH (CYP2E1) was decreased in SW (M 31%, F 38%) but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH), CYP1A (EROD, MROD) and 2B (BROD, PROD). In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

Role of nitric oxide in the inhibition of liver cytochrome P450 during sepsis

Nitric oxide (NO) plays an important role in the pathophysiology of sepsis and septic shock but the mechanism is not well understood. The aim of this study was to investigate the role of NO in the cytochrome P450 (CYP) isozyme activity and the expression of its gene during polymicrobial sepsis. The rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Aminoguanidine (AG, 100 mg/kg body weight) or N ω -nitro- l-arginine methyl ester ( l-NAME, 100 mg/kg body weight) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after CLP. The plasma nitrite/nitrate concentration increased 24 h after CLP, and this increase was almost completely abolished by AG and l-NAME. Sepsis increased the serum aminotransferase and lipid peroxidation levels, which were attenuated by AG but augmented by l-NAME. The hepatic concentration of the reduced gluthathione decreased in the CLP rats, which was inhibited by AG but augmented by l-NAME. The total CYP content decreased after CLP, which was restored by AG and l-NAME. The CYP1A1, 1A2, and 2E1 activities, along with their protein levels, decreased 24 h after CLP but these decreases were reversed by AG and l-NAME. The CYP1A1, 1A2, 2B1, and 2 E1 mRNA expression levels decreased 24 h after CLP, and l-NAME inhibited this decrease. NO plays a key role in the sepsis-mediated decrease in CYP via the interplay of two different mechanisms: NO-dependent suppression of protein via the enhanced inducible NO synthase, and NO-dependent transcriptional suppression via endothelial NO synthase.

Effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits

We studied the effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits. Total cytochrome P450 content, protein concentration, and catalytic activity of CYP2B, CYP3A, and CYP2C isoforms were measured. Triphenyldioxane significantly increases specific activity of CYP2B and CYP2C in the liver of rats and rabbits, respectively. Immunoblotting analysis of microsomal enzymes in the liver of animals showed that the increase in specific activity of CYP is related to high content of apoenzymes. We showed for the first time that rats and rabbits are characterized by interspecies differences in the induction of cytochrome P450 isoforms under the influence of triphenyldioxane.

Protein responses in blue mussels ( Mytilus edulis) exposed to organic pollutants: A combined CYP-antibody/proteomic approach

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel ( Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size ≈57 kDa, p I 4.5–4.6, whereas the CYP4 antibody cross-reacted with a protein of size ≈55 kDa, p I 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.

Cloning and expression of koala ( Phascolarctos cinereus) liver cytochrome P450 CYP4A15

In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala ( Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not ingest eucalyptus leaves as food [Ngo, S., Kong, S., Kirlich, A., Mckinnon, R.A., Stupans, I., 2000. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comp. Biochem. Physiol., C 127, 327–334]. A 1544 bp koala liver CYP4A cDNA, designated CYP4A15, was cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CYP4A15 cDNA encodes a protein of 500 amino acids and shares 69% nucleotide and 65% amino acid sequence identity to human CYP4A11. Transfection of the koala CYP4A15 cDNA into Cos-7 cells resulted in the expression of a protein with lauric acid hydroxylase activity. The koala CYP4A15 cDNA-expressed enzyme catalysed lauric acid hydroxylation at the rates of 0.45 ± 0.18 nmol/min/mg protein and 4.79 ± 1.91 nmol/min/nmol CYP (mean ± SD, n = 3), which were comparable to that of rat CYP4A subfamilies. Total CYP content for koala CYP4A15-expressed protein in Cos-7 cells was 0.094 ± 0.001 nmol/mg protein (mean ± SD, n = 3) with negligible CYP content in untransfected Cos-7 cells lysate. Immunoblot analysis, using a sheep anti-rat CYP4A polyclonal antibody, detected multiple CYP4A immunoreactive bands in the liver from all species studied. The koala bands were found to be fainter and less confined but appeared much broader as compared to rat, human and wallaby. Northern blot analysis, utilising the koala CYP4A15 cDNA 417 bp probe, detected a mRNA species of approximately 2.6 kb in the koala liver and a mRNA species of approximately 2.4 kb in other species studied. Relative to the intensity of the β-actin mRNA species, much stronger CYP4A mRNA signal was detected for koala liver relative to rat and human. In Southern blot analysis of EcoR 1-digested genomic DNAs, using the same koala CYP4A15 cDNA probe, the size of CYP4A gene fragments observed for the koala and other species were different, suggested a different CYP4A gene organization across species. Collectively, this study provides primary molecular data regarding koala CYP4A15 gene. The possibility that there may be higher CYP4A15 expression in the koala liver could not be excluded.

In vivo effects of antiviral acyclic nucleoside phosphonate 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) on cytochrome P450 system of rat liver microsomes

Interference of antiviral agent adefovir, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) with microsomal drug metabolizing system was investigated in rats. The content of total liver cytochrome P450 (CYP) was lowered while that of its denaturated form, P420, was elevated in animals intraperitoneally treated with PMEA (25 mg/kg). Similar effect was observed after treatment with a prodrug of adevofir, adefovir dipivoxil (bisPOM-PMEA). The CYP2E1-dependent formation of 4-nitrocatechol from p-nitrophenol was diminished, though the specific activity of p-nitrophenol hydroxylase remained unchanged. PMEA had no influence on expression of CYP2E1 protein and mRNA and mRNAs of other P450 isoenzymes (1A1, 1A2, 2C11, 3A1, 3A2, and 4A1). It may be concluded that repeated systemic administration of higher doses of PMEA results in a partial degradation of rat CYP protein to inactive P420.

Effect of Sulfur Dioxide Inhalation on CYP2B1/2 and CYP2E1 in Rat Liver and Lung

Sulfur dioxide (SO2) is a ubiquitous air pollutant, present in low concentrations in the urban air and in higher concentrations in the working environment. In this study, we investigated the effects of inhaled SO2 on the O-dealkylase of pentoxyresorufin (PROD) and p-nitrophenol hydroxylases (p-NP) activities and mRNA levels of CYP2B1/2 and CYP2E1 in the lung and liver of Wistar rats. Male Wistar rats were housed in exposure chambers and treated with 14.11 ± 1.53, 28.36 ± 2.12, and 56.25 ± 4.28 mg /m3 SO2 for 6 h/day for 7 days, while control rats were exposed to filtered air in the same condition. The mRNAs of CYP2B1/2 and -2E1 were analyzed in livers and lungs by using reverse-transcription polymerase chain reaction (RT-PCR). Our results showed that the PROD activities and mRNA of CYP2B1/2 were decreased in livers and lungs of rats exposed to SO2. The p-NP activities and mRNA of CYP2E1 were decreased in lungs but not in livers of rats exposed to SO2. Total liver microsomal cytochrome P-450 (CYP) contents were diminished in SO2-exposed rats. These results lead to two conclusions: (1) SO2 exposure can suppress CYP2B1/2 and CYP2E1 in lungs and CYP2B1/2 in livers of rats, thus modifying the liver and lung toxication/detoxication potential, and (2) the total liver microsomal CYP contents were diminished, although the activity and mRNA expression of CYP2E1 in rat livers were not affected by SO2 exposure.

Assessment of Induction of Cytochrome P450 by NO-1886 (Ibrolipim), a Lipoprotein Lipase-Promoting Agent, in Primary Cultures of Human Hepatocytes and in Female Rat Liver

The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA. A higher concentration (50 microM) of NO-1886 induced an increase in CYP2C8 mRNA and a decrease in CYP2C19 mRNA, and these changes continued after additional culture for 24 hr in fresh medium without NO-1886. The expression level of CYP3A4 mRNA after exposure to NO-1886 for 24 hr at 50 microM was about twice that in controls. Following additional culture for 24 hr in fresh medium without NO-1886, the expression of CYP3A4 mRNA was comparable to that in controls. On the other hand, the expression levels of CYP2C9 and CYP3A5 mRNA showed small and variable changes in each donor even at a high concentration (50 microM) of NO-1886. Furthermore, the pharmacokinetics of NO-1886 during repeated oral administration for 14 days was studied in female rats. The pharmacokinetic parameters of NO-1886 were nearly the same on days 1, 7, and 14 of repeated administration. The hepatic microsomal content of CYP isoforms was not affected by repeated administration for 7 days at a dose of 1 to 30 mg/kg in female rats, although the total CYP content was increased at a dose of 30 mg/kg. The expression levels of CYP1A2, CYP2B2, CYP2C12, and CYP2E1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2, 10, or 50 microM. The expression levels of CYP3A1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2 or 10 microM, but were increased, with large individual variation, by exposure at 50 microM. The mRNA expression levels in rat hepatocytes exposed to concentrations comparable to free plasma levels did not change significantly, which was consistent with the equivalence in the in vivo plasma concentrations observed on days 1 and 14 of repeated administration. These results suggest that repeated administration of NO-1886 at clinical doses does not significantly affect the expression levels of CYP isoforms in human liver, although the mRNA levels of the CYP isoforms involved in the metabolism of NO-1886 were increased by exposure to higher concentrations of NO-1886 in human hepatocytes in vitro.

Earthworm cytochrome P450 determination and application as a biomarker for diagnosing PAH exposure

We developed a new microsome purification method and used the Omura and Sato method to measure the total content of cytochrome (Cyt) P450 in earthworm (Eisenia fetida) microsomes. In method development, two different pretreatments, i.e. solubilization or manual separation were used to purify worm microsomes. Solubilization was more effective than manual separation and difference spectra showed a peak at 450 +/- 1 nm in microsomes received solubilization pretreatment. We conducted a 48 h contact test by exposing worms on pyrene (Py) and benzo[a]pyrene (BaP) spiked filter paper. A dose-response relationship was established between total P450 content and the concentration of Py or BaP ranging from 10(-6) mg mL(-1) to 10(-2) mg mL(-1). Results show that total Cyt P450 content in earthworms is a promising biomarker for diagnosing PAHs exposure at sublethal dose ranges.

Possible implication of macrophages in the regulation of cytochrome P450 activities in carp ( Cyprinus carpio)

Macrophages play a key role in the regulation of cytochrome P450 activity induced by immunostimulants in mammals. We investigated the effects of immunostimulants (LPS, dextran sulfate and tilorone) on biotransformation and macrophage activities in carp. The major effect of LPS was its capacity to inhibit 3-MC-induced cytochrome P450 activities in the liver and head kidney. Basal phase I activities were reduced by tilorone and dextran sulfate in immune organs. Tilorone and dextran sulfate differently modulated total cytochrome P450 contents and P4501A activities suggesting differential sensitivity for P450 classes. In immune organs, tilorone and dextran sulfate inhibited basal EROD activity. Tilorone inhibited 3-MC-induced EROD activity whereas dextran sulfate enhanced this activity. LPS and dextran sulfate increased ROS production by macrophages and all the immunostimulants induced macrophage activating factor (MAF) production. This study demonstrates for the first time in fish the capacity of CYP-regulated immunostimulants to activate macrophages and provides initial insight into the capacity of macrophages to regulate CYP activity induced by immunostimulants in fish.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) – a breakdown product of haem with cytoprotective and toxic properties – is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl 2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5–10 h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC 50 approximately equal to the substrate concentration. The 7-methoxyresorufin 7- O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

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 2α, 7α and 16 α-hydroxylase activities were higher, but testosterone 6β-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR − rats had higher 7α-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 6β-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7α-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.

SUPPRESSION OF HEPATIC CYTOCHROME P450-MEDIATED DRUG METABOLISM DURING THE LATE STAGE OF SEPSIS IN RATS

The effects of polymicrobial sepsis on the activity and gene expression of hepatic microsomal cytochrome P450 (CYP) were examined. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Liver and blood samples were taken 2, 6, and 24 h after CLP. The serum aminotransferase levels and lipid peroxidation increased 24 h after CLP. The hepatic concentrations of reduced glutathione and total CYP content decreased 24 h after CLP. The CYP1A1 activity and its protein level decreased 24 h after CLP. The CYP1A2 activity decreased 2 h and 24 h after CLP. Although the CYP2B1 mRNA expression level decreased 6 h and 24 h after CLP, the CYP2B1 activity and its protein level did not change in any of the experimental groups. The CYP2E1 activity and its protein level decreased 24 h after CLP. The CYP2E1 mRNA levels were lower at both 6 h and 24 h after CLP. The TNF-alpha mRNA expression level increased 2, 6, and 24 h after CLP. The iNOS mRNA expression level increased 24 h after CLP. These findings suggest that sepsis causes abnormalities in the microsomal drug-metabolizing function, particularly in the late stage, which is associated with higher level of oxidant stress and lipid peroxidation.

Effects of pregnancy on CYPs protein expression in rat liver

A body of evidence suggests that pregnancy may be responsible for the depression in the microsomal enzyme activity and the reduction in the total content of cytochrome P450 (CYP) in the rat liver. However, changes in expression of individual CYP isozyme remain poorly known. The current study was designed to examine the changes in CYPs protein expression in the liver of F344 rats in midpregnancy and late pregnancy by Western blot analysis and immunohistochemistry. Total nine antirat CYPs antibodies (CYP1A1, CYP2B1/CYP2B2, CYP2C6, CYP2C12, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP4A1) were used. In comparison with age-matched nonpregnant control rats, there were significant decreases in hepatic levels of CYP2B2, CYP2C6, and CYP4A1 in midpregnancy (day 13) and CYP2B2, CYP2C6, CYP4A1, CYP1A1, CYP2B1, and CYP2E1 in late pregnancy (day 19). The expression of CYP2C12, CYP2D1, and CYP 3A1 did not differ between nonpregnant and pregnant rats, and CYP2D4 was not detectable in microsomal proteins obtained from nonpregnant and pregnant rats at a protein loading of 20 μg total protein per lane. Immunohistochemistry showed that there were no differences in the distribution and degree of immunostainability for the abovementioned antibodies to nine CYPs between pregnant and nonpregnant rats.

Barramundi as an indicator species for environmental monitoring in north Queensland, Australia: Laboratory versus field studies

The dose-response relationship for hepatic 7-ethoxyresorufin-O-deethylase (EROD) induction in barramundi (Lates calcarifer) was examined under controlled laboratory conditions for 15 d using farm-reared barramundi. These results were compared with EROD activity measured in barramundi collected from two rivers catchments (impacted and nonimpacted) in northern Queensland, Australia. Barramundi were dosed by intraperitoneal injection with a known cytochrome P4501A (CYP1A) inducer, beta-naphthoflavone (beta-NF), at 5, 10, and 50 mg kg(-1) using two controls: A vehicle control (corn-oil injected) and an experimental control (no injection). The EROD induction occurred within 4 h in the 5, 10, and 50 mg beta-NF kg(-1) exposures, reaching mean maximum activities of 88.6 (+/-51.9), 85.5 (+/-91.7), and 149.1 (+/-106.4) pmol min(-1) mg protein(-1), respectively. Mean EROD activities remained low in the corn-oil controls (2.1+/-1.8 pmol min(-1) mg protein(-1)) and experimental controls (5.3+/-4.4 pmol min(-1) mg protein(-1)) throughout the study. Barramundi demonstrated a rapid response curve, which was dose dependent (50 > 10 > 5 mg beta-NF kg(-1)) and decreased progressively over time from induction. Measurement of total cytochrome P450 content (nmol mg protein(-1)) was not dose dependent. The EROD activities from field-collected barramundi from the Johnstone River (impacted) and Olive River (nonimpacted) suggest exposure to low-level contaminants in the Johnstone River fish only. With more controlled laboratory and field studies, barramundi have the potential to become a major indicator species in assessing exposure to environmental contaminants in coastal areas throughout northern Queensland, Australia.

Effect of long-term cold exposure on activities of cytochrome P450-containing monooxygenases and glutathione S-transferase in rat liver microsomes

Long-term cold exposure (5 degrees C) was followed by induction of rat liver monooxygenases. We revealed an increase in activity of NADPH-cytochrome C reductase, total content of cytochrome P450 (CYP), and activities of its molecular forms CYP1A1, 1A2, 2B1/B2, 2E1, and 3A1/A2 in microsomes. These indexes reached maximum by the 10th day, but decreased with lengthening of cold exposure. Glutathione S-transferase activity decreased under these conditions. Changes in enzyme activity could be related to the increase in blood corticosterone concentration.

Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on development, reproduction, and microsomal cytochrome P450.

A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr(low) allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr(low/low) mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr(low) allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr(low) females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr(low/low) mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.