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Many people convicted for drunken driving suffer from an alcohol use disorder and some traffic offenders consume denatured alcohol for intoxication purposes. Venous blood samples from people arrested for driving under the influence of alcohol were analyzed in triplicate by headspace gas chromatography (HS-GC) using three different stationary phases. The gas chromatograms from this analysis sometimes showed peaks with retention times corresponding to acetone, ethyl methyl ketone (2-butanone), 2-propanol, and 2-butanol in addition to ethanol and the internal standard (1-propanol). Further investigations showed that these drink-driving suspects had consumed an industrial alcohol (T-Red) for intoxication purposes, which contained > 90% w/v ethanol, acetone (~ 2% w/v), 2-butanone (~ 5% w/v) as well as Bitrex to impart a bitter taste. In n = 75 blood samples from drinkers of T-Red, median concentrations of ethanol, acetone, 2-butanone, 2-propanol and 2-butanol were 2050 mg/L (2.05 g/L), 97 mg/L, 48 mg/L, 26 mg/L and 20 mg/L, respectively. In a separate GC analysis, 2,3-butanediol (median concentration 87 mg/L) was identified in blood samples containing 2-butanone. When the redox state of the liver is shifted to a more reduced potential (excess NADH), which occurs during metabolism of ethanol, this favors the reduction of low molecular ketones into secondary alcohols via the alcohol dehydrogenase (ADH) pathway. Routine toxicological analysis of blood samples from apprehended drivers gave the opportunity to study metabolism of acetone and 2-butanone without having to administer these substances to human volunteers. [ABSTRACT FROM AUTHOR]

With more than 80 cytochrome P450 (CYP) encoding genes found in the nematode Caenorhabditis elegans (C. elegans), the cyp35 genes are one of the important genes involved in many biological processes such as fatty acid synthesis and storage, xenobiotic stress response, dauer and eggshell formation, and xenobiotic metabolism. The C. elegans CYP35 subfamily consisted of A, B, C, and D, which have the closest homolog to human CYP2 family. C. elegans homologs could answer part of the hunt for human disease genes. This review aims to provide an overview of CYP35 in C. elegans and their human homologs, to explore the roles of CYP35 in various C. elegans biological processes, and how the genes of cyp35 upregulation or downregulation are influenced by biological processes, upon exposure to xenobiotics or changes in diet and environment. The C. elegans CYP35 gene expression could be upregulated by heavy metals, pesticides, anti-parasitic and anti-chemotherapeutic agents, polycyclic aromatic hydrocarbons (PAHs), nanoparticles, drugs, and organic chemical compounds. Among the cyp35 genes, cyp-35A2 is involved in most of the C. elegans biological processes regulation. Further venture of cyp35 genes, the closest homolog of CYP2 which is the largest family of human CYPs, may have the power to locate cyps gene targets, discovery of novel therapeutic strategies, and possibly a successful medical regime to combat obesity, cancers, and cyps gene-related diseases. [ABSTRACT FROM AUTHOR]

Several N-vinyl compounds are produced in high volumes and are widely employed in the production of copolymers and polymers used in chemical, pharmaceutical, cosmetic and food industry. Hence, information on their genotoxicity and carcinogenicity is requisite. This review presents hitherto available information on the carcinogenicity and genotoxicity of N-vinyl compounds as well as their metabolism potentially generating genotoxic and carcinogenic derivatives. The genotoxicity and carcinogenicity of the investigated N-vinyl compounds vary widely from no observed carcinogenicity tested in lifetime bioassays in two rodent species (up to very high doses) to carcinogenicity in rats at very low doses in the absence of apparent genotoxicity. Despite of the presence of the vinyl group potentially metabolized to an epoxide followed by covalent binding to DNA, genotoxicity was observed for only one of the considered N-vinyl compounds, N-vinyl carbazole. Carcinogenicity was investigated only for two, of which one, N-vinyl pyrrolidone was carcinogenic (but not genotoxic) and ranitidine was neither carcinogenic nor genotoxic. As far as investigated, neither a metabolically formed epoxide nor a therefrom derived diol has been reported for any of the considered N-vinyl compounds. It is concluded that the information collected in this review will further the understanding of the carcinogenic potentials of N-vinyl compounds and may eventually allow approaching their prediction and prevention. A suggestion how to prevent genotoxicity in designing of N-vinyl compounds is presented. However, the available information is scarce and further research especially on the metabolism of N-vinyl compounds is highly desirable. [ABSTRACT FROM AUTHOR]

Given the strong trend to implement zebrafish (Danio rerio) embryos as translational model not only in ecotoxicological, but also toxicological testing strategies, there is an increasing need for a better understanding of their capacity for xenobiotic biotransformation. With respect to the extrapolation of toxicological data from zebrafish embryos to other life stages or even other organisms, qualitative and quantitative differences in biotransformation pathways, above all in cytochrome P450-dependent (CYP) phase I biotransformation, may lead to over- or underestimation of the hazard and risk certain xenobiotic compounds may pose to later developmental stages or other species. This review provides a comprehensive state-of-the-art overview of the scientific knowledge on the development of the CYP1-4 families and corresponding phase I biotransformation and bioactivation capacities in zebrafish. A total of 68 publications dealing with spatiotemporal CYP mRNA expression patterns, activities towards mammalian CYP-probe substrates, bioactivation and detoxification activities, as well as metabolite profiling were analyzed and included in this review. The main results allow for the following conclusions: (1) Extensive work has been done to document mRNA expression of CYP isoforms from earliest embryonic stages of zebrafish, but juvenile and adult zebrafish have been largely neglected so far. (2) There is insufficient understanding of how sex- and developmental stage-related differences in expression levels of certain CYP isoforms may impact biotransformation and bioactivation capacities in the respective sexes and in different developmental stages of zebrafish. (3) Albeit qualitatively often identical, many studies revealed quantitative differences in metabolic activities of zebrafish embryos and later developmental stages. However, the actual relevance of age-related differences on the outcome of toxicological studies still needs to be clarified. (4) With respect to current remaining gaps, there is still an urgent need for further studies systematically assessing metabolic profiles and capacities of CYP isoforms in zebrafish. Given the increasing importance of Adverse Outcome Pathway (AOP) concepts, an improved understanding of CYP capacities appears essential for the interpretation and outcome of (eco)toxicological studies. [ABSTRACT FROM AUTHOR]

Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models. [ABSTRACT FROM AUTHOR]

Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes. [ABSTRACT FROM AUTHOR]

The xenobiotic metabolism in the lung, an organ of first entry of xenobiotics into the organism, is crucial for inhaled compounds entering this organ intentionally (e.g. drugs) and unintentionally (e.g. work place and environmental compounds). Additionally, local metabolism by enzymes preferentially or exclusively occurring in the lung is important for favorable or toxic effects of xenobiotics entering the organism also by routes other than by inhalation. The data collected in this review show that generally activities of cytochromes P450 are low in the lung of all investigated species and in vitro models. Other oxidoreductases may turn out to be more important, but are largely not investigated. Phase II enzymes are generally much higher with the exception of UGT glucuronosyltransferases which are generally very low. Insofar as data are available the xenobiotic metabolism in the lung of monkeys comes closed to that in the human lung; however, very few data are available for this comparison. Second best rate the mouse and rat lung, followed by the rabbit. Of the human in vitro model primary cells in culture, such as alveolar macrophages and alveolar type II cells as well as the A549 cell line appear quite acceptable. However, (1) this generalization represents a temporary oversimplification born from the lack of more comparable data; (2) the relative suitability of individual species/models is different for different enzymes; (3) when more data become available, the conclusions derived from these comparisons quite possibly may change. [ABSTRACT FROM AUTHOR]

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time. [ABSTRACT FROM AUTHOR]

Microsomal epoxide hydrolase (mEH) is a detoxifying enzyme for xenobiotic compounds. Enzymatic activity of mEH can be greatly increased by a point mutation, leading to an E404D amino acid exchange in its catalytic triad. Surprisingly, this variant is not found in any vertebrate species, despite the obvious advantage of accelerated detoxification. We hypothesized that this evolutionary avoidance is due to the fact that the mEH plays a dualistic role in detoxification and control of endogenous vascular signaling molecules. To test this, we generated mEH E404D mice and assessed them for detoxification capacity and vascular dynamics. In liver microsomes from these mice, turnover of the xenobiotic compound phenanthrene-9,10-oxide was four times faster compared to WT liver microsomes, confirming accelerated detoxification. mEH E404D animals also showed faster metabolization of a specific class of endogenous eicosanoids, arachidonic acid-derived epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Significantly higher DHETs/EETs ratios were found in mEH E404D liver, urine, plasma, brain and cerebral endothelial cells compared to WT controls, suggesting a broad impact of the mEH mutant on endogenous EETs metabolism. Because EETs are strong vasodilators in cerebral vasculature, hemodynamics were assessed in mEH E404D and WT cerebral cortex and hippocampus using cerebral blood volume (CBV)-based functional magnetic resonance imaging (fMRI). Basal CBV levels were similar between mEH E404D and control mice in both brain areas. But vascular reactivity and vasodilation in response to the vasodilatory drug acetazolamide were reduced in mEH E404D forebrain compared to WT controls by factor 3 and 2.6, respectively. These results demonstrate a critical role for mEH E404D in vasodynamics and suggest that deregulation of endogenous signaling pathways is the undesirable gain of function associated with the E404D variant. [ABSTRACT FROM AUTHOR]

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics can have a major impact in multiple research fields, especially when combined with other technologies, such as stable isotope tracers and genetically modified mice. This review highlights recent applications of metabolomic technology in the study of xenobiotic metabolism and toxicity, and the understanding of disease pathogenesis and therapeutics. Metabolomics has been employed to study metabolism of noscapine, an aryl hydrocarbon receptor antagonist, and to determine the mechanisms of liver toxicities of rifampicin and isoniazid, trichloroethylene, and gemfibrozil. Metabolomics-based insights into the pathogenesis of inflammatory bowel disease, alcohol-induced liver diseases, non-alcoholic steatohepatitis, and farnesoid X receptor signaling pathway-based therapeutic target discovery will also be discussed. Limitations in metabolomics technology such as sample preparation and lack of LC-MS databases and metabolite standards, need to be resolved in order to improve and broaden the application of metabolomic studies. [ABSTRACT FROM AUTHOR]

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure. [ABSTRACT FROM AUTHOR]

The metabolic competence of cultured bovine colon epithelial cells was evaluated by determining activities of phase I and II enzymes in colonocytes cultured for different intervals (maximum of 10 days) compared with activities measured in freshly isolated cells. Cytochrome P450 1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity was detectable in freshly isolated colonocytes and in colon cells maintained in culture for up to 5 days. In contrast to liver samples, cytochrome P450 3A4-associated 7-benzyloxyresorufin O-debenzylase (BROD) activity was not detectable in bovine colon cells. Prostaglandin H synthase-mediated production of prostaglandin E2 was found in freshly isolated and also in cultured colonocytes. Both isoenzymes (COX 1 and COX 2) were detected in cultured cells. To examine phase II metabolic potency, activities of N-acetyltransferases 1 and 2, of phenol and amino sulfotransferases, of glutathione S-transferases alpha, mu, pi and theta and of UDP-glucuronyltransferase were measured. N-Acetyltransferase (NAT) activity (substrate p-aminobenzoic acid, PABA, a diagnostic substrate for the human NAT-1 enzyme) was stable under culture conditions and during the observed culture period comparable to that of freshly isolated cells. In contrast, sulfamethazine, a specific substrate for NAT-2, was not acetylated, neither in bovine colon cells nor in bovine liver samples. Whereas activity of amino sulfotransferase (substrate 2-naphthylamine) decreased continuously during the entire culture period, the activity of phenol sulfotransferase (substrate 1-naphthol) decreased only slowly. Activity of total glutathione S-transferases (alpha, mu, and pi) (substrate 1-chloro-2,4-dinitrobenzene) decreased after 2 days in culture, but was stable during the following culture period. Activity of glutathione S-transferase theta (substrate epoxy-3-nitrophenoxypropane) changed during the culture period. At the beginning and the end (after 10 days) of the culture period maximum activity was measured. Activity of UDP-glucuronyltransferase increased during the culture period reaching a maximum after 7 days. The results show that cultured bovine epithelial colon cells express several enzyme activities required for the biotransformation of xenobiotics. [ABSTRACT FROM AUTHOR]

Drug metabolizing enzyme activities have been determined in cultured porcine urinary bladder epithelial cells (PUBEC) in order to evaluate this system as an in vitro model for studies of urinary bladder carcinogens. Activities of several phase I and II enzymes were measured in cells cultured for various periods and compared with the activities determined in freshly isolated PUBEC. Prostaglandin H synthase mediated production of prostaglandin E was found both in freshly isolated and in cultured PUBEC, whereas cytochrome P450 1A1-associated EROD activity was only detectable in freshly isolated bladder cells. The latter activity was not inducible by benz( a)anthracene or 3-methylcholanthrene in PUBEC cultures. N-acetyltransferase (NAT) activity measured with p-aminobenzoic acid, a diagnostic substrate for human NAT-1, was stable and even higher during the culture period compared to freshly isolated cells. In contrast, isoniazid (a substrate for NAT-2) was not acetylated either in fresh or cultured PUBEC. Glutathione S-transferases activity determined with 1-chloro-2,4-dinitrobenzene decreased gradually to 50% after 1 week and to 20% after 4 weeks in culture compared to fresh cells. A similar decline was also observed for UDP-glucuronyltransferase activities measured with 1-naphthol. In accordance with the reported lack of sulfotransferases in pigs, no sulfation of 1-naphthol or 2-naphthylamine was detected in PUBEC. Our results show that cultured porcine urinary bladder epithelial cells maintain several enzyme activities required for the biotransformation of xenobiotics. In future investigations on the mechanism of action of bladder carcinogens PUBEC cultures may thus provide a useful in vitro model for this target tissue. [ABSTRACT FROM AUTHOR]