Cytochrome P450 Protein Expression Research Articles

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Hypolipidemic and insulin sensitizing effects of salsalate beyond suppressing inflammation in a prediabetic rat model.

Background and aims: Low-grade chronic inflammation plays an important role in the pathogenesis of metabolic syndrome, type 2 diabetes and their complications. In this study, we investigated the effects of salsalate, a non-steroidal anti-inflammatory drug, on metabolic disturbances in an animal model of prediabetes-a strain of non-obese hereditary hypertriglyceridemic (HHTg) rats. Materials and Methods: Adult male HHTg and Wistar control rats were fed a standard diet without or with salsalate delivering a daily dose of 200mg/kg of body weight for 6weeks. Tissue sensitivity to insulin action was measured ex vivo according to basal and insulin-stimulated 14C-U-glucose incorporation into muscle glycogen or adipose tissue lipids. The concentration of methylglyoxal and glutathione was determined using the HPLC-method. Gene expression was measured by quantitative RT-PCR. Results: Salsalate treatment of HHTg rats when compared to their untreated controls was associated with significant amelioration of inflammation, dyslipidemia and insulin resistance. Specificaly, salsalate treatment was associated with reduced inflammation, oxidative and dicarbonyl stress when inflammatory markers, lipoperoxidation products and methylglyoxal levels were significantly decreased in serum and tissues. In addition, salsalate ameliorated glycaemia and reduced serum lipid concentrations. Insulin sensitivity in visceral adipose tissue and skeletal muscle was significantly increased after salsalate administration. Further, salsalate markedly reduced hepatic lipid accumulation (triglycerides -29% and cholesterol -14%). Hypolipidemic effects of salsalate were associated with differential expression of genes coding for enzymes and transcription factors involved in lipid synthesis (Fas, Hmgcr), oxidation (Pparα) and transport (Ldlr, Abc transporters), as well as changes in gene expression of cytochrome P450 proteins, in particular decreased Cyp7a and increased Cyp4a isoforms. Conclusion: These results demonstrate important anti-inflammatory and anti-oxidative effects of salsalate that were associated with reduced dyslipidemia and insulin resistance in HHTg rats. Hypolipidemic effects of salsalate were associated with differential expression of genes regulating lipid metabolism in the liver. These results suggest potential beneficial use of salsalate in prediabetic patients with NAFLD symptoms.

Effect of antibiotic-administration period on hepatic bile acid profile and expression of pharmacokinetic-related proteins in mouse liver, kidney, and brain capillaries

Antibiotic administration affects pharmacokinetics through changes in the intestinal microbiota, and bile acids are involved in this regulation. The purpose of the present study was to clarify the effect of different periods of antibiotic administration on the hepatic bile acid profile and expression of pharmacokinetic-related proteins in mouse liver, kidney, and brain capillaries. Vancomycin and polymyxin B were orally administered to mice for either 5- or 25-days. The hepatic bile acid profile of the 25-day treatment group was distinct. In the liver, the protein expression of cytochrome P450 (Cyp)3a11 showed the greatest reduction to 11.4% after the 5-day treatment and further reduced to 7.01% after the 25-day treatment. Similar reductions were observed for sulfotransferase 1d1, Cyp2b10, carboxylesterase 2e, UDP-glucuronosyltransferase (Ugt)1a5, and Ugt1a9. In the kidney and brain capillaries, no drug-metabolizing enzymes or drug transporters were changed with >1.5-fold or <0.66-fold statistical significance in either period. These results suggest that bile acids and metabolizing enzymes in the liver are affected in a period-dependent manner by antibiotic treatment, while the blood-brain barrier and kidneys are less affected. Drug-drug interactions of antibiotics via the intestinal microbiota should be considered by changing drug metabolism in the liver.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Ecotoxicological Characterization of Type C Killer Whales From Terra Nova Bay (Ross Sea, Antarctica): Molecular Biomarkers, Legacy, and Emerging Persistent Organic Contaminants

Among killer whale forms, type C is a fish-eating form and is the most common in the Ross Sea. In the austral summer 2015, a study was conducted to evaluate the toxicological hazard these marine mammals face in the Antarctic ecosystem. Seven biopsy samples were collected from adult individuals (five males and two females) in the surroundings of the Italian Research Station Mario Zucchelli, Terra Nova Bay, by remote dart sampling from the pack ice. The accumulation levels of persistent organic pollutants (POPs) such as legacy (DDTs, PCBs, and HCB) and emerging (PBDEs and DP) were measured. Moreover, the protein expression of cytochrome P450 (CYP1A1 and 2B) and the mRNA level variations of the peroxisome proliferator-activated receptors α and γ (PPARα-γ) and the estrogen receptor α (ERα), aryl hydrocarbon receptor (AhR), andCyp1awere evaluated. Twenty PCB congeners, six DDTs, HCB, three HCHs, and fourteen brominated BDEs and DP-syn and anti-isomers were analyzed on freeze-dried blubber biopsy samples by GC-MS. The protein expression was evaluated by Western Blot and the mRNA levels were quantified by quantitative real-time PCR. The average abundance pattern for the contaminants was DDTs &amp;gt; PCBs &amp;gt; HCB &amp;gt; HCHs ≈ PBDEs &amp;gt;&amp;gt; DP. Contaminant levels resulted to be lower when compared to the existing data from the Antarctic type C killer whales from the McMurdo Sound (Ross Sea) and those reported for fish-eating killer whales worldwide. The mRNA levels of the five target genes were successfully quantified, but no statistical correlation was found with POP levels, suggesting that either the low levels of quantified POPs in blubber may not significantly affect the biological responses investigated, or that other stressors could contribute to the alterations of the molecular biomarkers. Although the results showed a lower risk related to contamination compared to more impacted areas, this study provides baseline data for the conservation of this species in an area with high ecological value, recently declared as the largest Marine Protected Area in Antarctica, where pollutants should remain at minimum levels despite increasing multiple stresses existing in the region.

In a Prediabetic Model, Empagliflozin Improves Hepatic Lipid Metabolism Independently of Obesity and before Onset of Hyperglycemia.

Recent studies suggest that treatment with SGLT-2 inhibitors can reduce hepatic lipid storage and ameliorate non-alcoholic fatty liver disease (NAFLD) development beyond their glycemic benefits. However, the exact mechanism involved is still unclear. We investigated the hepatic metabolic effect of empagliflozin (10 mg/kg/day for eight weeks) on the development of NAFLD and its complications using HHTg rats as a non-obese prediabetic rat model. Empagliflozin treatment reduced neutral triacylglycerols and lipotoxic diacylglycerols in the liver and was accompanied by significant changes in relative mRNA expression of lipogenic enzymes (Scd-1, Fas) and transcription factors (Srebp1, Pparγ). In addition, alterations in the gene expression of cytochrome P450 proteins, particularly Cyp2e1 and Cyp4a, together with increased Nrf2, contributed to the improvement of hepatic lipid metabolism after empagliflozin administration. Decreased circulating levels of fetuin-A improved lipid metabolism and attenuated insulin resistance in the liver and in peripheral tissues. Our results highlight the beneficial effect of empagliflozin on hepatic lipid metabolism and lipid accumulation independent of obesity, with the mechanisms understood to involve decreased lipogenesis, alterations in cytochrome P450 proteins, and decreased fetuin-A. These changes help to alleviate NAFLD symptoms in the early phase of the disease and before the onset of diabetes.

Echinacoside alleviates acetaminophen-induced liver injury by attenuating oxidative stress and inflammatory cytokines in mice.

This study evaluates the protective effect of Echinacoside on acute liver toxicity induced by acetaminophen in mice and the mechanism behind it. Echinacoside and N-Acetyl Cysteine were intragastrically administrated for 7 days, and acetaminophen was intraperitoneally injected into mice 1 h after the last treatment on day 7. At the end of the experimental period, histological examination, parameters for the level of oxidative damage, hepatic malondialdehyde, serum pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β), UDP-glucuronosyltransferases, and sulfotransferases changes were examined using enzyme-linked immunosorbent assay and standard biochemical procedures. The expression of cytochrome P450 2E1 protein was assessed by western blot, followed by in silico molecular docking. Acetaminophen treatment obviously increased the levels of ALT and AST, changed hepatic histopathology, promoted oxidative stress, decreased antioxidant enzyme activities, and elevated the pro-inflammatory cytokines. Echinacoside significantly attenuated Acetaminophen-induced liver damage in a dose-dependent manner, with the most effective dose at 100 mg/kg. The pretreatments of Echinacoside in different concentrations altered the Acetaminophen-induced hepatotoxicity levels by decreasing the level of liver enzymes, reducing the liver necrosis with vacuolization, decreasing the hepatic malondialdehyde formation, increasing hepatic antioxidants activities, suppressing the pro-inflammatory cytokines (Tumor Necrosis Factor, Interleukin-6 and Interleukin-1beta), inhibiting Nitric Oxide production, enhancing sulfotransferases and UDP-glucuronosyltransferases activities. Notably, the expression of cytochrome P450 2E1 was inhibited by Echinacoside in a dose-dependent manner and the binding energy was -214.3 MeV. Echinacoside showed a significant protective effect against Acetaminophen-induced hepatotoxicity through the inhibition of oxidative stress, the expression of pro-inflammatory cytokines and cytochrome P450 2E1 protein expression.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

Evaluation of cytochrome P450-based drug metabolism in hemorrhagic shock rats that were transfused with native and an artificial red blood cell preparation, Hemoglobin-vesicles

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs.

Assessing cytochrome P450-based drug-drug interactions with hemoglobin-vesicles, an artificial red blood cell preparation, in healthy rats

Hemoglobin-vesicles (Hb-V), hemoglobin encapsulated within a liposome, were developed as an artificial red blood cell (RBC). When Hb-V becomes clinically available in the future, patients would presumably be co-administered with one or more drugs. Since drug-drug interactions can cause serious adverse effects and impede overall curative effects, evidence regarding the risk associated with drug-drug interactions between Hb-V and such simultaneously administered drugs is needed. Therefore, we report on cytochrome P450 (CYP)-based drug interactions with Hb-V in healthy rats. At 1 day after the saline, Hb-V or packed RBC (PRBC) administration, the blood retention of CYP-metabolizing drugs (caffeine, chlorzoxazone, tolbutamide and midazolam) were moderately prolonged in the case of the Hb-V group, but not the PRBC group, compared to saline group. The results of a proteome analysis revealed that the Hb-V administration had only negligible effects on the protein expression of CYPs in the liver. Hb-V administration, however, clearly suppressed the CYP metabolic activity of the four target CYP isoforms compared with the saline and PRBC group. However, these alterations were nearly recovered at 7 day after the Hb-V administration. Taken together, these results suggest that the administration of Hb-V slightly and transiently affects the CYP-based metabolism of the above drugs.

Downregulation of mitogen-activated protein kinases (MAPKs) in chronic ethanol-induced fatty liver

Mitogen-activated protein kinases (MAPKs) are versatile proteins that have been suggested to be involved in the regulation of lipid metabolism. This study was designed to investigate the responses of MAPK signaling to chronic ethanol exposure in vivo and in vitro, and try to explore its role in the pathogenesis of alcoholic fatty liver (AFL). Mice were fed with Lieber-Decarli liquid diet (5% ethanol, w/v) for 4 weeks to induce fatty liver, and the chronological changes of MAPK phosphorylation were measured using western blotting. We found that chronic ethanol feeding led to accumulation of triglyceride (TG), decreased phosphorylation of MAPKs, decreased protein level of peroxisomal proliferator activation receptor α (PPARα), and increased protein expression of cytochrome P4502E1 (CYP2E1) in mice liver. In vitro study showed that overexpression of CYP2E1 blunted the response of MAPKs to ethanol, and MAPK phosphatase 1 (MKP-1) knockdown by siRNA led to upregulation of PPARα protein level. Lastly, epidermal growth factor (EGF), a well-known MAPK activator, significantly suppressed chronic ethanol-induced hepatic fat accumulation and decline of PPARα expression in mice liver. Collectively, MAPK suppression, possibly due to the activation of hepatic CYP2E1, may be involved in chronic ethanol-induced hepatic steatosis.

Temperature determines the rate at which retene affects trout embryos, not the concentration that is toxic

The toxicity of waterborne retene (7-isopropyl-1-methyl phenanthrene) to post-hatch embryos of rainbow trout (Oncorhynchus mykiss) was assessed at 5 and 11 °C. Survival times of retene-exposed embryos were 70 % longer at 5 °C than at 11 °C, but survival times and LC50 s did not vary when time was expressed as degree-days (thermal units), i.e., at a common stage of development. The size of survivors decreased with increasing retene concentrations, but not with temperature. Retene did not bioconcentrate to any extent (bioconcentration factors < 2) at either temperature, indicating effective biotransformation by embryos. However, concentrations of retene metabolites were slightly higher at 5 °C, suggesting slower excretion rates than at 11 °C. The relative expression of cytochrome P450 proteins (CYP1A) did not vary with temperature but increased with retene concentration, as indicated by cyp1a mRNA concentrations. The induction of CYP1A protein by retene exposure was evident in the vasculature of eye, brain, heart, kidney, liver, gill, mouth, intestine, muscle, and yolk-sac. However, immunohistochemical staining was greater at 5 than at 11 °C for all tissues except liver and muscle. Overall, temperature effects on retene toxicity disappeared when the duration of embryo development and retene exposure were expressed as thermal units (degree-days). Temperature controlled the rate of embryo development and the rate of toxicity (time to a toxic endpoint), but not the concentrations that were toxic.

Synonymous mutation rs2515641 affects CYP2E1 mRNA and protein expression and susceptibility to drug-induced liver injury.

Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 (CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results:CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.

The effects of non-esterified fatty acids and β-hydroxybutyrate on the hepatic CYP2E1 in cows with clinical ketosis.

Dairy cows with ketosis display severe oxidative stress as well as high blood concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB). Cytochrome P4502E1 (CYP2E1) plays an important role in the induction of oxidative stress. The aim of this study was to investigate CYP2E1 expression and activity in the liver of clinically ketotic cows (in vivo) and the effects of NEFA and BHB on CYP2E1 expression and activity in hepatocytes (in vitro). Dairy cows with clinical ketosis exhibited a low blood concentration of glucose but high concentrations of NEFA and BHB. Hepatic mRNA, protein expression, and activity of CYP2E1 were significantly higher in cows with clinical ketosis than in control cows. In vitro, both NEFA and BHB treatment markedly up-regulated the mRNA and protein expressions as well as activity of CYP2E1 in cow hepatocytes. Taken together, these results indicate that high levels of NEFA and BHB significantly up-regulate the expression and activity of hepatic CYP2E1, and may be influential in the induction of oxidative stress in cows with clinical ketosis.

Prophylactic Administration of Nanocurcumin Abates the Incidence of Liver Toxicity Induced by an Overdose of Copper Sulfate: Role of CYP4502E1, NF-κB and Bax Expressions.

Background:The consequences of excess copper in human tissue are the alterations in the oxidative stress markers and peroxidative damage of membrane lipids. Unselective copper binding may be the clue to damaging impact to protein construction and hence modifying their biological functions. The aim of this study is to match the hepatoprotective efficacy of curcumin (CM) or nanocurcumin (NCM) with that of desferrioxamine (DSF; standard heavy metal chelator) against toxic doses of copper sulphate (CuSO4).Method:All treatments were given simultaneously with CuSO4 for 7 days.Result:CuSO4 administration elevated serum alanine transaminase, and hepatic nitric oxide (NO), lipid peroxide, and caspase-3 as well as protein expression of cytochrome P4502E1, and nuclear factor-κB (NF-κB) and Bax gene expressions. On the other hand, hepatic levels of reduced glutathione, superoxide dismutase, and interleukin-10 were decreased, whereas DNA degradation was increased as well compared with the control group. The administration of the aforementioned antioxidants ameliorated all the previous altered measured parameters. Interestingly, NCM achieved the most pronounced hepatoprotective effect nearly equivalent to that of DSF.Conclusion:It was concluded that NCM is considered a promising candidate against CuSO4 toxicity, and cytochrome P450, NF-κB, and Bax are involved in its toxicity and treatment.

Sagittaria sagittifolia polysaccharide protects against isoniazid- and rifampicin-induced hepatic injury via activation of nuclear factor E2-related factor 2 signaling in mice

Ethnopharmacological relevanceThe Sagittaria sagittifolia L. polysaccharide (SSP) is a purified form of a homogeneous polysaccharide isolated from the root tubers of S. sagittifolia, which has been used as a protectant against hepatotoxicity induced by coadministration of isoniazid and rifampicin. However, the protective effect of SSP against isoniazid- and rifampicin-induced liver injury has never been studied. Aim of the studyIn this study, the hepatoprotective effect of SSP and its underlying mechanism were investigated in mice with isoniazid- and rifampicin-induced liver injury. Materials and methodsLiver injury was induced in mice by intragastric administration of isoniazid and rifampicin, and the mice were divided into the following six groups: standard control (administration of saline by gavage), model (intragastric administration of isoniazid and rifampicin at 100 mg/kg/day each), positive control (100 mg/kg/day silymarin by gavage 4 h after isoniazid and rifampicin administration), and SSP-treated (200, 400, or 800 mg/kg/day SSP by gavage after isoniazid and rifampicin administration). Subsequently, blood and liver samples were collected from all the animals and were assessed. ResultsSSP significantly alleviated the liver injury, as evidenced by decreased activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase in the serum and a decreased level of malondialdehyde in the liver, as well as by an increased level of glutathione and increased activities of superoxide dismutase and catalase in the liver. SSP also effectively reduced the pathological tissue damage. The gene and protein expression of cytochrome P450 (CYP) 2E1 and CYP3A4 was inhibited by SSP. The gene and protein expression of nuclear factor erythroid 2-related factor 2 (NRF2), glutamate-cysteine ligase, and heme oxygenase-1 were induced by SSP, whereas that of Kelch-like ECH-associated protein 1 was inhibited. ConclusionsSSP exerts a protective effect against isoniazid- and rifampicin-induced liver injury in mice. The underlying mechanisms may involve activation of NRF2 and its target antioxidant enzymes and inhibition of the expression of CYPs.

Tissue Distribution and Gender-Specific Protein Expression of Cytochrome P450 in five Mouse Genotypes with a Background of FVB.

To systematically investigate tissue distribution and gender-specific protein expression of Cytochrome P450 (Cyps) in five mouse genotypes with a background of Friend virus B (FVB). The Cyps were extracted from the tissue S9 fractions of the main metabolic organs and then absolutely quantified by applying the UHPLC-MS/MS method. The liver had the highest expression of Cyps, followed by the small intestine and kidney. In the liver, Cyp1a2, Cyp2c29, Cyp2c39, Cyp2d22, Cyp2e1, and Cyp3a11 were the main isoforms. Cyp1a2 and Cyp2c29 were male-specific, while Cyp2c39 was female-specific. Compared with the expression in Wild-type (WT) FVB mice, the expression of Cyp1a2, Cyp1b1, Cyp2d22, and Cyp3a25 significantly decreased in female efflux transporter (ET) knockout mice. In the small intestine, Cyp2c29 and Cyp3a11 were the major isoforms. Knockout of ET didn't alter the expression levels of most Cyps. However, female ET knockout mice presented higher Cyp2c29 expression than WT FVB mice. The Cyp7a1 expression was markedly decreased in ET knockout mice except Bcrp1-/- mice. In the kidney, Cyp2e1 was the main isoform and exhibited male specificity. Knockout of ET slightly affected the protein expression of Cyps in the brain, heart, lung, spleen and stomach. A comprehensive understanding of the distribution characteristics and gender-specific expression of Cyps in major metabolic organs of WT and ET knockout FVB mice should contribute to a better understanding of drug efficacy and toxicity, and drug-drug interactions.

Protective effect of acidic polysaccharide from Schisandra chinensis on acute ethanol-induced liver injury through reducing CYP2E1-dependent oxidative stress

AimSchisandra chinensis is a well-known traditional Chinese medicine used mainly as a recipe for hepatoprotection. Modern researches have revealed that the hepatoprotection is related to its lignans and crude polysaccharide. In this study, we examined the effect and mechanism of Schisandra chinensis acidic polysaccharide (SCAP) on the liver injury induced by ethanol. Main methodsSCAP was extracted with water extraction and ethanol precipitation. Liver injury models of both mice and HepG2 cells were produced by ethanol. The liver index, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum of the mice and cell culture supernatant were examined; HE staining was performed for observing pathological changes of liver. The malondialdehyde (MDA) level and superoxide dismutase (SOD) activities in serum, liver tissue and HepG2 cells, and triglyceride (TG) content in liver tissue were tested. Western blot was conducted to determine cytochrome P450 2E1 (CYP2E1) expression in liver tissue of mice and HepG2 cells. Key findingsSCAP significantly reduced serial AST and ALT levels in the injuried liver and HepG2 cells induced by ethanol and also decreased TG level in the liver tissue. SCAP also obviously improved the hepatopathological changes and decreased MDA level as well as increased SOD activities in the serum, liver tissue and HepG2 cells induced by ethanol. Furthermore, Western blot analysis indicated that SCAP significantly inhibited the upregulation of CYP2E1 protein. SignificanceSCAP has a protective effect on ethanol-induced liver injury in mice and cells, and the mechanism underlying may be via inhibiting the expression of CYP2E1 protein and then alleviating oxidative stress injury induced by ethanol.

Effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in rat pancreatic endocrine cells

Oxidative stress is one of the principle mechanism of acrylamide-induced toxicity. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide or by direct conjugation with glutathione. Bearing in mind that up to now the effects of acrylamide on oxidative stress status and CYP2E1 level in endocrine pancreas have not been studied we performed qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT) and CYP2E1 expression in islets of Langerhans of rats subchronically treated with 25 or 50mg/kg bw of acrylamide. Since the majority of cells (>80%) in rodent islets are beta cells, in parallel studies, we employed the Rin-5F beta cell line to examine effects of acrylamide on redox status and the activity of CAT, SOD and glutathione-S-transferase (GST), their gene expression, and CYP2E1, NF-E2 p45-related factor 2 (Nrf2) and iNOS expression. Immunohistochemically stained pancreatic sections revealed that acrylamide induced increase of iNOS and decrease of CYP2E1 protein expression, while expression of antioxidant enzymes was not significantly affected by acrylamide in islets of Langerhans. Analysis of Mallory-Azan stained pancreatic sections revealed increased diameter of blood vessels lumen in pancreatic islets of acrylamide-treated rats. Increase in the GST activity, lipid peroxidation and nitrite level, and decrease in GSH content, CAT and SOD activities was observed in acrylamide-exposed Rin-5F cells. Level of mRNA was increased for iNOS, SOD1 and SOD2, and decreased for GSTP1, Nrf2 and CYP2E1 in acrylamide-treated Rin-5F cells. This is the first report of the effects of acrylamide on oxidant/antioxidant parameters and CYP2E1 expression in pancreatic endocrine cells.

Comparison of short- and long-term exposure effects of cruciferous and apiaceous vegetables on carcinogen metabolizing enzymes in Wistar rats

Cruciferous and apiaceous vegetables may be chemopreventive due to their ability to modulate carcinogen-metabolizing enzymes but whether the effects on such enzymes are sustained over time is unknown. To examine the short- and long-term effects of the vegetables, rats were fed one of four diets for 7, 30, or 60 d: AIN-93G, CRU (21% cruciferous vegetables-fresh broccoli, green cabbage, watercress), API (9% apiaceous vegetables - fresh parsnips, celery), or API + CRU (10.5% CRU + 4.5% API). Although CRU increased activity and protein expression of cytochrome P450 (CYP) 1A1 and CYP1A2 after 7 d, only activity was sustained after 30 and 60 d. There was a trend towards an interaction between the length of feeding period and CRU for CYP1A1 activity; activity increased with greater time of feeding. API increased CYP1A2 activity but decreased sulfotransferase 1A1 activity after 7 d, although not at later times. Altogether, increased CYP1A activity by CRU was maintained with long term feeding while protein amount decreased, suggesting influence by mechanisms other than, or in addition to, transcriptional regulation. Thus, response patterns and interactions with length of feeding may differ, depending upon the types of vegetables and enzymes, requiring caution when interpreting the results of short-term feeding studies.