Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure.

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC-MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants.

The high resolution, accurate mass, and fast scanning features of the Orbitrap(TM) mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, ß-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds.

Alternative splicing interference by xenobiotics.

The protein coding sequence of most eukaryotic genes (exons) is interrupted by non-coding parts (introns), which are excised in a process termed splicing. To generate a mature messenger RNA (mRNA) hundreds of combinatorial protein-protein and RNA-protein interactions are required to splice out often very large introns with high fidelity and accuracy. Inherent to splicing is the use of alternative splice sites generating immense proteomic diversity from a limited number of genes. In humans, alternative splicing is a major mode of regulating gene expression, occurs in over 90% of genes and is particularly abundant in the brain. Only recently, it has been recognized that the complexity of the splicing process makes it susceptible to interference by various xenobiotics. These compounds include antineoplastic substances, commonly used drugs and food supplements and cause a spectrum of effects ranging from deleterious inhibition of general splicing to highly specific modifications of alternative splicing affecting only certain genes. Alterations in splicing have been implicated in numerous diseases such as cancer and neurodegeneration. Splicing regulation plays an important role in the execution of programmed cell death. The switch between anti- and pro-apoptotic isoforms by alternative splice site selection and misregulation of a number of splicing factors impacts on cell survival and disease. Here, our current knowledge is summarized on compounds interfering with general and alternative splicing and of the current methodology to study changes in these processes relevant to the field of toxicology and future risk assessments.

Identification and pathway mapping of furan target proteins reveal mitochondrial energy production and redox regulation as critical targets of furan toxicity.

Furan, a heat-generated food contaminant, is hepatotoxic and carcinogenic in rodents. Furan is oxidized by cytochrome P450 2E1 to cis-2-butene-1,4-dial, a chemically reactive α,ß-unsaturated dialdehyde, which has been identified as the key toxic metabolite of furan based on its ability to interact with tissue nucleophiles. In addition to genotoxicity, sustained cytotoxicity mediated through covalent binding of cis-2-butene-1,4-dial to critical target proteins is thought to play a key role in furan carcinogenicity. To identify putative protein targets of reactive furan metabolites, male F344/N rats (n = 5 per dose) were administered a single dose of [3,4-(14)C]-furan (20 mCi/mmol) at doses associated with hepatotoxicity following long-term exposure (0.1 and 2 mg/kg body weight [bw]). Liver proteins were separated by two-dimensional gel electrophoresis and protein spots carrying radiolabel were located by fluorography. In total, 83 discrete protein spots containing (14)C were consistently detected in livers of animals given [3,4-(14)C]-furan at 2.0 mg/kg bw, accounting for 4-5% of the proteome covered by our analyses. Protein spots were excised and digested in gel with trypsin for identification by protein mass spectrometry. Protein database search and subsequent pathway mapping identified 61 proteins localized predominantly in the cytosol and mitochondria, including structural proteins, mitochondrial enzymes involved in glucose metabolism, mitochondrial ß-oxidation, and adenosine triphosphate synthesis, and proteins that participate in the maintenance of redox homeostasis and protein folding. Collectively, our data suggest that functional loss of several individual proteins and interference with pathways, most notably mitochondrial energy production, redox regulation, and protein folding, may combine to disrupt cell homeostasis and cause hepatocyte cell death.

Gene expression and epigenetic changes by furan in rat liver.

Furan, a widely used industrial compound, has been found in a number of heated food items. Furan is carcinogenic to rats and mice, but the mechanism behind its carcinogenic effect is still not well understood. In this study, we tested the hypothesis that alteration of gene expression relating to cell cycle, apoptosis, DNA damage and of epigenetic modifications including miRNA and DNA methylation may contribute to rodent carcinogenicity of furan. Using quantitative PCR arrays specific to cell cycle-, apoptosis- and DNA damage-related genes, we found that three months furan treatment at 30 mg/kg (5 daily doses per week) induced extensive mRNA expression changes (largely up-regulation) in male Sprague Dawley rat liver, and the gene expression changes did not fully recover after a one month withdrawal of furan. We also found 18 miRNAs were up-regulated and 12 were down-regulated by PCR arrays. Many of these deregulated miRNAs were also found to have similar changes in furan-induced tumour samples. Both hyper- and hypo-methylation of specific gene promoter regions were identified and validated in the 3-month samples and tumour samples by microarray and COBRA (combined bisulfite restriction analysis). No global DNA methylation change was found in the 3 month treatment groups by LC-MS/MS, while furan-induced tumour samples showed global hypomethylation compared to non-tumour tissues. In conclusion, three months furan treatment at a carcinogenic dose resulted in irreversible gene expression changes, miRNA modulation and DNA methylation alteration in combination with a DNA-damage response, which suggests that non-genotoxic mechanisms are important for furan carcinogenicity.

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis.

BACKGROUND: DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. RESULTS: A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. CONCLUSION: The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.

Low doses of the carcinogen furan alter cell cycle and apoptosis gene expression in rat liver independent of DNA methylation.

BACKGROUND: Evidence of potent rodent carcinogenicity via an unclear mechanism suggests that furan in various foods [leading to an intake of up to 3.5 microg/kg body weight (bw)/day] may present a potential risk to human health. OBJECTIVES: We tested the hypothesis that altered expression of genes related to cell cycle control, apoptosis, and DNA damage may contribute to the carcinogenicity of furan in rodents. In addition, we investigated the reversibility of such changes and the potential role of epigenetic mechanisms in response to furan doses that approach the maximum estimated dietary intake in humans. METHODS: The mRNA expression profiles of genes related to cell cycle, apoptosis, and DNA damage in rat liver treated with furan concentrations of 0.1 and 2 mg/kg bw were measured by quantitative polymerase chain reaction (PCR) arrays. We assessed epigenetic changes by analysis of global and gene-specific DNA methylation [methylation-specific PCR, combined bisulfite restriction analysis (COBRA), and methylated DNA immunoprecipitation chip] and microRNA (miRNA) analyses. RESULTS: The expression profiles of apoptosis-related and cell-cycle-related genes were unchanged after 5 days of treatment, although we observed a statistically significant change in the expression of genes related to cell cycle control and apoptosis, but not DNA damage, after 4 weeks of treatment. These changes were reversed after an off-dose period of 2 weeks. None of the gene expression changes was associated with a change in DNA methylation, although we detected minor changes in the miRNA expression profile (5 miRNA alterations out of 349 measured) that may have contributed to modification of gene expression in some cases. CONCLUSION: Nongenotoxic changes in gene expression may contribute to the carcinogenicity of furan in rodents. These findings highlight the need for a more comprehensive risk assessment of furan exposure in humans.

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure 'biomarker' signals and induction of inflammatory, innate immune and apoptotic pathways.

The effects of chronic long-term exposure to multiply polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long-term chronic exposure to a multiply polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic programme, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants.

Induction of DNA strand breaks by genotoxicants in the alga Chlamydomonas reinhardtii.

The use of unicellular algae in ecotoxicity testing is well established, particularly regarding whole-organism and population-level end points such as lethality and population growth. Conflicting information exists, however, on the potential for genetic toxicity to be incorporated into the safety studies in this test organism. In the present study, DNA strand breaks (Comet assay) and ethoxyresorufin-O-deethylase (EROD) activity were used as indicators of genetic toxicity and cytochrome P450 1A baseline xenobiotic metabolism, respectively, in the unicellular green alga Chlamydomonas reinhardtii. DNA strand breaks were quantified following exposure to the direct-acting genotoxic agents 4-nitroquinoline-1-oxide (NQO) and the N-hydroxy metabolite of 2-acetylaminofluorene (N-OH-2-AAF) and the indirect-acting genotoxin chrysoidine. Following compound exposure, chrysoidine and N-OH-2-AAF produced statistically significant increases in DNA strand breaks at both 0.1 and 10 microM and 0.05 and 5 microM, respectively (p < 0.05 and p < 0.01). Different light sources were also found to influence DNA strand breaks, the minimum response being observed using a source that omits the ultraviolet range. Compared to many mammalian cells, both DNA damage responses and EROD activity were relatively weak. EROD activity was 0.03 pmol/min/10(6) cells in control cells, and the maximum level of DNA strand breaks observed was 14.1% at a 5 nM concentration of NQO. The responses exhibited were not enhanced by the use of a cell wall-free mutant strain. In conclusion, C reinhardtii responded, albeit weakly, to selected direct- and indirect-acting genotoxicants and also exhibited measurable EROD activity.

Changes in gene expression and assessment of DNA methylation in primary human hepatocytes and HepG2 cells exposed to the environmental contaminants-Hexabromocyclododecane and 17-beta oestradiol.

We evaluated the effects of two putative non-genotoxic hepatic carcinogens, hexabromocyclododecane (HBCD) and 17-beta oestradiol (E(2)) on global and CpG promoter DNA methylation in both primary human hepatocytes and hepatocellular carcinoma (HepG2) cells. The mRNA gene expression levels of genes involved particularly in cell cycle were also evaluated and potential correlation with DNA methylation status examined. HBCD at 0.03 and 0.3 ng/mL did not produce statistically significant differences in global genomic methylation. However, E(2) (0.1 ng/mL) significantly lowered global DNA methylation levels in HepG2 cells by approximately 65% (P<0.01). In primary hepatocytes, the promoter regions of N-cym and ERalpha were methylated in both control and treated groups, signifying lack of promoter demethylation by both HBCD and E(2). Furthermore, CpG promoter methylation of RB1 was observed in HepG2 cells but this was unaffected by treatments. The remaining genes (p16, C-myc, H-ras, THRalpha, histone H3, TBK1 and TNFRalpha) were unmethylated in their CpG promoter regions in both test systems. Quantitative RT-PCR showed that HBCD at 0.03 ng/mL up-regulated the expression of N-cym whereas E(2) up-regulated the expression of ERalpha and THRalpha genes in primary hepatocytes. In HepG2 cells, the mRNA gene expression levels of p16, RB1 and N-cym were significantly down regulated by HBCD (0.03 ng/mL) and E(2) (0.1 ng/mL) while HBCD at 0.3 ng/mL, significantly down regulated the expression levels of N-cym, ERalpha and ERbeta genes. Thus, while both HBCD and E(2) may alter the expression of certain genes involved in proliferation, the mechanisms appear unrelated to DNA methylation.

Supramolecular iron cylinder with unprecedented DNA binding is a potent cytostatic and apoptotic agent without exhibiting genotoxicity.

The supramolecular iron cylinder, [Fe(2)L(3)]Cl(4) (L = C(25)H(20)N(4)), shows unprecedented DNA binding in vitro, inducing intramolecular DNA coiling and also targeting Y-shaped DNA junctions. We investigated its effects on proliferation and survival in both tumor and normal cell lines. Iron cylinder reduced mitochondrial activity of cultures with potency similar to cisplatin, inhibited the cell cycle, and increased cell death by apoptosis. Associated with this, we observed a lowering of the association of propidium iodide with cellular DNA consistent with an observed competitive displacement of PI from naked DNA by cylinders. Importantly, and in contrast to existing anticancer drugs such as cisplatin, the iron cylinder [Fe(2)L(3)](4+) was not genotoxic. In summary, the design of metal complexes such as [Fe(2)L(3)](4+) with potential anticancer properties in the absence of genotoxicity may represent a significant step toward therapeutic advancement.

A quantitative inverse relationship between connexin32 expression and cell proliferation in a rat hepatoma cell line.

Gap junctions comprised of connexin proteins are involved in direct intercellular communication and the regulation of cell behaviour and homeostasis. Reduced connexin expression and loss of gap junction function is a characteristic of many cancer cells and of the effect of many non-genotoxic carcinogens that induce cell proliferation. Moreover, when certain cancer cell lines are transfected with specific connexin genes, cells can regain control over proliferation. We have employed RNA interference and dexamethasone to modulate connexin32 expression in MH(1)C(1) cells to a range of concentrations. This allowed the determination of the quantitative relationship between connexin32 protein expression and cell proliferation. The magnitude of cell proliferation, measured by bromodeoxyuridine incorporation, was inversely proportional to the level of connexin32 expression. Q-PCR indicated a lack of change of expression of a range of cell cycle-related genes at 24h. The inverse relationship between Cx32 expression and proliferation was continuous, and a threshold level of reduction of connexin32 was not observable for an influence on proliferation.

Reactive oxygen species from the uncoupling of human cytochrome P450 1B1 may contribute to the carcinogenicity of dioxin-like polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are classified by the International Agency for Research on Cancer as probable human carcinogens. A subset of PCBs are described as 'dioxin like' because of similarities to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dioxin-like PCBs have been shown to tightly bind the active site of cytochrome P450 (CYP) 1A isoforms, primarily CYP1A1, resulting in inhibition of CYP activity and the generation of reactive oxygen species (ROS) as a result of uncoupling of the catalytic cycle. Human CYP1B1 (hCYP1B1) is an extrahepatic CYP closely related to hCYP1A1 and is overexpressed in the lungs of smokers. Moreover, hCYP1B1 has been found to be overexpressed in cancers derived from a number of tissue types, as well as in pre-malignant prostate tumours, implicating overexpression of hCYP1B1 as a risk factor for extrahepatic carcinogenesis. It has been demonstrated previously that hCYP1B1 is inhibited by dioxin-like PCBs, but whether or not it is uncoupled has not been investigated. In the current study, the ability of three dioxin-like PCBs 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5-pentachlorobiphenyl and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169) to inhibit hCYP1B1 and stimulate the formation of ROS in V79MZ cells (which lack endogenous CYPs) expressing hCYP1B1 was demonstrated. Moreover, the generation of ROS was also associated with increases in parameters of oxidative stress related to genotoxicity (DNA oxidation and lipid peroxidation). For PCB169, these effects were time and concentration dependent. These data identify a novel mechanism of genotoxicity for dioxin-like PCBs, as well as providing further evidence that overexpression of hCYP1B1 is a risk factor for extrahepatic carcinogenesis.

Global genomic methylation levels in the liver and gonads of the three-spine stickleback (Gasterosteus aculeatus) after exposure to hexabromocyclododecane and 17-beta oestradiol.

Alteration of DNA methylation is a major epigenetic mechanism associated with the effects of nongenotoxic carcinogens. We evaluated the effects of two environmental pollutants, hexabromocyclododecane (HBCD), 17-beta oestradiol (E(2)) as well as 5-aza 2' deoxycytidine (5AdC) on global DNA methylation levels (5-methyl 2' deoxycytidine) in the liver and gonads of the three-spine stickleback (Gasterosteus aculeatus). HBCD at 30 and 300 ng/L of water did not produce statistically significant differences in global genomic methylation in liver of female stickleback. On the other hand, the methylation inhibitor, 5-aza-2'-deoxycytidine, significantly lowered hepatic global methylation levels in these fish by 14% (P<0.05). The naturally occurring oestrogen, 17-beta oestradiol (E(2)) at 100 ng/L also decreased global DNA methylation levels in female liver but this effect was not statistically significant. In contrast, both E(2) and 5AdC caused statistically significant (P<0.001 and P<0.01 respectively) global genomic hypermethylation in the gonads of male sticklebacks although the increase seen in the female gonads was not statistically significant. The male gonad effect though unexplained may potentially be an indirect response to hypomethylation in other tissues (such as the liver) and may have important implications regarding oestrogenic effects in fish. The contrasting effects of HBCD and E(2) on global DNA methylation in stickleback should contribute to the integrated risk assessment of these environmental chemicals.

Subcellular compartmentalization of glutathione: correlations with parameters of oxidative stress related to genotoxicity.

Glutathione (GSH) is a major component of the antioxidant defence system of mammalian cells and is found in subcellular pools within the cytoplasm, nucleus and mitochondria. To evaluate the relationships between these pools and parameters of oxidative stress related to genotoxicity, wild type (WT) and 8-oxo-2'-deoxyguanosine glycosylase 1 (OGG1)-null (mOGG1(-/-)) mouse embryonic fibroblasts (MEF) were treated with buthionine sulphoximine (BSO; 0-1000 microM, 24 h), an inhibitor of GSH biosynthesis. BSO treatment resulted in a concentration-dependent depletion of GSH from the cytoplasm, but depletion of mitochondrial and nuclear GSH occurred only at concentrations > or =100 microM. GSH levels were correlated with reactive oxygen species (ROS), lipid peroxidation (measured as the increase in the genotoxic end-product malondialdehyde (MDA)) and oxidative DNA modifications, measured as both frank DNA strand-breaks (FSB) and oxidized purine lesions (OxP) using the alkaline comet assay with formamidopyrimidine DNA glycosylase (FPG) modification; this system allowed for the identification of BSO-induced DNA modifications as primarily mutagenic 8-oxo-2'-deoxyguanosine lesions. A number of significant correlations were observed. First, negative linear correlations were observed between mitochondrial GSH and ROS (r = -0.985 and r = -0.961 for WT and mOGG1(-/-) MEF, respectively), and mitochondrial GSH and MDA (r = -0.967 and r = -0.963 for WT and mOGG1(-/-) MEF, respectively). Second, positive linear correlations were observed between ROS and MDA (r = 0.996 and r = 0.935 for WT and mOGG1(-/-) MEF, respectively), and ROS and OxP (r = 0.938 and r = 0.981 for WT and mOGG1(-/-) MEF, respectively). Finally, oxidative DNA modifications displayed a negative linear correlation with nuclear GSH (r = -0.963 and -0.951 between nuclear GSH and FSB and OxP, respectively, for WT MEF and r = -0.960 between nuclear GSH and OxP in mOGG1(-/-) MEF), thus, demonstrating the genotoxic potential of compounds that deplete GSH. The findings highlight the critical roles of the mitochondrial and nuclear GSH pools in protecting cellular components, particularly DNA, from oxidative modification.

Does exhaustive exercise result in oxidative stress and associated DNA damage in the chub (Leuciscus cephalus)?

DNA strand breaks [as determined by the conventional and formamidopyrimidine glycosylase (FPG)-modified Comet assay] and antioxidant defense status [as indicated by superoxide dismutase (SOD) activity and reduced glutathione (GSH) concentration] were evaluated in healthy adult chub (Leuciscus cephalus) after exhaustive exercise [swimming to their critical swimming speed (U(crit)), twice in succession with a 40 min rest period between] vs. confined (unexercised) control fish. The conventional Comet assay revealed significantly higher DNA strand breaks in all the tissues (blood, liver, and gill), with the highest increase over background evident in the epithelial gill cells of swum fish compared to the controls. Moreover, when the FPG-modified Comet assay was conducted to reveal specific oxidative lesions, the gill cells of exercised fish sustained the highest level of oxidative DNA damage in comparison to the control. Data on tissue antioxidant defense mechanism were less conclusive, with no significant differences in the tissue levels of SOD or GSH. This suggests that either the degree of oxidative stress was not great enough to evoke a response in terms of defense mechanisms or the timescale of antioxidant defense response was somewhat different from the time between the application of stress and subsequent tissue sampling. From the swimming data, U(crit) was significantly lower on the second assessment compared to the first (repeat ratio: 0.76), suggesting that the fish were exercised to a level which was not sustainable. Overall, these findings support the theory that acute extreme exercise could result in oxidative stress and associated DNA damage in fish. These observations suggest that fish living in fast flowing and polluted rivers are at increased risk of DNA damage.

Oxidative stress response of European flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray.

The monitoring of the impact of chemical pollutants upon marine ecosystems commonly employs a multi-biomarker approach. Functional genomics, using cDNA microarrays, allows for a comprehensive view of how an organism is responding to an exposure, with respect to changes in gene expression. Differentially expressed mRNAs were first isolated from livers of European flounder by means of suppressive, subtractive hybridisation. A clone set containing a total of 284 different potentially differentially expressed mRNAs was produced, of which 84 were tentatively identified. These were combined with previously cloned known stress genes isolated by degenerate PCR to produce a custom 500-clone microarray platform with each clone arrayed to four spots. Subsequent array experiments using cadmium-treated flounder detected up-regulation of 27 transcripts, including Cu/Zn superoxide dismutase, thioredoxin, a peroxiredoxin and a glutathione-S-transferase, reflecting oxidative stress in exposed flounder, while CYP1A expression was down-regulated. These changes were confirmed by real-time PCR. The array experiment highlighted a number of candidate genes for further analysis as potential novel biomarkers of cadmium exposure and demonstrated the applicability of the custom microarray approach in the study of the effects of toxicants.

Tissue levels and biomarkers of organic contaminants in feral and caged chub (Leuciscus cephalus) from rivers in the West Midlands, UK.

The Birmingham conurbation (West Midlands, UK) has traditionally been a major centre of UK industry and population and consequently has a legacy of pollution, which is reflected in the water quality of local rivers. Three of these rivers, exhibiting good, intermediate and poor overall water quality, were the subject of a study in which the effects of contamination on hepatic biomarkers and tissue contaminant loads in feral and caged chub (Leuciscus cephalus) were investigated. Muscle polychlorinated biphenyls and organochlorine pesticides (PCBs and OCPs), as well as bile pyrene and benzo[a]pyrene-like metabolite levels, were variable in both caged and feral fish, but were generally higher in tissue from the more polluted sites. OCPs were, in most cases, higher in the feral fish than in the caged fish, although the opposite was true of bile PAH metabolites, possibly due to differences in relative accumulation rates. Hepatic CYP1A activity (via ethoxyresorufin-O-deethylase (EROD) activity) in both feral and caged fish was also generally higher at the more polluted sites. EROD activity in feral and caged fish was statistically associated with polycyclic aromatic hydrocarbon (PAH) contamination and specific PCB congeners. Other biomarkers measured (reduced glutathione in liver, and serum aspartate aminotransferase) showed little consistent evidence of relationships with water quality. The assessment of these parameters during different seasons also revealed relatively little evidence of temporal variation. Overall, the caged chub appeared to reflect the pattern of general water quality more accurately than did feral fish, particularly with respect to EROD activity and to some degree bile PAH metabolites, thus supporting their use as sentinel species. However, the fact that muscle OCPs were generally higher in the feral fish suggests that the use of feral fish may be more indicative of exposure to certain classes of contaminant, and so biological monitoring programs should be designed with such considerations in mind.

Identification of functional regulatory regions of the connexin32 gene promoter.

Connexin32 (Cx32) is the predominant gap junction protein expressed in adult rat hepatocytes. This study investigated transcriptional regulation of the rat Cx32 gene in MH(1)C(1) rat hepatoma cells using transient expression assays in conjunction with promoter mutagenesis and 5' nested deletion analysis. Site-directed mutagenesis of the -736 and -187 hepatocyte nuclear factor-1 (HNF-1) sites, the -196 and -116 Sp1 sites, and the -729 and -329 Yin Yang 1 (YY1) sites all significantly reduced promoter activity. We have defined the contribution of each individual site to promoter activity in the intact cell. A novel upstream region of the Cx32 promoter (-1042 to -758) was cloned and shown to contain negative regulatory elements. The transcription factors HNF-1 and Sp1 have important functional roles in the transcriptional regulation of basal and cell-specific Cx32 expression. The multifunctional transcription factor YY1 is also implicated.

Chloroform, carbon tetrachloride and glutathione depletion induce secondary genotoxicity in liver cells via oxidative stress.

Chemical carcinogens are generally classified as genotoxic or non-genotoxic. However, weak genotoxicity at high concentrations is sometimes observed and interpretation is often problematic. In addition, certain rodent carcinogens exert their effects at doses associated with cytotoxicity and compensatory hyperplasia may be a contributing factor to tumourogenesis. We hypothesise that certain substances, at high concentrations, can induce an oxidative stress via the depletion of glutathione (GSH) and other antioxidant defences and that this may lead to indirect genotoxicity, that could contribute to carcinogenicity. In support of this, human HepG2 cells treated with buthionine sulphoximine (BSO) to deplete GSH, exhibited DNA strand breaks alongside elevated 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde deoxyguanosine (M(1)dG) adducts under conditions associated with lipid peroxidation. Chloroform and carbon tetrachloride are rodent carcinogens with characteristics as described above. In female rat hepatocytes, chloroform treatment resulted in a small dose-dependent increase in M(1)dG adducts (4 mM and above), DNA strand breakage (8 mM and above) and lipid peroxidation, in the absence of any associated increase in DNA oxidation. GSH depletion only occurred in association with cytotoxicity (20 mM; lactate dehydrogenase release). Alongside lipid peroxidation, carbon tetrachloride (1 and 4 mM) produced a small elevation in M(1)dG adducts and DNA strand breaks and increases in 8-oxodG were observed at the threshold of, and concomitant with, cytotoxicity (4 mM). These effects may contribute to high dose genotoxicity and carcinogenicity. Non-linearity in the dose response is expected on the basis of depletion of antioxidants, and therefore, a pragmatic threshold for biologically relevant responses should exist.