Xenobiotic-metabolizing Activities Research Articles

Insights into the differences related to the resistance mechanisms to the highly toxic fruit Hippomane mancinella (Malpighiales: Euphorbiaceae) between the larvae of the sister species Anastrepha acris and Anastrepha ludens (Diptera: Tephritidae) through comparative transcriptomics.

The Manchineel, Hippomane mancinella ("Death Apple Tree") is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites.

Compositional variation of the human fecal microbiome in relation to azo-reducing activity: a pilot study

BackgroundThrough an arsenal of microbial enzymes, the gut microbiota considerably contributes to human metabolic processes, affecting nutrients, drugs, and environmental poisons. Azoreductases are a predominant group of microbiota-derived enzymes involved in xenobiotic metabolism and drug activation, but little is known about how compositional changes in the gut microbiota correlate with its azo-reducing activity.ResultsTo this end, we used high-throughput 16S rRNA amplicon sequencing, with Illumina MiSeq, to determine the microbial community composition of stool samples from 16 adults with different azo-reducing activity. High azo-reducing activity positively correlated with the relative abundance of phylum Firmicutes (especially genera Streptococcus and Coprococcus) but negatively with phylum Bacteroidetes (especially genus Bacteroides). Typical variations in the Firmicutes-to-Bacteroidetes and Prevotella-to-Bacteroides ratios were observed among samples. Multivariate analysis of the relative abundance of key microbial taxa and other diversity parameters confirmed the Firmicutes proportion as a major variable differentiating high and non-azo-reducers, while Bacteroidetes relative abundance was correlated with azo-reduction, sex, and BMI.ConclusionsThis pilot study showed that stool samples with higher azo-reducing activity were enriched in Firmicutes but with relatively fewer Bacteroidetes. More samples and studies from different geographical areas are needed to bolster this conclusion. Better characterization of different azoreductase-producing gut microbes will increase our knowledge about the fate and differential human responses to azodye-containing drugs or orally consumed chemicals, thus contributing to efforts towards implementing microbiome testing in precision medicine and toxicology.

Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Engineering glutathione S-transferase with a point mutation at conserved F136 residue increases the xenobiotic-metabolizing activity

Glutathione S-transferases (GSTs) are multifunctional enzymes that play major roles in a wide range of biological processes, including cellular detoxification, biosynthesis, metabolism, and transport. The dynamic structural scaffold and diverse functional roles of GSTs make them important for enzyme engineering and for exploring novel biotechnological applications. The present study reported a significant gain-of-function activity in GST caused by a point mutation at the conserved F136 residue. The fluorescence quenching and kinetic data suggested that both binding affinity and catalytic efficiency of the mutant enzyme to the substrates 1-chloro-2,4-dinitrobenzene (CDNB), as well as the glutathione (GSH), is increased. Molecular docking showed that the mutation improves the binding interactions of the GSH with several binding-site residues. The simulation of molecular dynamics revealed that the mutant enzyme gained increased structural rigidity than the wild-type enzyme. The mutation also altered the residue interaction network (RIN) of the GSH-binding residues. These phenomena suggested that mutations led to conformational alterations and dominant differential motions in the enzyme that lead to increased rigidity and modifications in RIN. Collectively, engineering GST with a single point mutation at conserved F136 can significantly increase its xenobiotic activity by increasing the catalytic efficiency that may be exploited for biotechnological applications.

Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitro has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

The constitution and functional prediction of the microbiota in necrotizing enterocolitis with a gestational age of over 28 weeks.

Background:To explore the features and function of gut microbiota in necrotizing enterocolitis patients over 28 gestational age weeks through a case-control study.Methods:Fecal samples from patients with NEC over 28 gestational week age and matched control cases were collected. DNA of the fecal samples was extracted for 16 s rRNA sequencing to estimate the composition of the microbiota. Functional inference analyses were conducted through PICRUSt based on the sequencing raw data.Results:There was no significant difference in the total diversity of microbiota between the fecal samples from the patients with NEC and the controls (P = .40). Propionibacterium was more abundant in the NEC cases than in the controls. Conversely, Lactobacillus, Phascolarctobacterium, and Streptococcus_salivarius were found to be more plentiful in the controls through LEfSe analysis. Functional inference analysis revealed that the xenobiotic biodegradation and metabolic activity was lower in the NEC cases than in the controls (P < .05).Conclusion:The NEC cohort with a gestational age of over 28 weeks has a different pattern of microbiota compared with the controls. Functional inference analysis indicated that the potential function of the microbiota may also differ between these groups.

Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing.

The evaluation of genotoxicity plays an important role within hazard identification and risk assessment of chemicals and consumer products. For genotoxicity assessment, in vitro hepatic cells are often used as they have retained certain level of xenobiotic metabolic activity. However, current protocols are designed for the use on 2D monolayer models that are associated with several limitations due to the lack of numerous biological functions, which results in the loss of many hepatic properties. In this respect, an attractive alternative are three-dimensional (3D) models. The aim of our study was to develop physiologically more relevant 3D cell model (spheroids) from the human hepatocellular carcinoma (HepG2) cell line for genotoxicity testing. The spheroids were prepared by the forced floating method, which had been optimized for the production of a large number of uniform spheroids. The sensitivity of the spheroids to detect genotoxicity was determined by the comet assay after the exposure of spheroids to non-cytotoxic concentrations of model indirect acting genotoxic compounds, namely polycyclic aromatic hydrocarbon (B(a)P), mycotoxin (AFB1), two heterocyclic aromatic amines (PhIP and IQ) and a direct acting etoposide (ET). All five tested compounds concentration dependently induced DNA damage. Higher sensitivity of 3D cell model compared to 2D monolayer culture was noticed particularly for detection of the genotoxicity of the heterocyclic aromatic amines and BaP. Deregulation of mRNA expression (qPCR) by genotoxic compounds revealed that HepG2 cells in 3D express important genes encoding phase I and II metabolic enzymes, as well as DNA damage responsive genes in an inducible form. The newly developed HepG2 3D model shows improved sensitivity for detecting genotoxic compounds compared to 2D cultures and can provide a suitable experimental model for genotoxicity assessment.

NPA: an R package for computing network perturbation amplitudes using gene expression data and two-layer networks

BackgroundHigh-throughput gene expression technologies provide complex datasets reflecting mechanisms perturbed in an experiment, typically in a treatment versus control design. Analysis of these information-rich data can be guided based on a priori knowledge, such as networks of related proteins or genes. Assessing the response of a specific mechanism and investigating its biological basis is extremely important in systems toxicology; as compounds or treatment need to be assessed with respect to a predefined set of key mechanisms that could lead to toxicity. Two-layer networks are suitable for this task, and a robust computational methodology specifically addressing those needs was previously published.The NPA package (https://github.com/philipmorrisintl/NPA) implements the algorithm, and a data package of eight two-layer networks representing key mechanisms, such as xenobiotic metabolism, apoptosis, or epithelial immune innate activation, is provided.ResultsGene expression data from an animal study are analyzed using the package and its network models. The functionalities are implemented using R6 classes, making the use of the package seamless and intuitive. The various network responses are analyzed using the leading node analysis, and an overall perturbation, called the Biological Impact Factor, is computed.ConclusionsThe NPA package implements the published network perturbation amplitude methodology and provides a set of two-layer networks encoded in the Biological Expression Language.

Sensitization of Human Liver Cells Toward Fas-Mediated Apoptosis by the Metabolically Activated Pyrrolizidine Alkaloid Lasiocarpine.

Pyrrolizidine alkaloids (PAs) are common phytotoxins. Intoxication can lead to liver damage. Previous studies showed PA-induced apoptosis in liver cells. However, the exact role of the extrinsic apoptotic pathway has not been investigated yet. This study aims to analyze whether the PA representative lasiocarpine sensitizes human liver cells toward extrinsic Fas-mediated apoptosis. HepG2 cells with limited xenobiotic metabolic activity are used to analyze metabolism-dependent effects. External in vitro metabolism is simulated using rat or human liver enzymes. Additionally, metabolically competent HepaRG cells are used to confirm the observed effects in a human liver cell system with internal xenobiotic metabolism. Metabolized lasiocarpine decreases cell viability and induces Fas receptor gene expression in both cell lines. Increased Fas receptor protein expression on the cell surface is demonstrated by flow cytometry. The addition of a Fas ligand-simulating antibody induces apoptosis. Induction of extrinsic Fas-mediated apoptosis is verified by Western blotting for cleaved caspase 8, the initiator caspase of extrinsic apoptosis. All effects are dependent on lasiocarpine metabolism. The results demonstrate that metabolically metabolized lasiocarpine sensitizes human liver cells toward Fas-mediated apoptosis. They broaden our knowledge on the hepatotoxic molecular mechanisms of PA as widely distributed food contaminants.

The Effect of Environmental Enrichment on Glutathione-Mediated Xenobiotic Metabolism and Antioxidation in Normal Adult Mice.

Olfactory bulb (OB) plays an important role in protecting against harmful substances via the secretion of antioxidant and detoxifying enzymes. Environmental enrichment (EE) is a common rehabilitation method and known to have beneficial effects in the central nervous system. However, the effects of EE in the OB still remain unclear. At 6 weeks of age, CD-1® (ICR) mice were assigned to standard cages or EE cages. After 2 months, we performed proteomic analysis. Forty-four up-regulated proteins were identified in EE mice compared to the control mice. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes Pathway demonstrated that the upregulated proteins were mainly involved in metabolic pathways against xenobiotics. Among those upregulated proteins, 9 proteins, which participate in phase I or II of the xenobiotic metabolizing process and are known to be responsible for ROS detoxification, were validated by qRT-PCR. To explore the effect of ROS detoxification mediated by EE, glutathione activity was measured by an ELISA assay. The ratio of reduced glutathione to oxidized glutathione was significantly increased in EE mice. Based on a linear regression analysis, GSTM2 and UGT2A1 were found to be the most influential genes in ROS detoxification. For further analysis of neuroprotection, the level of iNOS and the ratio of Bax to Bcl-2 were significantly decreased in EE mice. While TUNEL+ cells were significantly decreased, Ki67+ cells were significantly increased in EE mice, implicating that EE creates an optimal state for xenobiotic metabolism and antioxidant activity. Taken together, our results suggested that EE protects olfactory layers via the upregulation of glutathione-related antioxidant and xenobiotic metabolizing enzymes, eventually lowering ROS-mediated inflammation and apoptosis and increasing neurogenesis. This study may provide an opportunity for a better understanding of the beneficial effects of EE in the OB.

Filling the gaps: need for research on cell-specific xenobiotic metabolism in the skin

The removal and metabolism of organic molecules is apivotal body function, usually performed by phase I andphase II enzymes. While detoxification is beneficial andnecessary, at the same time, the metabolic intermediatesand breakdown products generated in the process can haveimmunogenic, cancerogenic or apoptogenic potential,which differs from the respective parental chemical.Whether and which adverse health effects ensue dependson the affected cell types and tissue. For instance, ifdamage caused by a chemical induces apoptosis, easilyregenerating tissues may cope better than slowly prolifer-ating or rare precursor cells. On the other hand, potentialextent of damage is conceivably dependent on the xeno-biotic metabolizing activity of cell types, which in turnmight be governed by exposure, that is, tissues with highexposure could be more competent in xenobiotic metabo-lism. There is, however, a surprising lack of research toidentify cell-specific xenobiotic metabolism. Research onthe molecular activity of xenobiotic metabolizing enzymes(XME) has traditionally a strong focus on the liver as themain metabolizing and detoxifying organ of the body. Theskin is less well studied, albeit it forms a major barrieragainst the environment (including the chemical environ-ment) and is a target of occupational, accidental andintended-use (i.e., cosmetics and pharmaceuticals) chemi-cal exposure. Risks of exposure are irritation, inflamma-tion, allergies and cancer. Conceivably, skin cells maypossess yet underestimated strategies to balance XME-mediated danger.In a recent paper, the team around Michael Girardi(Modi et al. 2012) presented the exciting finding that dif-ferential expression of phase I enzymes CYP4501A1 and1B1 in skin Langerhans cells versus keratinocytes leads togeneration of DNA-modifying metabolites from the car-cinogen 7,12-Dimethylbenz[a]anthracen (DMBA) in theskin and eventually non-melanoma skin cancer. Theyfound that skin Langerhans cells efficiently metabolizeDMBA to DMBA-3,4-diol which leads to oncogenic Hrasmutations. Mice lacking skin Langerhans cells had fewerskin tumours upon DMBA treatment. They conclude‘‘ Thus, tissue-associated dendritic cells can enhancechemical carcinogenesis via PAH metabolism, highlightingthe complex relation between immune cells and carcino-genesis.’’ While the paper of Girardi and colleagues looksat chemical-induced (DMBA-induced) cancer, it has alsoimportant implications for understanding skin physiologyand skin toxicology.Sun light, that is, its UV radiation component, is themost important stressor of skin (Fritsche et al. 2007) andhas long been known to be genotoxic and a major riskfactor in skin cancer (Narayanan et al. 2010). UV and UV-induced DNA damage are the primary cause not only formelanoma with increasing incidence and new casesworldwide every year (Little and Eide 2012), but also ofskin basal cell carcinoma, squamous cell carcinoma,Kaposi

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

Organic solvents, such as dimethylsulfoxide (DMSO) and methanol are widely used as vehicles to solubilise lipophilic test compounds in toxicity testing. However, the effects of such solvents upon innate detoxification processes in aquatic organisms are poorly understood. This study assessed the effect of solvent exposure upon cytochrome P450 (CYP)-mediated xenobiotic metabolism in Daphnia magna and zebrafish larvae (4d post fertilisation). Adult D. magna were demonstrated to have a low, but detectable, metabolism of ethoxyresorufin in vivo and this activity was not modulated by pre-exposure to DMSO or methanol (24h, up to 0.1% and 0.05% v/v, respectively). In contrast, the metabolism of ethoxyresorufin in zebrafish larvae was significantly reduced by both solvents (0.1% and 0.05% v/v, respectively) after 24h of exposure. In zebrafish, these observed decreases in activity towards ethoxyresorufin were accompanied by decreased expression of a variety of genes coding for drug metabolising enzymes (corresponding to CYP1, CYP2, CYP3 and UDP-glucuronyl transferase [UGT] family enzymes), measured by quantitative PCR. Reduction of gene expression and CYP1 enzyme activities by methanol (0.05% v/v) in zebrafish larvae was partially reversed by co-exposure with Aroclor 1254 (100μgL−1). Overall this study suggests that relatively low concentrations of organic solvents can impact upon the biotransformation of certain xenobiotics in zebrafish larvae, and that this warrants consideration when assessing compounds for metabolism and toxicity in this species.

Temporal patterns in the transcriptomic response of rainbow trout, Oncorhynchus mykiss, to crude oil

Time is often not characterized as a variable in ecotoxicogenomic studies. In this study, temporal changes in gene expression were determined during exposure to crude oil and a subsequent recovery period. Juvenile rainbow trout, Oncorhynchus mykiss, were exposed for 96 h to the water accommodated fractions of 0.4, 2 or 10 mg l −1 crude oil loadings. Following 96 h of exposure, fish were transferred to recovery tanks. Gill and liver samples were collected after 24 and 96 h of exposure, and after 96 h of recovery for RNA extraction and microarray analysis. Fluorescently labeled cDNA was hybridized against matched controls, using salmonid cDNA arrays. Each exposure scenario generated unique patterns of altered gene expression. More genes responded to crude oil in the gill than in the liver. In the gill, 1137 genes had altered expression at 24 h, 2003 genes had altered expression levels at 96 h of exposure, yet by 96 h of recovery, no genes were significantly altered in expression. In the liver at 10 mg l −1, only five genes were changed at 24 h, yet 192 genes had altered expression after 96 h recovery. At 2 mg l −1 in the liver, many genes had altered regulation at all three time points. The 0.4 mg l −1 loading also showed 289 genes upregulated at 24 h after exposure. The Gene Ontology terms associated with altered expression in the liver suggested that the processes of protein synthesis, xenobiotic metabolism, and oxidoreductase activity were altered. The concentration-responsive expression profile of cytochrome P450 1A, a biomarker for oil exposure, did not predict the majority of gene expression profiles in any tissue or dose, since direct relationships with dose were not observed for most genes. While the genes and their associated functions agree with known modes of toxic action for crude oil, the gene lists obtained do not match our previously published work, presumably due to array analysis procedures. These results demonstrate that changes in gene expression with time and dose may be complicated, and should be characterized in controlled laboratory settings before attempts are made to interpret responses in field-collected organisms. Further, processes for analyzing microarray data need to be developed such that standardized gene lists are developed, or that analysis does not rely on lists of significantly altered genes before arrays can be further evaluated as a monitoring tool.

Effects of semi-static exposition to carbofuran on xenobiotic metabolizing activities in common carp (Cyprinus carpio)

Effects of semi-static exposition to carbofuran on xenobiotic metabolizing activities in common carp (Cyprinus carpio)

Abstract 1860: Association of esophageal cancer risk with genetic polymorphisms of xenobiotic metabolizing genes, DNA repair genes, and inflammation-regulating genes in Huaian, China

Abstract Accumulating evidences suggest that both exposure to environmental carcinogens and individual susceptibility factors contribute to the formation of esophageal cancer. Huaian City is one of the highest risk areas of esophageal cancer in China with the annual incidence over 100/100,000 people. To explore etiological risk factors contributing to the high rate of esophageal cancer, a population-based case-control study was conducted in 185 esophageal squamous cell carcinoma (ESCC) cases and 515 age-, gender-, and residency-matched healthy controls in Huaian. Our previous study found an association between dietary fumonisin exposure and ESCC risk in this population. In the present study, we further investigated the association between ESCC and polymorphic genes involving in xenobiotic metabolizing activities, DNA repair, and inflammation regulation including GSTM1, GSTT1, XPD (Lys751Gln), hOGG1 (Ser326Cys), TNF-α (G308A), NF-κB (−94ins/delATTG), COX-2 (G765C), and iNOS (Ser608Leu). Genetic polymorphisms of GSTM1 and GSTT1 were determined by multiplex PCR and other polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP) analysis using genomic DNA samples obtained from lymphocytes of study subjects. The prevalence of GSTT1 null genotype was significantly higher in cases (53.51%) than in controls (41.94%) (p=0.0067), with an age- and gender-adjusted odds ratio (OR) of 1.68 and 95% confidence interval (CI) of 1.18 − 2.39. After stratifying by gender, GSTT1 null type was found more prevalent in male cases (57.14%) than in male controls (42.64%) (p=0.0117). XPD Lys751Gln polymorphism was associated with increased ESCC risk (age- and gender-adjusted OR=1.74, 95% CI: 1.11-2.74). XPD heterozygous and mutant genotypes (Lys/Gln+Gln/Gln) were more prevalent in cases (20.54%) than in controls (14.26%) (p=0.0453), and in female cases (24.32%) than in female controls (13.01%) (p=0.0187). Heterozygous genotype of TNF-α was found in 12.97% of cases and 7.65% of controls, showing a marginal increased risk of ESCC (adjusted OR=1.68, 95% CI: 0.95-2.98). These results suggest that polymorphisms of GSTT1, XPD and TNF-α gene may contribute to ESCC risk in this high-risk population. (Supported by NIH/NCI grants, CA94683 and CA90997). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1860.

Effects of short-term heat stress on endophytic ergot alkaloid-induced alterations in rat hepatic gene expression

Exposure to ergot alkaloids in endophyte-infected fescue (E+) is associated with impaired animal productivity, especially during heat stress, which is commonly referred to as fescue toxicosis. To elucidate the pathogenesis of this condition, the effects of short-term heat stress (HS) on hepatic gene expression in rats exposed to endophytic ergot alkaloids were evaluated. Rats implanted with telemetric transmitters to continuously measure core temperature were fed an E+ diet and maintained under thermoneutral (TN) conditions (21 degrees C) for 5 d, followed by TN or 31 degrees C (HS) conditions for 3 d. Feed intake (FI) and BW were monitored daily. The E+ and HS-induced alterations in hepatic genes were evaluated using DNA microarrays and PCR analyses. Hepatic antioxidant enzyme activities, as well as the incidence of apoptosis, were determined. As expected, intake of E+ reduced FI and BW from pretreatment levels under TN conditions, with greater reductions during short-term HS. Genes involved in gluconeogenesis and apoptosis were upregulated, whereas genes associated with oxidative phosphorylation, xenobiotic metabolism, antioxidative mechanisms, immune function, cellular proliferation, and chaperone activity were all downregulated with short-term HS. Hepatocytic apoptosis was increased and antioxidant enzyme activity decreased in the livers of rats exposed to HS. The hypothesized, exacerbating effects of HS on the direct, endophytic toxin-related and indirect, reduced caloric intake-associated alterations in hepatic gene expression were clearly demonstrated in rats and may help to elucidate the pathogenesis of fescue toxicosis in various animal species.

Trends in the study of genetic susceptibility

Genotoxicology investigates the molecular basis of cellular responses to DNA damage. During more than three decades of genetic toxicology testing, a large number of tests with varying sensitivity and specificity have been developed. These genotoxicity tests can predict: the likelihood of a substance to be a (rodent) carcinogen, the mechanism of carcinogenic activity of different substances, if the results of genotoxicity studies only predict carcinogenicity or they are a distinct hazard endpoint (Nohynek, 2005). The current genetic toxicity testing batteries represent: Ames test which is a component of all genetic testing batteries. A mammalian cell mutagenicity assay which should confirm or complete the Ames test. Chromosomal aberrations tests which are based on a different endpoint than gene mutations. Positive in vitro results need to be confirmed by in vivo tests; results from test batteries have higher predictive value than results of a single test (Nohynek, 2005). The human molecular epidemiology represents a new trend in the study of genetic susceptibility and interactions between genetic and environmental factors of risk. Doses and biochemical effects biomonitoring have nowadays a tremendous utility providing an efficient means of measuring human exposure to chemical substances. Human biomonitoring considers all routes of uptake and all sources which are relevant making it an ideal instrument for risk assessment and risk management; blood is by far the most approved matrice. Human biomonitoring can be done for most chemical substances which are in the focus of the worldwide discussion of environmental medicine (Angerer et al., 2007). Molecular epidemiology studies of human populations exposed to potential mutagens have the aim to assess the risk of genetic disease or cancer by analysing the relationship between internal exposure and biological effects in target cells under consideration of confounding factors (Faust et al., 2004). Human biomonitoring can identify new chemical exposures, trends and changes in exposure, establish distribution of exposure among the general population, identify vulnerable groups and populations with higher exposures and identify environmental risks at specific contaminated sites. The sensitivity of methods moreover enables the elucidation of human metabolism and toxic mechanisms of the pollutants (Angerer et al., 2007). Biomarkers of effect that indicate exposure to a causative agent and that reflect the individual risk of disease are numerous. The most commonly used biomarkers in cancer epidemiology include: measurements of DNA damage, such as DNA breaks, altered bases, bulky adducts; chromosomal aberrations (CA), micronuclei (MN) and sister chromatid exchange (SCE), which are also the results of DNA damage (Kassie et al., 2000; Collins, 1998). Chromosomal aberrations and micronuclei are biomarkers of damage due to genetic instability or exposure to environmental mutagens or carcinogens. A recent approach is to associate the biomarkers of genetic susceptibility which take into account cancer susceptibility and interindividual differences in the response to a genotoxic exposure, and the analysis of CA and/or MN, which serves as a biomarker of interactions between the environment and the genetic material of the cell. Information is being gathered on how DNA damage and more particularly the frequency of chromosomal aberrations and/or micronuclei depend on the polymorphisms in genes implicated in xenobiotic metabolism (activation and/or detoxification) and DNA lesion repair. For biomonitoring purposes, numerous confounding factors (age, sex, tobacco consumption, etc.) influence the CA and MN biomarker, and thus associating genetic polymorphisms to CA and MN would be useful to better define the prevention and prediction of risk (Iarmarcovai et al., 2007a; Iarmarcovai et al., 2007b). The in vitro genetic toxicology tests used for regulatory purposes measure formation of gene mutations and chromosomal changes following DNA damage induced by the compounds under test, and are used to predict the carcinogenic potential of pharmaceuticals, industrial chemicals, food additives and cosmetic ingredients (Kirkland et al., 2007). In vitro test systems will aid in the identification of the most sensitive species and strains. Also, in vitro systems are good models for studying qualitative and quantitative species differences in toxicity. Further, in vitro systems are excellent models for characterisation of the mode of action/mechanism for critical effects, but findings need to be validated in vivo. In vitro systems will aid in the extrapolation from high to low dose and from experimental animals to humans (Holme et al., 2002).

Micronuclei and genetic polymorphisms: from exposure to susceptibility

The concept of genetic susceptibility and interactions between genetic and environmental factors of risk is a new trend in molecular epidemiology studies of cancers. Micronuclei are biomarkers of chromosome damage due to genetic instability or exposure to environmental mutagens or carcinogens. The micronucleus assay in combination with fluorescent in situ hybridization discriminates between micronuclei containing acentric chromosome fragments (chromosome breakage) and micronuclei containing whole chromosomes (chromosome loss). A recent approach is to associate the biomarkers of genetic susceptibility, which take into account cancer susceptibility and interindividual differences in the response to a genotoxic exposure, and the micronucleus assay, which serves as a biomarker of interactions between the environment and the genetic material of the cell. Information is being gathered on how DNA damage and more particularly the frequency and centromeric content of micronuclei depend on the polymorphisms of genes implicated in xenobiotic metabolism (activation or detoxication), DNA lesion repair, or folate metabolism. For biomonitoring purposes, numerous confounding factors (age, sex, tobacco consumption) influence the micronucleus biomarker, and thus associating genetic polymorphisms to micronuclei would be useful to better define the prevention and prediction of cancer risk.

Loss of detoxification in inflammatory bowel disease: dysregulation of pregnane X receptor target genes

Background & Aims: Phase 1, phase 2, and cellular efflux transporters are critical components in intestinal barrier function against xenobiotics and bacteria. We therefore performed global gene expression profiling in patients with ulcerative colitis (UC) and Crohn’s disease as well as control specimens, with a special emphasis on genes involved in detoxification and epithelial membrane integrity. Methods: Mucosal biopsy specimens from nonaffected regions of the colon and the terminal ileum were subjected to DNA microarray analysis and pathway-related data mining. Real-time reverse-transcription polymerase chain reaction was used for verification of selected regulated candidate genes in larger inflammatory bowel disease sample numbers and intestinal cell lines. Results: Several dysregulated genes were identified in both disease groups and tissues. A set of genes coordinately down-regulated in the colon of patients with UC was composed of cellular detoxification and defense genes, which are target genes for the transcription factor pregnane X receptor (PXR). Messenger RNA expression of ABCB1 (MDR1) and PXR was significantly reduced in the colon of patients with UC but was unaffected in patients with Crohn’s disease. In contrast to some of its target genes, the expression of PXR was not sensitive to tumor necrosis factor α stimulation of intestinal cell lines. Conclusions: A disease- and tissue-specific decrease in the expression of detoxification enzymes and ABC transporters was observed, which may be explained by a loss of PXR expression. Thus, dysregulation of xenobiotic metabolism and PXR activity in the gut is likely to contribute to the pathophysiology of UC.

Effects of endocrine active substances in wildlife species: Genetic, biochemical, and physiological factors in variable susceptibility to endocrine disruptors

Responses to endocrine active substances (EASs) in animals are various, and differences between the responses among individuals, populations and species are well known. These differences are observed not only in EASs but in most environmental chemicals including synthetic and naturally occurring ones. The basic differences in sensitivity to EASs are attributed to that of affinity or specificity of the receptors to EASs at the cellular level. Although the nucleotide sequences encoding for estrogen receptor proteins have been documented in several species and the functions of the receptors are the same, the ability to bind the natural hormones and the estrogenic xenobiotics is not necessarily identical. The reproductive endocrine system is basically common among vertebrates, but chemical types of hormones, physiological roles of hormones and the basal blood levels of hormones differ among each species, especially in sex steroids. These differences cause various types of responses and sensitivity to EASs among animal species. Xenobiotic metabolism is important for the genetical, biochemical and physiological factors concerning the influence of EASs. Some EASs directly inhibit cytochrome P450 (CYP) activity as was reported in tributyltin that inhibits CYP19 (aromatase) activity causing imposex in neogastropods. Some organochlorines including dioxins stimulate aryl hydrocarbon (Ah) receptor-mediated xenobiotic metabolism, and result in the metabolic disruption of steroid hormones such as estrogen as were reported in eggshell thinning in birds of prey and uterus occlusion in seals. CYP activity greatly differs among wildlife species in both terrestrial and aquatic organisms, and these differences are significantly responsible for the multiple effects or toxicity of EASs. Sex and age differences also cause different responses to EASs and are largely due to the differences in xenobiotic metabolizing activities.