Expression Of Cytochrome P450 Isoforms Research Articles

Urinary bladder carcinogenic potential of 4,4'-methylenebis(2-chloroaniline) in humanized-liver mice.

Occupational exposure to 4,4'-methylenebis(2-chloroaniline) (MOCA) has been linked to an increased risk of bladder cancer among employees in Japanese plants, indicating its significance as a risk factor for urinary bladder cancer. To investigate the role of MOCA metabolism in bladder carcinogenesis, we administered MOCA to non-humanized (F1-TKm30 mice) and humanized-liver mice for 4 and 28 weeks. We compared MOCA-induced changes in metabolic enzyme expression, metabolite formation, and effects on the urinary bladder epithelium in the two models. At week 4, MOCA exposure induced simple hyperplasia, cell proliferation, and DNA damage in the urothelium of the humanized-liver mice, while in the non-humanized mice these effects were not observed. Notably, the concentration of 4-amino-4'-hydroxylamino-3,3'-dichlorodiphenylmethane (N-OH-MOCA) in the urine of humanized-liver mice was more than 10 times higher than that in non-humanized mice at the 4-week mark. Additionally, we observed distinct differences in the expression of cytochrome P450 isoforms between the two models. Although no bladder tumors were detected after 28 weeks of treatment in either group, these findings suggest that N-OH-MOCA significantly contributes to the carcinogenic potential of MOCA in humans.

Evaluation of Zuo-Gui Yin Decoction Effects on Six CYP450 Enzymes in Rats Using a Cocktail Method by UPLC-MS/MS.

Background Zuo-Gui Yin Decoction (ZGYD), a traditional Chinese prescription, is mainly used in various kinds of andrology and gynecology diseases. However, the study on the interaction of ZGYD and drugs has not been reported. Therefore, evaluating the interaction between ZGYD and metabolic enzymes is helpful to guide rational drug use. Objective This study was conducted to explore the effects of ZGYD on the activity and mRNA expressions of six Cytochrome P450 (CYP450) enzymes in rats and to provide a basis for its rational clinical use. Methods Sprague-Dawley rats were randomly divided into control, ZGYD high, medium, and low-dose group (n = 6). The concentrations of six probe substrates in plasma of rats in each group were determined by UPLC-MS/MS. In addition, RT-PCR and Western blot were used to determine the effects of ZGYD on the expression of CYP450 isoforms in the liver. Results Compared with the control group, the main pharmacokinetic parameters AUC(0-t), AUC (0~∞), of omeprazole, dextromethorphan, and midazolam in the high-dose group were significantly decreased, while the CL of these were significantly increased. The gene expressions of CYP2C11 and CYP3A1 were upregulated in the ZGYD medium, high-dose group. The protein expression of CYP2C11 was upregulated in the high-dose group, and the protein expression of CYP3A1 was upregulated in the medium, high-dose group. Conclusion The results showed that ZGYD exhibited the induction effects on CYP2C11 and CYP3A1 (CYP2C19 and CYP3A4 in humans) in rats. However, no significant change in CYP1A2, CYP2B1, CYP2C7, and CYP2D2 activities was observed. It would be useful for the safe and effective usage of ZGYD in clinic.

Aryl hydrocarbon receptors as potential therapeutic targets

Aryl hydrocarbon receptors (AhR) are regulators of the expression of cytochrome P-450 isoforms, mediating a wide variety of the effects of substances from the endogenous or exogenous origin, including those produced from the microbiome. An exciting new aspect of their activity is their localization in the brain and their potential to modulate the action of the immune system. AhR is emerging as an essential toxicological and therapeutic target for neuromodulation. Further studies are needed for elucidating their utility as drug-targets.

Atrazine-xenobiotic nuclear receptor interactions induce cardiac inflammation and endoplasmic reticulum stress in quail (Coturnix coturnix coturnix)

Atrazine (ATR) is one of the most extensively used herbicide that eventually leaches into groundwater and surface water from agricultural areas. Exposure to ATR does harm to the health of human and animals, especially the heart. However, ATR exposure caused cardiotoxicity in bird remains unclear. To evaluate ATR-exerted potential cardiotoxicity in heart, quail were exposed with 0, 50, 250, and 500 mg/kg BW/day ATR by gavage treatment for 45 days. Cardiac histopathological alternation was observed in ATR-induced quail. ATR exposure increased the Cytochrome P450s and Cytochrome b5 contents, Cytochrome P450 (CYP) enzyme system (APND, ERND, AH, and NCR) activities and the expression of CYP isoforms (CYP1B1, CYP2C18, CYP2D6, CYP3A4, CYP3A7, and CYP4B1) in quail heart. The expression of nuclear xenobiotic receptors (NXRs) was also influenced in the heart by ATR exposure. ATR exposure significantly caused the up-regulation of pro-inflammatory cytokines (TNF-α, IL-6, NF-κB, and IL-8), down-regulation of anti-inflammatory cytokines (IL-10) expression levels and increased NO content and iNOS activity. The present research provides new insights into the mechanism that ATR-induced cardiotoxicity through up-regulating the expression levels of GRP78 and XBP-1s, triggering ER stress, activating the expression of IRE1α/TRAF2/NF-κB signaling pathway related factors (IRE1α, TRAF2, IKK, and NF-κB) and inducing an inflammatory response in quail hearts. In conclusion, ATR exposure could induce cardiac inflammatory injury via activating NXRs responses, disrupting CYP homeostasis and CYP isoforms transcription, altering NO metabolism and triggering ER stress and inflammatory response by activating IRE1α/TRAF2/NF-κB signaling pathway.

Arachidonic acid metabolites of CYP4A and CYP4F are altered in women with preeclampsia

Few studies exist on cytochrome P450 (CYP450) metabolites of arachidonic acid (AA) pertaining to the pathophysiological events in pregnancy. We hypothesized that metabolism of AA via the CYP450 pathways is altered within the placenta in women with preeclampsia (PE) and contributes to the pathophysiology of the disease. Thus, placental vascular CYP450 enzyme expression and activity were measured in normal pregnant (NP) and preeclamptic (PE) patients. CYP450 isoform expression (CYP4A11, CYP4A22, CYP4F2, and CYP4F3) was found to be elevated within the placenta of women with PE compared to normal pregnant (NP) women and chronic hypertensive (CHTN) pregnant women. In addition, placental production of 20-HETE was significantly increased in PE women compared to both NP and CHTN women. Moreover, there was an imbalance in circulating 20-HETE:EETs in PE women. To examine whether alterations in CYP450 AA metabolism contribute to the altered placentation in PE, trophoblast function, proliferation and migration were assessed in the presence of exogenous 20-HETE and a 20-HETE specific synthesis inhibitor, HET0016. Trophoblast proliferation was significantly increased in the presence of 20-HETE (1 μM) and reduced with 20-HETE blockade by HET0016 (1 mM, 5 mM, and 10 mM). On the contrary, administration of exogenous 20-HETE (1 μM) significantly reduced trophoblast migration. In conclusion, metabolism of AA via CYP450 is altered in PE, and increased placental production of 20-HETE may contribute to the pathophysiology of the disease.

Abstract 415: Cytochrome CYP3A4 expression may predict metastasis in Ewing's sarcoma family of tumors.

Abstract Background: Ewing's sarcoma family tumor (EFT) is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy in metastatic disease. Cytochrome P450 (CYP) monooxygenases are a multigene family of enzymes that are involved in phase I reactions of metabolism of a wide range of endogenous compounds and xenobiotics, including anticancer drugs. Expression of CYP isoforms may influence tumor development, progression and resistance to therapy. Overexpression of CYP3A isoforms was associated with more aggressive behavior in breast cancer, osteosarcoma and other tumors. The purpose of this study is to define the expression CYP3A isoforms in EFT and establish its prognostic significance. Design: Archived EFT cases and pertinent clinical data were compiled to include patient's age, sex, tumor location, tumor size, overall survival, disease-free survival and clinical stage. Two representative histologic sections of the tumors were selected for tissue microarray construction. Sections were processed for immunohistochemical labeling with CYP3A4, CYP3A5, and CYP3A7. Liver sections were used as positive control. The intensity of the staining was scored as negative, low (weak staining less than the positive control) or high (equal to or stronger than the positive control). The staining intensity was correlated with clinical data and the significance of any associations was statistically analyzed by one tailed and two tailed t-tests. Results: 36 cases of Ewing sarcoma family of tumors were selected, 22 were males and 12 females. Age ranged from 12-72 years. 4 cases were excluded from immunohistochemical interpretation due inadequate viable tissue. CYP3A4 staining was present in 27 (84%) cases with high expression noted in 6 (19%) of 32 cases. High expression was significantly associated with distant metastases (p< 0.05). There was no association of high CYP3A4 expression with age, sex, tumor size, or location (pelvic or extrapelvic). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases (15.6%, 40.6%) respectively, and were not associated with age, sex, tumor size, metastasis or location. None of the studied biomarkers showed any correlation with overall or disease free survival. Conclusion: CYP3A4, CYP3A5 and CYP3A7 are variably expressed in EFTs. High CYP3A4 expression may predict metastasis in EFT. Additional studies with higher number of cases are needed to further investigate the role of CYP3A4 in the prognosis of Ewing's sarcoma family tumors. Citation Format: Atif Ahmed, Hamid Zia, Graeme I. Murray, Carrie A. Vyhlidal, J. Steven Leeder, Marilyn M. Bui. Cytochrome CYP3A4 expression may predict metastasis in Ewing's sarcoma family of tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 415. doi:10.1158/1538-7445.AM2013-415

Effect of chondroitin sulfate on turpentine-induced down-regulation of CYP1A2 and CYP3A6

This study aimed to assess whether chronic administration of chondroitin sulfate (CS) affects baseline expression of cytochrome P450 isoforms and impedes the decrease in expression and activity of CYP1A2 and CYP3A6 in rabbits with a turpentine-induced inflammatory reaction (TIIR). Seven groups of 5 rabbits, 3 control groups and 4 receiving 20mg/kg/day of CS for 20 and 30days, were used. The rabbits of 1 control group and 2 groups receiving CS had a TIIR; finally, the rabbits of one of the control groups remained in the animal facilities for 30days to assess the effect of time and environment on cytochrome P450. In control rabbits, intake of CS for 20 and 30days did not affect CYP3A6, CYP1A2 and NADPH cytochrome P450 reductase (CPR) mRNA, protein expression and activity. Compared with control rabbits, the TIIR not only reduced mRNA, protein expression and activity of CYP3A6 and CYP1A2 but also that of CPR. In rabbits with TIIR, CS prevented the decrease of CYP3A6 expression but not the reduction in activity. CS did not impede TIIR-induced down-regulation of CYP1A2. Hepatic NO concentrations and NF-κB nuclear translocation were increased by the TIIR, effect reversed by CS. In vitro, in hepatocytes, CS did not alter the expression and activity of CYP3A6, CYP1A2, and CPR. In conclusion, oral CS elicits a systemic effect but does not affect CYP1A2, CYP3A6, and CPR in control rabbits, although in rabbits with TIIR, CS prevents CYP3A6 protein down-regulation but not that of CYP1A2.

OL-033 Etambutol-mediated changes in rat liver cytochrome P-450 isoforms expression and DNA-fragmentation processes

source for most antibiotics on the market today, including the first-line and salvage TB regimens, including rifampin, streptomycin, amikacin and etc. A set of Microbial Natural Product Library (MNPL) containing secondary metabolites from a unique collection of actinomycetes and fungal strains from unor under-explored ecological niches in China has been constructed. A pilot screen for potential anti-TB compounds was conducted with a selection of 5,000 extracts samples from this MNPL, utilizing a GFP labeled M. bovis BCG based HTS model (Z’-factor 0.8, CV 90% inhibition against logarithmically growing BCG were further evaluated on M. tb H37Rv strains at Broad Institute, as a dilution series ranging from 1× to 1/128×. The results showed that 46 extracts demonstrated anti-H37Rv activity, with 8 showing activity at 1/16×, and 1 showing activity at 1/128×. The following large scale fermentation and bioactivity guided compound isolation work lead to the discovery of diversified class of anti-TB compounds, including actinomycins (MIC 1 4 ug/ml), quinomycins (MIC 0.5 ug/ml), nanaomycins (MIC 8 ug/ml), cyclopeptides (MIC 2 8 ug/ml), anthraquinones (MIC 4 8 ug/ml), oligomycins, part of which were new compounds (not listed).

Effects of co-exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin and perfluorooctane sulfonate or perfluorooctanoic acid on expression of cytochrome P450 isoforms in chicken ( Gallus gallus) embryo hepatocyte cultures

In this study we investigated the effect of a single-compound exposure or two compound co-exposure to tetrachlorodibenzo- p-dioxin (TCDD) plus perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) on the mRNA expression of cytochromes P450 (CYP) 1A4, 4V2 and 3A37, ethoxyresorufin- O-deethylase (EROD) activity and cell viability in chicken ( Gallus gallus domesticus) embryo primary hepatocyte cultures. Cell viability after 24 h of incubation was significantly decreased in cells exposed to PFOS at concentrations between 30 µM and 60 µM with or without co-exposure to TCDD (0.3 nM at maximum). PFOA did not decrease cell viability even at maximum concentrations of 60 µM. TCDD induced CYP1A4 mRNA and EROD activity substantially as reported previously. PFOS also increased CYP1A4 mRNA in a concentration-dependent manner. Co-exposure of cells to PFOS plus TCDD did not change CYP1A4 mRNA levels compared to cells treated with TCDD alone. PFOS alone did not induce CYP4V2 mRNA, however 40–50 µM PFOS plus TCDD (0.3 nM) induced CYP4V2 mRNA compared to TCDD alone ( P < 0.05). This trend was similar to that observed with co-exposure to TCDD plus PFOA, suggesting that PFOA alone did not induce CYP4V2 mRNA, whereas co-exposure to TCDD plus PFOA induced the expression levels. PFOS alone decreased CYP3A37 mRNA by a maximum of 45%, however after co-exposure to TCDD, recovery of mRNA expression to levels measured in DMSO-treated cells was observed. Our data suggest a complex gene response to mixtures of dioxin-like and perfluorinated compounds.

Comparative analysis of family 1 cytochrome P-450 mRNA expression in human intestinal adenocarcinoma and intact portion of the intestine

The expression of mRNA of proteins involved in the transformations of cytostatics (cytochrome P-450 1A1 and 1B1 isoforms) and genes encoding proteins participating in their regulation (Ah receptor, AHRR and ARNT) in intestinal tumors and intact portions of the intestine were studied. The expression of cytochrome P-450 1A1 increased in poorly differentiated tumors in comparison with its expression in intact portions of the intestine (tumor/intact tissue=1.65). The expression of cytochrome P-450 1B1 was higher in well-differentiated tumors (tumor/intact tissue=1.62). The possibility of practical use of high expression of cytochrome P-450 isoforms in tumors in comparison with intact intestinal tissue is discussed.

Nitric oxide mediates an LPS-induced depression of cytochrome P450 (CYP1A) activity in astrocytes

During inflammatory responses in the brain, the expression of cytochrome P450 isoforms in the CNS are modulated and the capacity of the brain to metabolize drugs and to synthesize or degrade certain endogenous chemicals and drugs is diminished. While this response can be attributed in part, to the production and action of cytokines within the brain, it is also likely that other inflammatory mediators play an integral role. This paper investigates a potential role for nitric oxide (NO) in the loss of cytochrome P450 (CYP1A) in the brain during inflammation. Escherichia coli lipopolysaccharide (LPS), a commonly used proinflammatory endotoxin, was incubated with cultured rat astrocytes to provide a model of inflammation in the CNS. CYP1A activity was significantly decreased in cultured astrocytes incubated with LPS for 24 h. This loss in enzyme activity was accompanied by a substantial production of nitric oxide (NO) by these cells. Immunohistochemical examination demonstrated an upregulation of inducible nitric oxide synthase (iNOS) expression following the exposure of astrocytes to LPS. The addition of a selective iNOS blocker (1400W) caused a partial but significant reversal of the LPS-mediated loss in CYP1A. The incubation of astrocytes with the NO-generating compound (DETA NONOate) resulted in a loss of CYP1A. Taken together, these observations suggest that NO plays a pivotal role in the inflammation mediated loss in CYP1A activity in the brain.

Histomorphological changes and cytochrome P450 isoforms expression and activities in precision-cut liver slices from neonatal rats

Precision-cut rat liver slices are a widely accepted in vitro tool for the examination of drug metabolism, enzyme induction or hepatotoxic effects of xenobiotics. After prolonged incubation, however, distinct histopathological changes and increasing losses in function are seen with liver slices from adult animals. Since tissue from neonatal animals is expected to be less vulnerable, in the present study liver slices from 1-day-old rats were examined for morphological changes and for the expression of different cytochrome P450 (CYP) isoforms after incubation for up to 24 h and after a 24 h in vitro exposure to beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or pregnenolone 16alpha-carbonitrile (PCN). In parallel, CYP activities were assessed by different model reactions in slice homogenates and in intact slices. Histopathological changes were less pronounced in liver slices from 1-day-old rats than in those from adult animals. During the 24 h of incubation even a maturation of the tissue occurred, since the proportion of haemopoietic stem cells declined and the glycogen content of the hepatocytes increased. The CYP expression pattern after 2 and 24 h of incubation was similar to that of normal liver specimens from neonatal rats showing a moderate CYP1A1, 2B1 and 3A2 expression. The immunostaining for CYP1A1 and 2B1 was elevated after incubation with BNF. PB enhanced CYP2B1 and 3A2 expression, and DEX and PCN increased CYP3A2 immunostaining. This induction pattern was paralleled by respective effects on the corresponding model reactions. Thus, besides increased viability, slices from neonatal rats are excellently suited for the evaluation of an in vitro induction of CYP enzymes as well.

Effect of hypoxia on cytochrome P450 activity and expression.

In the last three decades, numerous reports have shown that patients with chronic pulmonary disease and with heart failure with hypoxemia cleared drugs at a lower rate than healthy volunteers. As a consequence decreased clearance, drug toxicity is frequent in these patients. The reduction in drug clearance is due to a decrease in activity of cytochrome P450 isoforms, partly associated to the hypoxemia. With in vivo animal models, acute moderate hypoxia (PaO2 of around 35-50 mm Hg) reduces the clearance of drugs biotransformed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2E1, although hypoxia does not affect the clearance of drugs biotransformed by CYP3A6. Ex vivo and in vitro experiments demonstrate that hypoxia down-regulates CYP1A1, CYP1A2, CYP2B6, CYP2C9 and CYP2C19, decrease preceded by a reduction in activity. On the other hand, acute moderate hypoxia up-regulates CYP3A6. The changes in protein expression are preceded by modifications in the mRNA coding for the proteins. The effect of hypoxia on hepatic cytochrome P450 is carried out by serum mediators, e.g. interferon-gamma, interleukin-1beta, and interleukin-2 are responsible for the decrease in activity and in expression of cytochrome P450 isoforms, and erythropoietin accounts for the increase in CYP3A6. Probably several mechanisms underlie and contribute to the decrease in activity and down-regulation of cytochrome P450 isoforms by hypoxia, e.g. reducing potentiation factors, inducing repressor elements and activating negative regulatory elements. The up-regulation of CYP3A6 implies a PTK- and p42/44MAPK-dependent stabilization/activation, nuclear translocation of HIF-1 and AP-1, binding to CYP3A6 promoter, and transactivation of the gene to induce CYP3A6 expression.

Differential expression of cytochrome P450 isoforms in the lungs of septic animals.

Sepsis is characterized by an early, hyperdynamic phase and a late, hypodynamic phase. Although studies have shown that cytochrome P450 (CYP) plays an important role in the regulation of vascular reactivity, alterations of vascular CYP isoforms in sepsis remain unknown. Since CYP2C11 and CYP2J4 convert arachidonic acid to vasodilative epoxyeicosatrienoic acids, and CYP4A3 metabolizes arachidonic acid to both epoxyeicosatrienoic acids and vasoconstrictive 19,20-hydroxyeicosatetraenoic acid, the aim of this study was to examine the expression of these isoforms in sepsis and their association with hemodynamic changes. Prospective, controlled, and randomized animal study. An institute research laboratory. Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). At 5 hrs (early sepsis) or 20 hrs (late sepsis) after cecal ligation and puncture, blood vessel-rich tissues (i.e., lungs) were harvested. The expression of CYP isoforms at both messenger RNA and protein levels was determined by reverse transcription polymerase chain reaction and Western blot analysis (CYP2C11), respectively. Hemodynamic variables were measured by radioactive microspheres. The results indicate that the gene expression of CYP2C11 and CYP2J4 was significantly down-regulated at 20 hrs after cecal ligation and puncture, whereas the expression of CYP4A3 was markedly up-regulated at 5 hrs. The protein concentrations of CYP2C11 also decreased significantly at 20 hrs after cecal ligation and puncture. Although total peripheral resistance markedly increased, mean arterial pressure did not change significantly at 20 hrs after the onset of sepsis. In contrast, cardiac output and pulmonary perfusion markedly decreased in late sepsis. Since the up-regulated CYP4A3 is associated with the early, hyperdynamic phase of sepsis and the down-regulated CYP2C11 and CYP2J4 are associated with the late, hypodynamic phase, vascular CYP isoforms that metabolize arachidonic acid may be involved in regulating the cardiovascular response during the progression of sepsis.

Influence of recipient gender on cytochrome P450 isoforms expression in intrasplenic fetal liver tissue transplants in rats

Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern which is regulated by a differential profile of growth hormone (GH) secretion. The aim of the present study was to elucidate whether liver cell transplants at an ectopic site are also subject to this influence. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant recipients and age-matched controls were treated with β-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the solvents and sacrificed 24 or 48 h thereafter. Livers and intrasplenic transplants were evaluated for the expression of the P450 subtypes 1A1, 2B1, 2E1, 3A2 and 4A1 by means of immunohistochemistry. The livers of both male and female rats displayed nearly no P450 1A1, but a distinct P450 2B1, 2E1, 3A2 and 4A1 expression. Whereas no sex differences were seen in the P450 1A1 expression, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in males and that for P450 2E1 in females. Similarly, in the intrasplenic liver cell transplants almost no P450 1A1, but a noticeable P450 2B1, 2E1, 3A2 and 4A1 expression was observed. Like in the respective livers, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in the transplants hosted by male than by female rats, whereas the opposite was the case for the P450 2E1 expression. Both in livers and transplants with some sex-specific differences P450 1A1 and 2E1 expression was induced by BNF, that of P450 2B1 by BNF and PB, and that of P450 3A2 by PB and DEX. These results indicate that the P450 system of ectopically transplanted liver cells is influenced by the gender of the recipient organism like that of the orthotopic livers.

Cryopreserved precision-cut rat liver slices: morphology and cytochrome P450 isoforms expression after prolonged incubation

Precision-cut liver slices are an accepted in vitro system for toxicological investigations. However, cryopreservation of slices would make a more efficient utilisation, particularly of human liver tissue possible. In the present study sections of cryopreserved male rat liver slices were examined immunohistochemically for cytochrome P450 (CYP) isoforms expression after prolonged incubation and after exposure to typical inducers. Morphologically, with just thawed slices no major alterations were seen, but remarkable cell damage was observed even after 2 h of incubation mainly in the middle of the slices and in the periportal and intermediate regions of the lobules. After 24 h of incubation, viable cells were only observed at the edges of the slices or around bigger vessels. In the viable cells of the cryopreserved liver slices after 2 h of incubation CYP expression pattern was similar to that in normal liver specimens: a low CYP1A1, but a strong CYP2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. After 24 h, the immunostaining for CYP2B1 and 3A2 in the viable cells was reduced, but that for CYP1A1 was increased. Incubation with β-naphthoflavone further elevated CYP1A1 and 2B1 expression. Phenobarbital caused an enhanced CYP2B1 and 3A2 and dexamethasone and pregnenolone 16α-carbonitrile an increased CYP3A2 immunostaining. These results show that also in cryopreserved liver slices and after a prolonged incubation, a distinct expression pattern and an in vitro induction of phase I enzymes can be demonstrated immunohistochemically.

Effect of xenoestrogen exposure on the expression of cytochrome P450 isoforms in rainbow trout liver

We studied the estrogenic effects of model chemicals in one-year-old juvenile rainbow trout. Methoxychlor (20 mg/kg), diethylstilbestrol (15 mg/kg), 4-tert-octylphenol (25 and 50 mg/kg), and biochanin A (25 and 50 mg/kg) were injected intraperitoneally on days 1, 4, and 7. Fish were sacrificed on day 9 and examined for multiple biomarkers. All of the test chemicals caused increases in plasma vitellogenin levels, a biomarker of estrogenicity. Treatment with the xenoestrogens decreased hepatic lauric acid hydroxylase activity and, as shown by Western blots, also generally reduced expression of hepatic cytochrome P450s 2K1 (CYP2K1), 2M1 (CYP2M1), and 3A27 (CYP3A27) at the protein level. Both doses of biochanin A also significantly induced P4501A (CYPIA) and greatly increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity. These findings suggest that methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biochanin A were all estrogenic and mimicked 17beta-estradiol (E2) in repressing the expression of cytochrome P450 isoforms (CYP2KI, CYP2M1, and CYP3A27) in the rainbow trout liver. Additionally, biochanin A was found to induce CYPIA in this fish species.

Downregulation of intestinal cytochrome p450 in chronic renal failure.

Chronic renal failure (CRF) is associated with a decrease in intestinal drug metabolism. The mechanisms remain poorly understood, but one hypothesis involves a reduction in cytochrome P450 levels. This study aimed to investigate the effects of CRF on intestinal cytochrome P450. Two groups of rats were defined, i.e., rats with CRF (induced by 5/6 nephrectomy) and control pair-fed rats. Total cytochrome P450 levels and protein and mRNA expression of cytochrome P450 isoforms, as well as in vitro N-demethylation of erythromycin (a probe for CYP3A activity) and 7-ethoxyresorufin o-deethylase activity (a probe for CYP1A), were assessed in intestinal microsomes. Body weights were similar in the two groups. Creatinine clearance was reduced by 77% (P < 0.001) in CRF rats, compared with control pair-fed animals. Total intestinal cytochrome P450 activity was reduced by 32% (P < 0.001) in CRF rats. CYP1A1 and CYP3A2 protein expression was considerably reduced (>40%, P < 0.001) in rats with CRF. CYP2B1, CYP2C6, and CYP2C11 levels were the same in the two groups. RT-PCR assays revealed marked downregulation of CYP1A1 and CYP3A2 gene expression in CRF rats (P < 0.001). Although intestinal cytochrome P450 levels were reduced in CRF, induction by dexamethasone was present. N-Demethylation of erythromycin and 7-ethoxyresorufin o-deethylase activity were decreased by 25% (P < 0.05) in CRF rats, compared with control rats. In conclusion, CRF in rats is associated with decreases in intestinal cytochrome P450 activity (mainly CYP1A1 and CYP3A2) secondary to reduced gene expression.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 in the periportal areas was even decreased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.

In vitro sulfoxidation of aldicarb by hepatic microsomes of channel catfish, Ictalurus punctatus.

The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in mammals, birds, and fish, and is readily biotransformed by most organisms studied. Metabolic products of aldicarb include the more toxic sulfoxide and the less toxic sulfone as two of the major products. Both the cytochrome P450 (CYP) and the flavin monooxygenase systems (FMO) are involved in this process. This study examined the capacities of liver microsomes of male channel catfish (Ictalurus punctatus), which lack FMO, to biotransform aldicarb in vitro. In addition, the acetylcholinesterase inhibitory potencies of aldicarb and its sulfoxide and sulfone derivatives were determined. For metabolism studies, incubations of [14C]-aldicarb (0.1mM) were carried out for up to 15-90 min using 1.0 mg/mL of hepatic microsomal protein. Total NADPH- dependent biotransformation was low (< 3.0% conversion to polar metabolites), and was inhibited by carbon monoxide. The only metabolite detected was aldicarb sulfoxide (Kmapp = 53.8 +/- 25.3 microM; Vmaxapp = 0.040 +/- 0.007 nmol/min/mg). Treatment of fish with the CYP modulators beta-naphthoflavone (BNF, 50 mg/kg) and ethanol (EtOH, 1.0% aqueous) had no effect on sulfoxide production. No correlation existed between CYP isoform expression (determined by western blot) and aldicarb sulfoxidation rates, suggesting the involvement of an unmeasured CYP isoform or involvement of several isoforms with low specificity. This study indicates that a low rate of bioactivation of aldicarb to aldicarb sulfoxide may be responsible for the resistance of channel catfish to aldicarb toxicity relative to that of other piscine species.