The effect of chronic kidney disease on CYP2B expression and activity in male Wistar rats.

Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy-bupropion, and bupropion were quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Level of mRNA and protein were determined by RT-PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (Vmax) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.

Effect of Nicotine on CYP2B1 Expression in a Glioma Animal Model and Analysis of CYP2B6 Expression in Pediatric Gliomas.

Cyclophosphamide (CPA) is a pro-drug commonly used in the chemotherapeutic schemes for glioma treatment but has high toxicity and the side effects include brain damage and even death. Since CPA is activated mainly by CY2B6, over-expression of the enzyme in the tumor cells has been proposed to enhance CPA activation. In this study, we explored the induction of the Cyp2b1 (homologous to CYP2B6) by nicotine in an animal rat model with glioma. Gene expression and protein levels were analyzed by RT-PCR and Western blot. Nicotine treatment increased CYP2B1 protein levels in the healthy animals' brain tissue. In the brain tissue of animals with glioma, the CYP2B1 showed a high expression, even before nicotine treatment. Nicotine did not increase significantly the CYP2B1 protein expression in the tumor, but increased its expression in the tumor vicinity, especially around blood vessels in the cortex. We also explored CY2B6 expression in glioma samples derived from pediatric patients. Tumor tissue showed a variable expression of the enzyme, which could depend on the tumor malignancy grade. Induction of the CYP2B6 in pediatric gliomas with lower expression of the enzyme, could be an alternative to improve the antitumoral effect of CPA treatment.

Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Chinese herbal formula Fuzheng Huayu alleviates CCl4-induced liver fibrosis in rats: a transcriptomic and proteomic analysis.

Liver fibrosis is a consequence of chronic liver disease that can progress to liver cirrhosis or even hepatocarcinoma. Fuzheng Huayu (FZHY), a Chinese herbal formula, has been shown to exert anti-fibrotic effects. To better understand the molecular mechanisms underlying the anti-fibrotic effects of FZHY, we analyzed transcriptomic and proteomic combination profiles in CCl4-induced liver fibrosis in rats, which were treated with extracted FZHY powder (0.35 g·kg-1·d-1, ig) for 3 weeks. We showed that FZHY administration significantly improved liver function, alleviated hepatic inflammatory and fibrotic changes, and decreased the hydroxyproline content in the livers of CCl4-treated rats. When their liver tissues were examined using microarray and iTRAQ, we found 255 differentially expressed genes (fold change ≥1.5, P<0.05) and 499 differentially expressed proteins (fold change ≥1.2, P<0.05) in the FZHY and model groups. Functional annotation with DAVID (The Database for Annotation, Visualization and Integrated Discovery) showed that 15 enriched gene ontology terms, including drug metabolic process, response to extracellular stimulus, response to vitamins, arachidonic acid metabolic process, response to wounding, and oxidation reduction might be involved in the anti-fibrotic effects of FZHY; whereas KEGG pathway analysis revealed that eight enriched pathways, including arachidonic acid metabolism, retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism might also be involved. Moreover, the protein-protein interaction network demonstrated that 10 core genes/proteins overlapped, with Ugt2a3, Cyp2b1 and Cyp3a18 in retinol metabolism pathway overlapped to a higher degree. Compared to the model rats, the livers of FZHY-treated rats had significantly higher mRNA and protein expression levels of Ugt2a3, Cyp2b1 and Cyp3a18. Furthermore, the concentration of retinoic acid was significantly higher in the FZHY-treated rats compared with the model rats. The results suggest that the anti-fibrotic effects of FZHY emerge through multiple targets, multiple functions, and multiple pathways, including FZHY-regulated retinol metabolism, xenobiotic metabolism by cytochrome P450, and drug metabolism through up-regulated Ugt2a3, Cyp2b1, and Cyp3a18. These genes may play important anti-fibrotic roles in FZHY-treated rats.

Evaluation of the human relevance of the constitutive androstane receptor-mediated mode of action for rat hepatocellular tumor formation by the synthetic pyrethroid momfluorothrin.

High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

Unmasking efavirenz neurotoxicity: Time matters to the underlying mechanisms.

Efavirenz is an anti-HIV drug that presents relevant short- and long-term central nervous system adverse reactions. Its main metabolite (8-hydroxy-efavirenz) was demonstrated to be a more potent neurotoxin than efavirenz itself. This work was aimed to understand how efavirenz biotransformation to 8-hydroxy-efavirenz is related to its short- and long-term neuro-adverse reactions. To access those mechanisms, the expression and activity of Cyp2b enzymes as well as the thiolomic signature (low molecular weight thiols plus S-thiolated proteins) were longitudinally evaluated in the hepatic and brain tissues of rats exposed to efavirenz during 10 and 36days. Efavirenz and 8-hydroxy-efavirenz plasma concentrations were monitored at the same time points. Cyp2b induction had a delayed onset in liver (p<0.001), translating into increases in Cyp2b activity in liver and 8-hydroxy-efavirenz plasma concentration (p<0.001). Moreover, an increase in S-cysteinyl-glycinylated proteins (p<0.001) and in free low molecular weight thiols was also observed in liver. A distinct scenario was observed in hippocampus, which showed an underexpression of Cyp2b as well as a decrease in S-cysteinylated and S-glutathionylated proteins. Additionally, the observed changes in tissues were associated with a marked increase of S-glutathionylation in plasma. Our data suggest that the time course of efavirenz biotransformation results from different mechanisms for its short- and long-term neurotoxicity. The difference in the redox profile between liver and hippocampus might explain why, despite being mostly metabolized by the liver, this drug is neurotoxic. If translated to clinical practice, this evidence will have important implications in efavirenz short- and long-term neurotoxicity prevention and management.

Effects of Nonalcoholic Fatty Liver Disease on Hepatic CYP2B1 and in Vivo Bupropion Disposition in Rats Fed a High-Fat or Methionine/Choline-Deficient Diet.

Nonalcoholic fatty liver disease (NAFLD) refers to hepatic pathologies, including simple fatty liver (SFL), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis, that may progress to hepatocellular carcinoma. These liver disease states may affect the activity and expression levels of drug-metabolizing enzymes, potentially resulting in an alteration in the pharmacokinetics, therapeutic efficacy, and safety of drugs. This study investigated the hepatic cytochrome P450 (CYP) 2B1-modulating effect of a specific NAFLD state in dietary rat models. Sprague-Dawley rats were given a methionine/choline-deficient (MCD) or high-fat (HF) diet to induce NASH and SFL, respectively. The induction of these disease states was confirmed by plasma chemistry and liver histological analysis. Both the protein and mRNA levels of hepatic CYP2B1 were considerably reduced in MCD diet-fed rats; however, they were similar between the HF diet-fed and control rats. Consistently, the enzyme-kinetic and pharmacokinetic parameters for CYP2B1-mediated bupropion metabolism were considerably reduced in MCD diet-fed rats; however, they were also similar between the HF diet-fed and control rats. These results may promote a better understanding of the influence of NAFLD on CYP2B1-mediated metabolism, which could have important implications for the safety and pharmacokinetics of drug substrates for the CYP2B subfamily in patients with NAFLD.

[Effect of xenobiotics on microRNA expression in rat liver].

Using bioinformatics analysis we selected microRNAs which could bind 3'-UTR-region of cytochrome P450 (CYP) genes. Three microRNA miR-21, -221, -222, their potential targets might be mRNA for CYP1A1, and two microRNA miR-143, miR-152 for CYP2B1 accordingly were selected for experimental verification. Expression level of these microRNAs in rat liver upon benzo(a)pyrene (BP), phenobarbital (PB), and DDT induction was determined using RT-qPCR method. In rats treated by both BP, and DDT the hepatic content of miR-21, -221, -222 significantly demonstrated a 2-3-fold decrease. The decrease in miR expression was accompanied by a considerable (5.5-8.7-fold) increase in the CYP1A1-mediated EROD activity. The expression of miR-143 remained unchanged after the PB treatment, while the expression of miR-152 increased by 2 times, however, the (10.5-fold) increase in PROD activity of CYP2B was much higher. In the DDT-treated liver PROD activity increased by 20 times, the expression of miR-152 didn't change, and the expression of miR-143 increased by 2 times. The bioinformatics analysis of interactions between microRNAs and targets showed that the studied miRs can potentially bind 3'-end of AhR, ESR1, GR, CCND1, PTEN mRNA. Thus, the expression profile of miR-21, -221, -222, -143, -152 might change under the xenobiotics exposure. In silico analysis confirmed, that microRNAs target not only cytochrome P450 mRNA but also other genes, including those involved in hormonal carcinogenesis, they also can be regulated with studied miRs.

Subacute nicotine co-exposure has no effect on 2,2',3,5',6- pentachlorobiphenyl disposition but alters hepatic cytochrome P450 expression in the male rat.

Polychlorinated biphenyls (PCBs) are metabolized by cytochrome P450 2B enzymes (CYP2B) and nicotine is reported to alter CYP2B activity in the brain and liver. To test the hypothesis that nicotine influences PCB disposition, 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and its metabolites were quantified in tissues of adult male Wistar rats exposed to PCB 95 (6mg/kg/d, p.o.) in the absence or presence of nicotine (1.0mg/kg/d of the tartrate salt, s.c.) for 7 consecutive days. PCB 95 was enantioselectively metabolized to hydroxylated (OH-) PCB metabolites, resulting in a pronounced enrichment of E1-PCB 95 in all tissues investigated. OH-PCBs were detected in blood and liver tissue, but were below the detection limit in adipose, brain and muscle tissues. Co-exposure to nicotine did not change PCB 95 disposition. CYP2B1 mRNA and CYP2B protein were not detected in brain tissues but were detected in liver. Co-exposure to nicotine and PCB 95 increased hepatic CYP2B1 mRNA but did not change CYP2B protein levels relative to vehicle control animals. However, hepatic CYP2B protein in animals co-exposed to PCB 95 and nicotine were reduced compared to animals that received only nicotine. Quantification of CYP2B3, CYP3A2 and CYP1A2 mRNA identified significant effects of nicotine and PCB 95 co-exposure on hepatic CYP3A2 and hippocampal CYP1A2 transcripts. Our findings suggest that nicotine co-exposure does not significantly influence PCB 95 disposition in the rat. However, these studies suggest a novel influence of PCB 95 and nicotine co-exposure on hepatic cytochrome P450 (P450) expression that may warrant further attention due to the increasing use of e-cigarettes and related products.

Phase I/II clinical trial of encapsulated, cytochrome P450 expressing cells as local activators of cyclophosphamide to treat spontaneous canine tumours.

Based upon promising preclinical studies, a clinical trial was performed in which encapsulated cells overexpressing cytochrome P450 enzyme isoform 2B1 were implanted around malignant mammary tumours arising spontaneously in dogs. The dogs were then given cyclophosphamide, one of the standard chemotherapeutic agents used for the treatment of mammary tumours. The dogs were assessed for a number of clinical parameters as well as for reduction in tumour size. The treatment was well tolerated with no evidence of adverse reactions or side effects being associated with the administration of the encapsulated cells. Reductions in tumour size of more than 50% were observed for 6 out of the 11 tumours analysed while 5 tumours showing minor responses, i.e. stable disease. In contrast, the tumours that received cyclophosphamide alone showed only stable disease. Taken together, this data suggests that encapsulated cytochrome P450 expressing cells combined with chemotherapy may be useful in the local treatment of a number of dog mammary tumours and support the performance of further clinical studies to evaluate this new treatment.

PCB153 and p,p'-DDE disorder thyroid hormones via thyroglobulin, deiodinase 2, transthyretin, hepatic enzymes and receptors.

Polychlorinated biphenyls (PCBs) and DDT are widespread environmental persistent organic pollutants that have various adverse effects on reproduction, development and endocrine function. In order to elucidate effects of PCBs and DDT on thyroid hormone homeostasis, Sprague-Dawley rats were dosed with PCB153 and p,p'-DDE intraperitoneally (ip) for five consecutive days and sacrificed within 24 h after the last dose. Results indicated that after combined exposure to PCB153 and p,p'-DDE, total thyroxine , free thyroxine, total triiodothyronine, and thyroid-stimulating hormone in serum were decreased, whereas free triiodothyronine and thyrotropin-releasing hormone were not affected. Thyroglobulin and transthyretin levels in serum were significantly reduced. mRNA expression of deiodinases 2 (D2) was also suppressed, while D1 and D3 levels were not significantly influenced after combined exposure. PCB153 and p,p'-DDE induced hepatic enzymes, UDPGTs, CYP1A1, CYP2B1, and CYP3A1 mRNA expressions being significantly elevated. Moreover, TRα1, TRß1, and TRHr expressions in the hypothalamus displayed increasing trends after combined exposure to PCB153 and p,p'-DDE. Taken together, observed results indicate that PCB153 and p,p'-DDE could disorder thyroid hormone homeostasis via thyroglobulin, deiodinase 2, transthyretin, hepatic enzymes, and hormone receptors.

Extravascular use of drug-eluting beads: a promising approach in compartment-based tumor therapy.

Intraperitoneal carcinomatosis (PC) may occur with several tumor entities. The prognosis of patients suffering from PC is usually poor. Present treatment depends on the cancer entity and includes systemic chemotherapy, radiation therapy, hormonal therapy and surgical resection. Only few patients may also benefit from hyperthermic intraperitoneal chemotherapy with a complete tumor remission. These therapies are often accompanied by severe systemic side-effects. One approach to reduce side effects is to target chemotherapeutic agents to the tumor with carrier devices. Promising experimental results have been achieved using drug-eluting beads (DEBs). A series of in vitro and in vitro experiments has been conducted to determine the suitability of their extravascular use. These encapsulation devices were able to harbor CYP2B1 producing cells and to shield them from the hosts immune system when injected intratumorally. In this way ifosfamide--which is transformed into its active metabolites by CYP2B1--could be successfully targeted into pancreatic tumor growths. Furthermore DEBs can be used to target chemotherapeutics into the abdominal cavity for treatment of PC. If CYP2B1 producing cells are proven to be save for usage in man and if local toxic effects of chemotherapeutics can be controlled, DEBs will become promising tools in compartment-based anticancer treatment.

The enhancing effect of ethanol on the mutagenic activation of N-nitrosomethylbenzylamine by cytochrome P450 2A in the rat oesophagus.

Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B(1) or other N-nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Maternal exposure to low doses of DES altered mRNA expression of hepatic microsomal enzymes in male rat offspring.

Our previous studies demonstrated that prenatal diethylstilbestrol (DES) treatment disrupts steroidogenesis but induces high-level expression of androgen receptor (AR) mRNA to inhibit the disruption of spermatogenesis. This study examined which prenatal DES treatment influenced hepatic microsomal enzymes, CYP3A1, CYP2B1/2, CYP2C11, UGT2B1 (UDP-glucuronosyltransferase 2B1), and IGF-1 (insulin-like growth factor-1), in male rat offspring. DES treatment decreased the mRNA expression levels of CYP3A1 and CYP2B1/2, but did not alter the expression of CYP2C11. At 6 weeks, DES treatment increasd the mRNA expression levels of UGT2B1 and IGF-1. These results suggest that prenatal DES treatment alters two hepatic enzymes (CYP3A1 and CYP2B1/2) and IGF-1 mRNA expression levels to counteract the low level of testosterone, but this disrupted UGT2B1 mRNA expression reduces the testosterone level.

Licorice extract increases cyclophosphamide teratogenicity by upregulating the expression of cytochrome P-450 2B mRNA.

BACKGROUND: Since cyclophosphamide is metabolically activated to teratogenic acrolein and cytotoxic phosphoramide mustard by cytochrome P-450 type 2B (CYP2B), we assessed the effects of licorice, a CYP2B inducer, on the fetal defects induced by cyclophosphamide. METHODS: Pregnant Sprague-Dawley rats were daily administered with licorice (100 mg/kg) by gavage for 7 days, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11 mg/kg) 1 hr after the final licorice treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. RESULTS: Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 93.8, 41.1, and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. When pre-treated with licorice, cyclophosphamide-induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with licorice greatly increased mRNA expression and activity of hepatic CYP2B. CONCLUSIONS: The results indicate that repeated intake of licorice may aggravate cyclophosphamide-induced body weight loss and malformations of fetuses by upregulating CYP2B.

N-Nitrosodiethylamine genotoxicity in primary rat hepatocytes: effects of cytochrome P450 induction by phenobarbital.

Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21-105 µg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21-21 µg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ~6-fold and ~2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105 µg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.

Changes in expression of hepatic cytochrome P450 subfamily enzymes during development of adjuvant-induced arthritis in rats.

An animal model of rheumatoid arthritis can be elicited in male Lewis rats by a single intradermal injection of liquid paraffin containing dead Mycrobacterium tuberculosis (MT adjuvant) into the planar surface of the right hind-foot. In the present study, we used this animal model to examine the changes in expression of hepatic cytochorme P450 (CYP) enzymes during the development of the arthritis. Swellings of the MT adjuvant-injected hind-foot initially occurred at 1-8 days after the injection. Thereafter, the swelling gradually become more severe up to 13 days later and was maintained for up to 25 days. Swellings of the other hind-foot was also observed after 12 days and gradually become more severe up to 15 days with maintenance of the severe swelling for up to 25 days. The gene expression levels and enzyme activities of hepatic CYP 3A and CYP2B subfamily enzymes at 1, 12, and 25 days after the MT adjuvant injection were significantly decreased, compared with the corresponding time-matched controls. The decreases in the gene expression levels and activities of all the enzymes examined were closely correlated with increases in the expression levels of the inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, interleukin-1ß and interleukin-6, which were produced in the liver. All of the present findings demonstrate that hepatic CYP3A and CYP2B subfamily enzymes are decreased during the development of MT adjuvant-induced arthritis and further suggest that the decreases are dependent on the production of inflammatory cytokines in the liver.

Cytochrome-P450 2B1 gene silencing attenuates puromycin aminonucleoside-induced cytotoxicity in glomerular epithelial cells.

Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.

Transcriptional and post-transcriptional response of drug-metabolizing enzymes to PAHs contamination in red mullet (Mullus barbatus, Linnaeus, 1758): a field study.

Aim of this study was to evaluate the responsiveness of red mullet (Mullus barbatus) liver detoxification enzymes to PAHs at transcriptional and post-transcriptional levels in the field. Fish were captured in the north-eastern Adriatic Sea, close to an oil refinery. Sixteen PAHs (EPA) were determined in sediments and fish fillets; transcription levels of cyp1a, cyp3a and abcc2 genes and EROD, BROD, B(a)PMO, BFCOD, GST and UDPGT enzymatic activities were measured. Levels of PAHs in sediments reflect the oil pollution gradient of the area, with weak correspondence in fish fillets. cyp1a gene transcription and EROD, B(a)PMO and BFCOD activities were significantly induced in the oil refinery site, and a slight up-regulation of cyp3a and abcc2 was also observed. GST and UDPGT remained unchanged. The present study provides the first data on detoxification responses at transcriptional levels in the liver of red mullet and confirms phase I enzymes as suitable biomarkers of exposure to PAHs in field studies.

Celecoxib activates Stat5 and restores or increases the expression of growth hormone-regulated genes in hepatocarcinogenesis.

We have previously evaluated the chemopreventive effect of celecoxib on preneoplastic lesions in rat liver. However, though the effects of celecoxib have been tested in a variety of carcinomas, there has not been a study on the modulation of gene expression in response to this drug. Here, we evaluated the effect of celecoxib on the gene expression profile associated with hepatocarcinogenesis. Male Sprague-Dawley rats underwent the modified resistant hepatocyte model and were fed a diet containing 1500 ppm of celecoxib. Gene expression profiles were evaluated using DNA microarrays and further validations were performed using quantitative PCR, western blotting and immunohistochemical staining. Celecoxib modulated the expression of 46 genes, and those regulated by growth hormone were selected for further analysis. Celecoxib significantly upregulated the expression of the Cyp2b1/2, Cyp3a1, and alpha2-urinary globulin (alpha2uG) genes and restored the expression of Cyp2b3 to normal. The protein expression of Cyp2b1/2 was increased, but the expressions of Cyp3a1 and alpha2uG were only restored to normal levels. The increased Cyp2b1/2 expression in response to celecoxib was mainly confined to preneoplastic lesions. A search for the upstream mediator of these genetic alterations found that carcinogenesis inactivated by 87% the signal transducer and activator of transcription 5 (Stat5), a transcription factor that is activated by growth hormone signaling, but celecoxib treatment restored its activation. In conclusion, these results suggest that celecoxib exerts anticancer effects on altered hepatic cells by restoring mRNA and the protein expression levels of specific genes, in part through the reactivation of Stat5.