Sex as a major determinant of gene expression in tissues of mice exposed to arsenate.

Inorganic arsenic, frequently found as contaminant of ground water used for drinking purposes in many areas of the world, is a well-known potent human toxicant and carcinogen. Chronic exposure to inorganic arsenic has been associated with cancer of skin, lung, bladder and kidney and, probably, liver. The mechanism of arsenic action in vivo is poorly understood, in particular in relation to dose, type of tissue and gender. To elucidate tissue- and gender dependent biological responses in the genome of mice, we have used cDNA macroarrays for investigation on the expression of 1185 cancer-related genes in mice after exposure to arsenate in drinking water. Continuous exposures of mice to arsenate in drinking water modulate the gene expression in tissues. Interestingly, there were remarkable sex differences: male and female mice show completely different changes in the expression of cancer-related genes. The main gene functional families modulated, were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover and proto-oncogens. This result demonstrates important gene-environmental interactions: the molecular mechanisms triggered by arsenic levels frequently experienced following exposure via drinking water, are totally different in males and females. The results obtained using cancer-related genes will be compared with the profiles of over 30.000 genes using the Applied Biosystems expression Array System, to clarify the sex-specific gene pathways

The arsenic in mice as experimental model for risk modifiers.

Studies on the relevance of host factors in modulating the physiological responses following chronic exposure to xenobiotics were carried out according to a \u201cToxicogenomic Model on Arsenic in Mice\u201d developed at thte JRC. This model is focused on chronic exposure to arsenate given alone or in combination with other xenobiotics, to assess potential \u201ccocktail effects\u201d and related cumulative risks. DNA-macroarrays technology is applied to evaluate physiological responses at transcriptional level and assessing possible biochemical responses. A cluster of 1200 cancer genes was used for screening purposes, while quantitative PCR on selected genes applied for validation. The exposure varied from in-utero and post-lactation up to adult age (4 months), the chemical forms (arsenate and dimethylarsenate) and doses from 0.1 up to 10 mg As/L in drinking water. Comparison between acute single doses and chronic exposure was also performed. Chronic exposure to arsenate and atrazine in drinking water was selected as an example of multiple chronic exposure. The liver, kidney, lung, bone marrow, adrenals, uterus, and testis were the tissues considered. In the tissues of mice chronically exposed to arsenate, the modulation of gene expression was not only depending on the levels, types and length of exposure, while differently regulated also by the sex, age and diet. The main gene functional families modulated were covering a wide range of biochemical and physiological regulations, like cell cycle modulation, cell adhesion, apoptosis, xenobiotic metabolism, DNA repair, protein turnover, and proto-oncogenes. The patterns of gene expression were strongly influenced by co-exposure to other xenobiotics like atrazine and naphthalene, particularly for genes involved in the metabolism and in neuroendocrine regulation. These effects varied according to the tissue considered, supporting the needs for coherent and specifically designed studies to assess relevant biomarkers of long-term exposure to low levels of xenobiotics and their mixtures

Dietary proteins modulates the gene expression in mice chronically exposed to arsenate.

In the frame of a project on the assessment of risk modifying factors modulating the health effects of environmental chemicals we are developing a toxicogenomic approach using an \u201carsenic in mice\u201d experimental model, considering multistressors exposure, genetics, age, levels and length of exposure, etc. In the present study, we used cDNA Macroarrays to investigate the effects of low protein intake on the expression of 1185 cancer-related genes in the liver of male and female mice transplacentary exposed to different levels of arsenate in drinking water during gestation and developmental age. The results of this study support the relevance of dietary factors in modulating the physiological responses in gene expression following chronic exposure to xenobiotics. In mice chronically exposed to arsenate in drinking water, the modulation of gene expression in different tissues was not only depending on the levels of the xenobiotic under investigation, but mainly regulated by the content of proteins in diet

Combined in-utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates the gene expression and clonogenicity of myeloid progenitors in bone marrow.

Increasing evidence proves that human fetuses are exposed to multiple risk factors and major concerns have been expressed towards exposure to potential endocrine modulating chemicals at early stage of life and during growth. Understanding that exposures occur as mixture of chemicals and that they converge on other inherent and environmental risk-modulating factors, there is a need to develop experimental models to assess the effects of exposure to multiple chemicals during different stage of life. In the present study, the clonogenicity of myeloid progenitors (CFU-GM) and the modulation of gene expression of 1197 cancer-related genes (DNA macroarrays) in bone marrow were used to investigate in male and female young mice the combined effects of continuous exposure to arsenate and atrazine in drinking water. Female adult mice were treated with arsenate in drinking water (1 mg As/L) for 10 days before mating and during the gestation. Offspring were randomly put into separate groups of males and females. One group of arsenic exposed offspring were exposed for 4 months to atrazine (1mg Atr/L) and arsenate (1 mg As/L) in drinking water (As+Atr). One group of each of arsenic unexposed offspring were exposed for 4 months to atrazine (1mg Atr/L) in drinking water (Atr). Additional arsenate (1 mg As/L) was given to one group of arsenic exposed offspring (As). Control mice without any treatment were also analysed (Ctrl). In male mice the exposure to arsenate or to atrazine alone did not result in significant changes on the gene expression in bone marrow cells, whereas, co-exposure to arsenic and atrazine (As+Atr) resulted in a significant up-modulation of gene expression. The percentage of CFU-GM weakly decreased after exposure to individual compounds, while the co-exposure did not change the clonogenicity of the progenitors. In female mice, the co-exposure to both chemicals resulted in a drastic up-modulation of gene expression, while in these cells the single treatments showed a up-modulation of few genes as well. The percentage of CFU-GM decreased significantly after atrazine exposure, did not change with arsenic treatment, but dramatically increased after the combined administration. These results indicate that in-utero and juvenile co-exposure of mice to atrazine and arsenate induce significant effects at the level of transcriptional activation of genes in bone marrow cells, as well as stimulating the myeloid progenitors to proliferate, particularly when co-administered in drinking water to female mice

Meeting Report: Validation of Toxicogenomics-Based Test Systems: ECVAM–ICCVAM/NICEATM Considerations for Regulatory Use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities

Nickel, Cobalt and Chromium-Induced Cytotoxicity and Intracellular Accumulation in Human HaCaT Keratinocytes.

Abstract not availableJRC.(EI)-Environment Institut

Sex-Dependent Expression of Cancer-Related Genes in Mice Exposed to Arsenic via Drinking Water.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Physiological and nutritional factors modulating the gene expression in the liver of mice exposed transpacentary to arsenate.

There are suggestions that inorganic arsenic, a major drinking water contaminants in several countries, could act as a transplacental carcinogen in mice (Walkees et al.2004). There are also evidences that toxic responses following exposure to arsenic are strongly influenced by nutritional, phisiological and genetic factors (Vahter, 2000). In the frame of a project on the assessment of risk modifying factors modulating the health effects of environmental chemicals we are using an \u201carsenic in mice\u201d experimental model to develop toxicogenomic approaches. In the present study, we used cDNA Macroarrays to investigate the effects of low protein intake, on the expression of 1185 cancer-related genes in the liver of male and female mice transplacentary exposed to arsenate by arsenate in drinking water during gestation. Female adult mice were fed either with standard rodent chow (18% protein rich) or with a protein deprived one (8%) forten days. Both groups of animals were also exposed to different concentrations of sodium arsenate in drinking water (0.1 mg As/L; 1 mg As/L; 10 mg As/L) for 10 days before mating and during gestation and the feeding period. Offspring were fed with the two different chows and exposed to different concentrations of arsenate in drinking water according to treatment of their mothers, up to two months of age. In the liver of newborn mice at two days of age, the exposure of pregnant mice had significant effects on the modulation of gene expression mainly in males, while in females some slight effects were observed in offspring from mothers exposed to the lowest As-concentration. For both sexes the deprived diet significantly modulates the hepatic gene expression. Major effects on the gene expression were observed in the liver of offspring whose exposure to arsenate in drinking water was continued for other two months after birth. The diet low in proteins affected the effects of As-exposure on gene expression and, at the same time, different concentrations of arsenate in drinking water caused different gene expression. These results suggest that physiological responses in tissues following semi-chronic exposure to arsenate are modulated not only by the levels of arsenic exposure, while differently influenced by host factors like age and gender. Furthermore, nutritional factors like the protein levels in the diet would significantly contribute to the overall toxic responses

Oncogene Transgenic Mice: A Useful Model to Study in vivo the Relationships between Gangliosides and Oncogenes.

Abstract not availableJRC.(EI)-Environment Institut