Hepatic Xenobiotic Metabolism Research Articles

Genome-wide analysis of hepatic DNA methylation reveals impact of epigenetic aging on xenobiotic metabolism and transport genes in an aged mouse model

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Transcriptomics and Metabolomics Reveal Tomato Consumption Alters Hepatic Xenobiotic Metabolism and Induces Steroidal Alkaloid Metabolite Accumulation in Mice.

Tomato consumption is associated with many health benefits including lowered risk for developing certain cancers. It is hypothesized that tomato phytochemicals are transported to the liver and other tissues where they alter gene expression in ways that lead to favorable health outcomes. However, the effects of tomato consumption on mammalian liver gene expression and chemical profile are not well defined. The study hypothesizes that tomato consumption would alter mouse liver transcriptomes and metabolomes compared to a control diet. C57BL/6J mice (n = 11-12/group) are fed a macronutrient matched diet containing either 10% red tomato, 10% tangerine tomato, or no tomato powder for 6 weeks after weaning. RNA-Seq followed by gene set enrichment analyses indicates that tomato type and consumption, in general, altered expression of phase I and II xenobiotic metabolism genes. Untargeted metabolomics experiments reveal distinct clustering between control and tomato fed animals. Nineteen molecular formulas (representing 75 chemical features) are identified or tentatively identified as steroidal alkaloids and isomers of their phase I and II metabolites; many of which are reported for the first time in mammals. These data together suggest tomato consumption may impart benefits partly through enhancing detoxification potential.

Potential Harm of IQOS Smoke to Rat Liver.

The Food and Drug Administration has recently classified the IQOS electronic cigarette as a modified-risk tobacco product. However, IQOS cigarettes still release various harmful constituents typical of conventional cigarettes (CCs), although the concentrations are markedly lower. Here, we investigated the damaging effects of IQOS smoking on the liver. Male Sprague Dawley rats were exposed, whole body, 5 days/week for 4 weeks to IQOS smoke (4 sticks/day), and hepatic xenobiotic metabolism, redox homeostasis and lipidomic profile were investigated. IQOS boosted reactive radicals and generated oxidative stress. Exposure decreased cellular reserves of total glutathione (GSH) but not GSH-dependent antioxidant enzymes. Catalase and xanthine oxidase were greater in the exposed group, as were various hepatic CYP-dependent monooxygenases (CYP2B1/2, CYP1A1, CYP2A1, CYP2E1-linked). Respiratory chain activity was unaltered, while the number of liver mitochondria was increased. IQOS exposure had an impact on the hepatic lipid profile. With regard to the expression of some MAP kinases commonly activated by CC smoking, IQOS increased the p-p38/p38 ratio, while erythroid nuclear transcription factor 2 (Nrf2) was negatively affected. Our data suggest that IQOS significantly impairs liver function, supporting the precautionary stance taken by the WHO toward the use of these devices, especially by young people and pregnant women.

Investigating the Acute Metabolic Effects of the N-Methyl Carbamate Insecticide, Methomyl, on Mouse Liver.

Many pesticides have been identified as endocrine and metabolism-disrupting chemicals with hepatotoxic effects. However, data are limited for insecticides in the n-methyl carbamate class, including methomyl. Here, we investigate the liver and systemic metabolic effects of methomyl in a mouse model. We hypothesize that methomyl exposure will disrupt xenobiotic and intermediary metabolism and promote hepatic steatosis in mice. Male C57BL/6 mice were exposed daily to 0-5 mg/kg methomyl for 18 days. Mice were fed water and regular chow diet ad libitum. Metabolic phenotyping was performed, and tissue samples were collected. Effects were generally greatest at the highest methomyl dose, which induced Cyp1a2. Methomyl decreased whole body weight while the liver:body weight and testes:body weight ratios were increased. Hepatic steatosis increased while plasma LDL decreased. Fasting blood glucose and the glucose tolerance test area under the curve decreased along with hepatic glycogen stores. Methomyl, however, did not increase liver oxidative stress or injury. Collectively, these data demonstrate that methomyl disrupts hepatic xenobiotic and intermediary metabolism while increasing the testes:body weight ratio, suggesting that it may be an endocrine disrupting chemical. Besides methomyl's known action in cholinesterase inhibition, it may be involved in aryl hydrocarbon receptor activation. The potential impact of n-methyl carbamate insecticides on metabolic health and diseases, including toxicant-associated steatotic liver disease (TASLD), warrants further investigation.

Effects of glyphosate residues and different concentrate feed proportions in dairy cow rations on hepatic gene expression, liver histology and biochemical blood parameters.

Glyphosate (GLY) is worldwide one of the most used active substances in non-selective herbicides. Although livestock might be orally exposed via GLY-contaminated feedstuffs, not much is known about possible hepatotoxic effects of GLY. As hepatic xenobiotic and nutrient metabolism are interlinked, toxic effects of GLY residues might be influenced by hepatic nutrient supply. Therefore, a feeding trial with lactating dairy cows was conducted to investigate effects of GLY-contaminated feedstuffs and different concentrate feed proportions (CFP) in the diets as tool for varying nutrient supply to the liver. For this, 61 German Holstein cows (207 ± 49 days in milk; mean ± standard deviation) were either fed a GLY-contaminated total mixed ration (TMR, GLY groups, mean GLY intake 122.7 μg/kg body weight/day) or control TMR (CON groups, mean GLY intake 1.2 μg/kg body weight/day) for 16 weeks. Additionally, both groups were further split into subgroups fed a lower (LC, 30% on dry matter basis) or higher (HC, 60% on dry matter basis) CFP resulting in groups CONHC (n = 16), CONLC (n = 16), GLYHC (n = 15), GLYLC (n = 14). Blood parameters aspartate aminotransferase, γ-glutamyltransferase, glutamate dehydrogenase, cholesterol, triglyceride, total protein, calcium, phosphorus, acetic acid and urea and histopathological evaluation were not influenced by GLY, whereas all mentioned parameters were at least affected by time, CFP or an interactive manner between time and CFP. Total bilirubin blood concentration was significantly influenced by an interaction between GLY and CFP with temporarily elevated concentrations in GLYHC, whereas the biological relevance remained unclear. Gene expression analysis indicated 167 CFP-responsive genes, while seven genes showed altered expression in GLY groups compared to CON groups. Since expression changes of GLY-responsive genes were low and liver-related blood parameters changed either not at all or only slightly, the tested GLY formulation was considered to have no toxic effects on the liver of dairy cows.

Gut Microbiome Critically Impacts PCB-induced Changes in Metabolic Fingerprints and the Hepatic Transcriptome in Mice.

Polychlorinated biphenyls (PCBs) are ubiquitously detected and have been linked to metabolic diseases. Gut microbiome is recognized as a critical regulator of disease susceptibility; however, little is known how PCBs and gut microbiome interact to modulate hepatic xenobiotic and intermediary metabolism. We hypothesized the gut microbiome regulates PCB-mediated changes in the metabolic fingerprints and hepatic transcriptome. Ninety-day-old female conventional and germ-free mice were orally exposed to the Fox River Mixture (synthetic PCB mixture, 6 or 30 mg/kg) or corn oil (vehicle control, 10 ml/kg), once daily for 3 consecutive days. RNA-seq was conducted in liver, and endogenous metabolites were measured in liver and serum by LC-MS. Prototypical target genes of aryl hydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor were more readily upregulated by PCBs in conventional conditions, indicating PCBs, to the hepatic transcriptome, act partly through the gut microbiome. In a gut microbiome-dependent manner, xenobiotic, and steroid metabolism pathways were upregulated, whereas response to misfolded proteins-related pathways was downregulated by PCBs. At the high PCB dose, NADP, and arginine appear to interact with drug-metabolizing enzymes (ie, Cyp1-3 family), which are highly correlated with Ruminiclostridium and Roseburia, providing a novel explanation of gut-liver interaction from PCB-exposure. Utilizing the Library of Integrated Network-based Cellular Signatures L1000 database, therapeutics targeting anti-inflammatory and endoplasmic reticulum stress pathways are predicted to be remedies that can mitigate PCB toxicity. Our findings demonstrate that habitation of the gut microbiota drives PCB-mediated hepatic responses. Our study adds knowledge of physiological response differences from PCB exposure and considerations for further investigations for gut microbiome-dependent therapeutics.

KLF15 regulates endobiotic and xenobiotic metabolism.

Hepatic metabolism and elimination of endobiotics (for example, steroids, bile acids) and xenobiotics (for example, drugs, toxins) is essential for health. While the enzymatic (termed phase I-II) and transport machinery (termed phase III) controlling endobiotic and xenobiotic metabolism (EXM) is known, understanding of molecular nodal points that coordinate EXM function in physiology and disease remains incomplete. Here we show that the transcription factor Kruppel-like factor 15 (KLF15) regulates all three phases of the EXM system by direct and indirect pathways. Unbiased transcriptomic analyses coupled with validation studies in cells, human tissues, and animals, support direct transcriptional control of the EXM machinery by KLF15. Liver-specific deficiency of KLF15 (Li-KO) results in altered expression of numerous phase I-III targets, and renders animals resistant to the pathologic effects of bile acid and acetaminophen toxicity. Furthermore, Li-KO mice demonstrate enhanced degradation and elimination of endogenous steroid hormones, such as testosterone and glucocorticoid, resulting in reduced male fertility and blood glucose levels, respectively. Viral reconstitution of hepatic KLF15 expression in Li-KO mice reverses these phenotypes. Our observations identify a previously unappreciated transcriptional pathway regulating metabolism and elimination of endobiotics and xenobiotics.

Toxicokinetics of Imidacloprid-Coated Wheat Seeds in Japanese Quail ( Coturnix japonica) and an Evaluation of Hazard.

Birds are potentially exposed to neonicotinoid insecticides by ingestion of coated seeds during crop planting. Adult male Japanese quail were orally dosed with wheat seeds coated with an imidacloprid (IMI) formulation at either 0.9 or 2.7 mg/kg body weight (BW) (∼3 and 9% of IMI LD50 for Japanese quail, respectively) for 1 or 10 days. Quail were euthanized between 1 and 24 h postexposure to assess toxicokinetics. Analysis revealed rapid absorption (1 h) into blood and distribution to the brain, muscle, kidney, and liver. Clearance to below detection limits occurred at both dose levels and exposure durations in all tissues within 24 h. Metabolism was extensive, with 5-OH-IMI and IMI-olefin detected at greater concentrations than IMI in tissues and fecal samples. There was no lethality or overt signs of toxicity at either dose level. Furthermore, no evidence of enhanced expression of mRNA genes associated with hepatic xenobiotic metabolism, oxidative DNA damage, or alterations in concentrations of corticosterone and thyroid hormones was observed. Application of the toxicokinetic data was used to predict IMI residue levels in the liver with reasonable results for some field exposure and avian mortality events. It would appear that some affected species of birds are either consuming larger quantities of seeds or exhibit differences in ADME or sensitivity than predicted by read-across from these data.

Dietary Iron Overload Abrogates Chemically-Induced Liver Cirrhosis in Rats.

Chronic liver disease is an intractable disease, which can progress to cirrhosis and hepatocellular carcinoma. Hepatic iron overload is considered to be involved in the progression of chronic liver diseases; however, the mechanism remains to be elucidated. Here we investigate the role of dietary iron overload using chemically-induced liver cirrhosis model. Rats were fed a high-iron or standard diet and were injected intraperitoneally with thioacetamide (TAA) or saline twice a week for 20 weeks. Rats with TAA treatment (TAA group) had progressive liver cirrhosis characterized by persistent hepatocellular injury, mononuclear cell inflammation and bridging fibrosis; these lesions were markedly reduced in rats with iron feeding and TAA treatment (Fe-TAA group). Rats with iron feeding alone (Fe group) had no evidence of liver injury. Hepatic expression of cleaved caspase-3, but not phospho-RIP3, was decreased in Fe-TAA group compared with that in TAA group. The number of TUNEL-positive (terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptotic hepatocytes was lower in the Fe-TAA group than in the TAA group. Hepatic xenobiotic metabolism and lipid peroxidation were shown to be less related to the abrogation of liver cirrhosis. Our results suggested that dietary hepatic iron overload abrogates chemically-induced liver cirrhosis in rats, which could partly involve decreased hepatocellular apoptosis.

Inhibition of cholinergic and non-cholinergic targets following subacute exposure to chlorpyrifos in normal and high fat fed male C57BL/6J mice

The effects of obesity on organophosphate pesticide-mediated toxicities, including both cholinergic and non-cholinergic targets, have not been fully elucidated. Therefore, the present study was designed to determine if high fat diet intake alters the effects of repeated exposure to chlorpyrifos (CPS) on the activities of both cholinergic and noncholinergic serine hydrolase targets. Male C57BL/6J mice were placed on either standard rodent chow or high fat diet for four weeks with CPS exposure (2.0 mg/kg) for the last 10 days of diet intake. Exposure to CPS did not alter acetylcholinesterase in the central nervous system, but it did significantly inhibit circulating cholinesterase activities in both diet groups. CPS significantly inhibited hepatic carboxylesterase and fatty acid amide hydrolase and this inhibition was significantly greater in high fat fed animals. Additionally, CPS exposure and high fat diet intake downregulated genes involved in hepatic de novo lipogenesis as well as cytochrome P450 enzymes involved in hepatic xenobiotic metabolism. In summary, the present study demonstrates that high fat diet intake potentiates CPS mediated inhibition of both carboxylesterase and fatty acid amide hydrolase in the liver of obese animals following subacute exposure and suggests obesity may be a risk factor for increased non-cholinergic hepatic CPS toxicity.

Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells’ activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug–drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.

Polychlorinated biphenyls disrupt hepatic epidermal growth factor receptor signaling

1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH).2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesized that PCBs may also diminish EGFR signaling.3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested.4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2–4 μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture.5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo.6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

Paternal nicotine exposure alters hepatic xenobiotic metabolism in offspring.

Paternal environmental conditions can influence phenotypes in future generations, but it is unclear whether offspring phenotypes represent specific responses to particular aspects of the paternal exposure history, or a generic response to paternal 'quality of life'. Here, we establish a paternal effect model based on nicotine exposure in mice, enabling pharmacological interrogation of the specificity of the offspring response. Paternal exposure to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring. This effect manifested as increased survival following injection of toxic levels of either nicotine or cocaine, accompanied by hepatic upregulation of xenobiotic processing genes, and enhanced drug clearance. Surprisingly, this protective effect could also be induced by a nicotinic receptor antagonist, suggesting that xenobiotic exposure, rather than nicotinic receptor signaling, is responsible for programming offspring drug resistance. Thus, paternal drug exposure induces a protective phenotype in offspring by enhancing metabolic tolerance to xenobiotics.

Dietary lipids differentially modulate the initiation of experimental breast carcinogenesis through their influence on hepatic xenobiotic metabolism and DNA damage in the mammary gland

Breast cancer is the most common malignancy among women worldwide. In addition to reproductive factors, environmental factors such as nutrition and xenobiotic exposure have a role in the etiology of this malignancy. A stimulating and a potentially protective effect on experimental breast cancer has been previously described for high corn oil and high extra-virgin olive oil diets, respectively. This work investigates the effect of these lipids on the metabolism of 7,12-dimethylbenz(a)anthracene (DMBA), a polycyclic aromatic hydrocarbon that can initiate carcinogenesis and its consequences in an experimental rat breast cancer model. The PUFA n-6-enriched diet increased expression of Phase I enzymes prior to DMBA administration and raised the activity of CYP1s in the hours immediately after induction, while reducing the activity of Phase II enzymes, mainly NQO1. The levels of reactive metabolites measured in plasma by GC–MS and DMBA-DNA adducts in the mammary gland of the animals fed the high corn oil diet were also higher than in the other groups. On the other hand, the high extra-virgin olive oil diet and the control low-fat diet exhibited better coordinated Phase I and Phase II activity, with a lower production of reactive metabolites and less DNA damage in the mammary gland. The concordance between these effects and the different efficacy of the carcinogenesis process due to the dietary treatment suggest that lipids may differently modify mammary gland susceptibility or resistance to cancer initiation over the exposure to environmental carcinogens. SummaryDietary lipids influence the initiation of DMBA-induced mammary cancer through the modulation of liver xenobiotic metabolism, formation of reactive metabolites and subsequent DNA damage in the target tissue.

Single Gene Inactivation with Implications to Diabetes and Multiple Organ Dysfunction Syndrome

Nutritional and environmental epigenetics are involved with the repression of anti-aging genes that are linked to the chronic disease epidemic. Unhealthy diets inactivate the calorie sensitive gene Sirtuin 1 (Sirt 1) involved in epigenetic processes that promote immune system alterations, mitochondrial apoptosis, Non-alcoholic Fatty Liver Disease (NAFLD), diabetes and Nitric Oxide (NO) modification with relevance to core body temperature involved with appetite regulation, glucose homeostasis and hepatic xenobiotic metabolism. The interplay between NO and epigenetics has attracted interest with relevance to autoimmune disease and mitophagy that has become of critical concern to diabetes and the development of MODS. Future research involved with nutritional research and the maintenance of Sirt 1 transcriptional control is critical to the prevention of MODS that is linked to the immune system and insulin resistance. In the developing world bacterial lipopolysaccharides a critical repressor of Sirt 1 is now involved with NAFLD and various organ diseases relevant to tissue accumulation of xenobiotics from various environments with relevance to MODS and the global chronic disease epidemic.

Insilco Modeling of Commonly Occurring Genetic Polymorphism of Human CYP3A4 on the Binding Affinity to UR-144

CYP3A4 accounts for about 30% in the human hepatic metabolism of xenobiotics. The defending mechanism of drug or xenobiotic metabolizing enzymes, particularly CYP superfamily has been found to be altered by both genetic polymorphisms and the environmental factors. The aim of this study was to examine UR-144 binding to CYP3A4 wild type and six different natural variants (I118V, R130Q, R162Q, D174H, T185S and L373F). A rigid ligand was docked by AutoDock Vina to flexible amino acid residues selected from the residues forming the binding pocket. The analysis of the docking results showed there is no difference in the binding affinities between the wild type and the natural variants. However, comparing the absolute binding affinity, the wild type (-12.8kcal/mol) has shown, among all, the lowest binding energy. Thus, this study depicts that the SNPs of CYP3A4 do not have any effects on the binding affinity of UR-144 to the active site.

Antiproliferation of berberine is mediated by epigenetic modification of constitutive androstane receptor (CAR) metabolic pathway in hepatoma cells

Constitutive androstane receptor (CAR) regulates hepatic xenobiotic and energy metabolism, as well as promotes cell growth and hepatocarcinogenesis. Berberine is an ancient multipotent alkaloid drug which derived from Coptis chinensis plants. Here we report that berberine is able to be cellular uptake and accessible to chromatin in human hepatoma HepG2 cells. Berberine induces more apoptosis, cell cycle arrest, but less ROS production in CAR overexpressed mCAR-HepG2 cells. Moreover, berberine inhibits expressions of CAR and its target genes CYP2B6 and CYP3A4. Furthermore, berberine enhances DNA methylation level in whole genome but reduces that in promoter regions CpG sites of CYP2B6 and CYP3A4 genes under the presence of CAR condition. These results indicated that the antiproliferation of berberine might be mediated by the unique epigenetic modifying mechanism of CAR metabolic pathway, suggesting that berberine is a promising candidate in anticancer adjuvant chemotherapy, due to its distinct pharmacological properties in clinic.

Hepatocytes as in vitro test system to investigate metabolite patterns of pesticides in farmed rainbow trout and common carp: Comparison between in vivo and in vitro and across species

In vitro tools using isolated primary fish hepatocytes have been proposed as a useful model to study the hepatic metabolism of xenobiotics in fish. In order to evaluate the potential of in vitro fish hepatocyte assays to provide information on in vivo metabolite patterns of pesticides in farmed fish, the present study addressed the following questions: Are in vitro and in vivo metabolite patterns comparable? Are species specific differences of metabolite patterns in vivo reflected in vitro? Are metabolite patterns obtained from cryopreserved hepatocytes comparable to those from freshly isolated cells? Rainbow trout and common carp were dosed orally with feed containing the pesticide methoxychlor (MXC) for 14days. In parallel, in vitro incubations using suspensions of freshly isolated or cryopreserved primary hepatocytes obtained from both species were performed. In vivo and in vitro samples were analyzed by thin-layer chromatography with authentic standards supported by HPLC-MS. Comparable metabolite patterns from a qualitative perspective were observed in liver in vivo and in hepatocyte suspensions in vitro. Species specific differences of MXC metabolite patterns observed between rainbow trout and common carp in vivo were well reflected by experiments with hepatocytes in vitro. Finally, cryopreserved hepatocytes produced comparable metabolite patterns to freshly isolated cells. The results of this study indicate that the in vitro hepatocyte assay could be used to identify metabolite patterns of pesticides in farmed fish and could thus serve as a valuable tool to support in vivo studies as required for pesticides approval according to the EU regulation 1107.

Disruption of redox homeostasis and carcinogen metabolizing enzymes changes by administration of vitamin E to rats

AimsA large meta-analysis of randomized clinical trials has seriously questioned chemoprevention based on vitamins including vitamin E (VE), and an increased risk for cancer among long-term users was actually seen. However, the mechanism underlying these findings still remain unknown. To clarify the mechanism, in an in vivo model we studied the putative disruption of redox homeostasis and the perturbation of carcinogen metabolizing enzymes determined by VE. Main methodsMale Sprague–Dawley rats were treated ip with either 100 or 200mg/kg b.w. daily for 7 or 14 consecutive days. Controls received vehicle only. Cytochrome P450 (CYP) content, CYP-reductase, CYP-linked monooxygenases, as well as phase-II and the antioxidant enzymes catalase and NAD(P)H:quinone reductase were investigated in both liver and kidney. Free radical species in tissue subcellular preparations were measured by electronic paramagnetic resonance (EPR) spectroscopy coupled to a radical probe technique. Key findingsNo substantial changes of hepatic xenobiotic metabolism enzymes were determined by VE. Conversely, a powerful booster effect of various renal phase-I carcinogen bioactivating enzymes at both dosages and observational times was recorded. While no relevant changes of post-oxidative phase-II reactions were found in the liver, a significant inactivating effect was caused by VE in renal tissues. Antioxidant enzymes were found mainly downregulated by the treatment. In the kidney, a marked free radical over-generation linked to CYP induction was observed. SignificanceThis study proved that VE acts as a co-carcinogen and pro-oxidant agent. Such epigenetic mechanisms may contribute to explain the harmful outcomes observed in humans.