The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA-developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real-time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition-induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity-induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell-cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro-apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells.

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity-induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T-lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity-induced DNA methylation changes were detected by methylation sensitive-random amplified polymorphic DNA (MS-RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real-time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS-RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time-dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity-induced gene expression changes and associated adverse health effects.

Arsenic is a known carcinogen, and its exposure is associated with cancers in multiple target organs including the prostate. Whether arsenic causes cancer by increased cell proliferation or cell survival is not clear. Additionally, mitochondria have been shown to play important roles in arsenic-induced DNA damage and carcinogenesis. However, the mechanism of mitochondrial involvement in arsenic-induced cancer is not clear. Therefore, the objectives of this study were to investigate the effect of arsenic on cell proliferation/survival and genotoxicity, and to determine the effect of arsenic on the expression of mitochondrial transcription factor A (mtTFA) in human prostate epithelial cells, RWPE-1. Results of this study revealed that chronic exposure to arsenic causes increased cell survival. Arsenic also induced nuclear DNA damage and mutations in mitochondrial DNA. Expressions of DNA repair genes ERCC6, XPC, OGG1, and reactive oxygen species (ROS) scavenger MnSOD was also altered in arsenic-exposed cells. Arsenic concentration-dependent increased expression of mtTFA and its regulator NRF-1 was observed in arsenic-exposed cells, suggesting that arsenic regulates mitochondrial activity through an NRF-1-dependent pathway. In summary, this study suggests that chronic exposure to arsenic causes DNA damage and increased cell survival that may ultimately result in neoplastic transformation of human prostate epithelial cells. Additionally, this study also provides evidence that arsenic controls mitochondrial function by regulating mtTFA expression.

Abstract. Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Genotoxicity of arsenic has been a topic of controversy. Both genetic alterations (mutations) and epigenetic changes (methylation) have been shown to play a crucial role in environmental carcinogenesis. Chronic exposure to arsenic has been shown to induce malignant transformation of mammalian cells. However, the genetic aberrations induced by arsenic in this process are unclear. The purpose of this study was to determine if both lower (1 pg/ml) and higher concentrations (100 ng/ml) of arsenic induces either mutations or methylation changes that could lead to the development of genomic instability in TM3 cells, immortalized Leydig cells derived from normal mouse testis. Two independent exposure times were used in this study which resulted in cells of 33 and 100 generations in age. Arsenic-induced genetic and epigenetic changes were screened at a genome-wide level by random amplified polymorphic DNA (RAPD), also known as AP-PCR method with undigested DNA as well as DNA digested by the methylation sensitive isosizomeric restriction enzymes MSPI and HpaII and untreated controls. Changes in the DNA fingerprint of both, the restriction enzyme digested DNA (indicating methylation changes) as well as undigested DNA (indicating mutations) from arsenic-treated (low as well as high dose) samples were observed as compared to their controls. Thus, this study provides the first evidence at DNA sequence level for mutagenic potential of arsenic. Further characterization of these altered genomic regions is underway. The understanding of these genetic and epigenetic changes in arsenic-induced carcinogenesis will provide a basis for better interventional approaches in both the treatment and prevention of arsenicinduced cancer.

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