Inorganic arsenic exposure-induced premature senescence and senescence-associated secretory phenotype (SASP) in human hepatic stellate cells.

Exposure to inorganic arsenic has been known to induce cancers in various organs, however, the underlying mechanisms remain unclear. Premature senescence refers to the irreversible growth arrest induced by stress stimuli. The senescence-associated secretory phenotype (SASP), particularly in fibroblasts, has been shown to promote cancer development. In this study, we examined whether arsenite exposure causes premature senescence and induction of SASP in liver fibroblasts using the human hepatic stellate cell line, LX-2. Exposure of LX-2 cells to 5 or 7.5 µM of sodium arsenite for 144 h induced the features of senescence in the cells, including morphological changes, growth inhibition, increased senescence-associated ß-galactosidase activity, increased P21 gene expression, and decreased LAMINB1 gene expression. The mRNA expressions of SASP factors, such as MMP1, MMP3, IL-8, IL-1ß, and CXCL1, were also highly upregulated. The wound healing assay revealed that the conditioned medium from LX-2 cells with arsenite-induced senescence increased the migration activity of cells of the human hepatoma cell line, Huh-7. Gene expression data of liver cancer samples from the Human Protein Atlas showed that high expression levels of the SASP factors that were upregulated in the cells with arsenite-induced senescence were strongly associated with a poor prognosis. In addition, the cellular levels of γ-H2AX, a DNA double-strand break marker, were increased by arsenite exposure, suggesting that DNA damage could contribute to premature senescence induction. These results show that arsenite exposure induces premature senescence in hepatic stellate cells and suggest that the SASP factors from the senescent cells promote hepatic carcinogenesis.

Gestational arsenic exposure and paternal intergenerational epigenetic inheritance.

A growing body of evidence has shown that gestational exposure to environmental factors such as imbalanced diet, environmental chemicals, and stress can lead to late-onset health effects in offspring and that some of these effects are heritable by the next generation and subsequent generations. Furthermore, altered epigenetic modifications in DNA methylation, histone modifications and small RNAs in a single sperm genome have been shown to transmit disease phenotypes acquired from the environment to later generations. Recently, our group found that gestational exposure of F0 pregnant dams to an inorganic arsenic, sodium arsenite, increases the incidence of hepatic tumors in male F2 mice, and the effects are paternally transmitted to the F2. Here, we first overview the epigenetic changes involved in paternal intergenerational and transgenerational inheritance caused by exposure to environmental factors. Then, we discuss our recent studies regarding paternal inheritance of the tumor-augmenting effects in F2 mice by gestational arsenite exposure, in which we investigated alterations of DNA methylation status in F2 tumors and causative F1 sperm. We also discuss the possible targets of the F2 effects. Finally, we discuss future perspectives on the studies that are needed to fully understand the health effects of arsenic exposure.

Gestational arsenite exposure augments hepatic tumors of C3H mice by promoting senescence in F1 and F2 offspring via different pathways.

Previous studies showed that gestational arsenite exposure increases incidence of hepatic tumors in the F1 and F2 male offspring in C3H mice. However, the mechanisms are largely unknown. In this study, we focused on whether cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to tumor formation in C3H mice, and whether gestational arsenite exposure augments hepatic tumors through enhancement of cellular senescence. Three senescence markers (p16, p21 and p15) and two SASP factors (Cxcl1 and Mmp14) were increased in hepatic tumor tissues of 74- or 100-weeks-old C3H mice without arsenite exposure, and treatment with a senolytic drug (ABT-263) diminished hepatic tumor formation. Gestational arsenite exposure enhanced the expression of p16, p21 and Mmp14 in F1 and p15 and Cxcl1 in F2, respectively. Exploring the mechanisms by which arsenite exposure promotes cellular senescence, we found that the expression of antioxidant enzymes (Sod1 and Cat) were reduced in the tumors of F1 in the arsenite group, and Tgf-ß and the receptors of Tgf-ß were increased in the tumors of F2 in the arsenite group. Furthermore, the analysis of the Cancer Genome Atlas database showed that gene expression levels of the senescence markers and SASP factors were increased and associated with poor prognosis in human hepatocellular carcinoma (HCC). These results suggest that cellular senescence and SASP have important roles in hepatic tumorigenesis in C3H mice as well as HCC in humans, and gestational arsenite exposure of C3H mice enhances senescence in F1 and F2 via oxidative stress and Tgf-ß activation, respectively.

Identification by TCGA database search of five genes that are aberrantly expressed and involved in hepatocellular carcinoma potentially via DNA methylation changes.

BACKGROUND: Various treatments for hepatocellular carcinoma (HCC) are utilized in clinical practice; however, the prognosis is still poor on account of high recurrence rates. DNA methylation levels of CpG islands around promoters (promoter CpGis) inversely regulate gene expression and closely involved in carcinogenesis. As a new strategy, several chemicals globally inhibiting DNA methylation have been developed aiming at reducing DNA methylation levels and maintaining the expression of tumor suppressor genes. On the other hand, since these drugs nonspecifically modify DNA methylation, they can cause serious adverse effects. In order to ameliorate the methods by targeting specific CpGs, information of cancer-related genes that are regulated by DNA methylation is required. METHODS: We searched candidate genes whose expressions were regulated by DNA methylation of promoter CpGi and which are involved in HCC cases. To do so, we first identified genes whose expression were changed in HCC by comparing gene expressions of 371 HCC tissues and 41 non-tumor tissues using the Cancer Genome Atlas (TCGA) database. The genes were further selected for poor prognosis by log-rank test of Kaplan-Meier plot and for cancer relevance by Pubmed search. Expression profiles of upregulated genes in HCC tissues were assessed by Gene Ontology (GO) analysis. Finally, using DNA methylation data of TCGA database, we selected genes whose promoter DNA methylation levels were inversely correlated with gene expression. RESULTS: We found 115 genes which were significantly up- or downregulated in HCC tissues and were associated with poor prognosis and cancer relevance. The upregulated genes were significantly enriched in cell division, cell cycle, and cell proliferation. Among the upregulated genes in HCC, we identified hypomethylation of CpGis around promoters of FANCB, KIF15, KIF4A, ERCC6L, and UBE2C. In addition, TCGA data showed that the tumor suppressor gene P16 is unexpectedly overexpressed in many types of cancers. CONCLUSIONS: We identified five candidate genes whose expressions were regulated by DNA methylation of promoter CpGi and associate with cancer cases and poor prognosis in HCC. Modification of site-specific DNA methylation of these genes enables a different approach for HCC treatment with higher selectivity and lower adverse effects.

DNA methylation changes involved in the tumor increase in F2 males born to gestationally arsenite-exposed F1 male mice.

Multigenerational adverse effects from the environment such as nutrition and chemicals are among important concerns in environmental health issues. Previously, we have found that arsenite exposure of only F0 females during their pregnancy increases hepatic tumors in the F2 males in C3H mice. In the current study, we investigated the association of DNA methylation with the hepatic tumor increase in the F2 males of the arsenite group. Reduced-representation bisulfite sequencing analysis newly identified that DNA methylation levels of regions around the transcriptional start sites of Tmem54 and Cd74 were decreased and the expression of these genes were significantly increased in the hepatic tumors of F2 males of the arsenite group. The associations between DNA methylation in these regions and gene expression changes were confirmed by treatment of murine hepatoma cell lines and hepatic stellate cell line with 5-aza-2'-deoxycytidine. Overexpression of Cd74 in Hepa1c1c7 cells increased Trib3 expression and suppressed the expression of tumor suppressor genes Id3 and Atoh8. Human database analysis using the Cancer Genome Atlas indicated that TMEM54, CD74, and TRIB3 were significantly increased and that ATOH8 was decreased in hepatocellular carcinoma. The data also showed that high expression of TMEM54 and TRIB3 and low expression of ATOH8 were associated with poor survival. These results suggested that an increase in Tmem54 and Cd74 expression via DNA methylation reduction was involved in the tumor increase in the F2 male offspring by gestational arsenite exposure of F0 females. This study also suggested that genes downstream of Cd74 were involved in tumorigenesis.

The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes.

BACKGROUND: C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. METHODS: We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. RESULTS: We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. CONCLUSIONS: Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information for further epigenetic studies of mice models of HCC. The present study particularly proposed novel DNA methylation-regulated pathways for Mst1r and Slpi, which may be applied not only to mouse HCC but also to human HCC.

Long-term arsenite exposure induces premature senescence in B cell lymphoma A20 cells.

Chronic arsenite exposure induces immunosuppression, but the precise mechanisms remain elusive. Our previous studies demonstrated that arsenite exposure for 24 h induces G0/G1 arrest in mouse B lymphoma A20 cells and the arrest is caused through induction of cyclin-dependent kinase inhibitor p16(INK4a) followed by accumulation of an Rb family protein, p130. In this study, we further investigated the consequences of long-term arsenite exposure of A20 cells. The results demonstrated that exposure to 10 µM sodium arsenite up to 14 days induces a great increase in G0/G1 arrest, irreversible cell growth suppression, cellular morphological changes and positive staining for senescence-associated ß-galactosidase. The long-term arsenite exposure also induced up-regulation of p16(INK4a) followed by robust accumulation of p130 and activation of the p53 pathway. Knockdown experiments with siRNA showed that p130 accumulation is essential for cell cycle arrest by long-term arsenite exposure. Since p16(INK4a) and the p53 pathway are known to be activated by DNA damage, we investigated the involvement of DNA damage formation by long-term arsenite exposure. We found that a variety of DNA repair-related genes were significantly down-regulated from 24 h of arsenite exposure and activation-induced cytidine deaminase was greatly up-regulated after long-term arsenite exposure. Consistent with these findings, long-term arsenite exposure increased a DNA double-strand break marker, γ-H2AX and increased mutation frequency in a Bcl6 gene region. These results revealed that long-term arsenite exposure induces premature senescence through DNA damage increase and p130 accumulation in lymphoid cells.

Augmenting effects of gestational arsenite exposure of C3H mice on the hepatic tumors of the F2 male offspring via the F1 male offspring.

Gestational exposure can affect the F2 generation through exposure of F1 germline cells. Previous studies reported that arsenite exposure of only F0 females during their pregnancy increases hepatic tumors in the F1 males in C3H mice, whose males are predisposed spontaneously to develop hepatic tumors later in life. The present study addressed the effects of gestational arsenite exposure on tumorigenesis of the F2 males in C3H mice. Expression analysis of several genes in the normal livers at 53 and 80 weeks of age clearly showed significant changes in the F2 males obtained by crossing gestational arsenite-exposed F1 (arsenite-F1) males and females compared to the control F2 males. Some of the changes were shown to occur in a late-onset manner. Then the tumor incidence was assessed at 75-82 weeks of age in the F2 males obtained by reciprocal crossing between the control and arsenite-F1 males and females. The results demonstrated that the F2 males born to arsenite-F1 males developed tumors at a significantly higher rate than the F2 males born to the control F1 males, irrespective of exposure of F1 females. Gene expressions of hepatocellular carcinoma markers ß-catenin (CTNNB1) and interleukin-1 receptor antagonist in the tumors were significantly upregulated in the F2 males born to arsenite-F1 males compared to those born to the control F1 males. These results show that arsenite exposure of only F0 pregnant mice causes late-onset changes and augments tumors in the livers of the F2 males by affecting the F1 male offspring.

[Biological effects of arsenic and diseases: The mechanisms involved in arsenic-induced carcinogenesis].

Chronic arsenic exposure is associated with many diseases, including cancers. Our study using in vivo assay in gpt-delta transgenic mice showed that arsenic particularly induces G : C to T : A transversions, a mutation type induced through oxidative-stress-induced 8-OHdG formation. Gestational arsenic exposure of C3H mice was reported to increase hepatic tumor incidence. We showed that gestational arsenic exposure increased hepatic tumors having activated oncogene Ha-ras by C to A mutation. We also showed that DNA methylation status of Fosb region is implicated in tumor augmentation by gestational arsenic exposure. We further showed that long-term arsenic exposure induces premature senescence. Recent studies reported that senescence is involved in not only tumor suppression, but also tumorgenesis. All these effects of arsenic might be involved in arsenic-induced carcinogenesis.

The effect of a methyl-deficient diet on the global DNA methylation and the DNA methylation regulatory pathways.

Methyl-deficient diets are known to induce various liver disorders, in which DNA methylation changes are implicated. Recent studies have clarified the existence of the active DNA demethylation pathways that start with oxidization of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine by ten-eleven translocation (Tet) enzymes, followed by the action of base-excision-repair pathways. Here, we investigated the effects of a methionine-choline-deficient (MCD) diet on the hepatic DNA methylation of mice by precisely quantifying 5meC using a liquid chromatography-electrospray ionization-mass spectrometry and by investigating the regulatory pathways, including DNA demethylation. Although feeding the MCD diet for 1 week induced hepatic steatosis and lower level of the methyl donor S-adenosylmethionine, it did not cause a significant reduction in the 5meC content. On the other hand, the MCD diet significantly upregulated the gene expression of the Tet enzymes, Tet2 and Tet3, and the base-excision-repair enzymes, thymine DNA glycosylase and apurinic/apyrimidinic-endonuclease 1. At the same time, the gene expression of DNA methyltransferase 1 and a, was also significantly increased by the MCD diet. These results suggest that the DNA methylation level is precisely regulated even when dietary methyl donors are restricted. Methyl-deficient diets are well known to induce oxidative stress and the oxidative-stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine (8OHdG), is reported to inhibit DNA methylation. In this study, we also clarified that the increase in 8OHdG number per DNA by the MCD diet is approximately 10 000 times smaller than the reduction in 5meC number, suggesting the contribution of 8OHdG formation to DNA methylation would not be significant.

Inorganic arsenic exposure induces E2F-dependent G0/G1 arrest via an increase in retinoblastoma family protein p130 in B-cell lymphoma A20 cells.

Inorganic arsenic exerts toxic effect on multiple systems including the immune system. We previously showed in a study on mouse thymocytes and B-cell lymphoma A20 cells that arsenite induces cell cycle arrest at G0/G1 by suppressing expression of E2F-target genes. In this study, we furthermore investigated the involvement of retinoblastoma (RB) family proteins in E2F-dependent cell cycle arrest by arsenite. Arsenite exposure of A20 cells was showed to increase the protein level of p130, a RB family member, without changing the mRNA level. Suppression of arsenite-induced p130 by siRNA reduced the G0/G1 phase, indicating that p130 accumulation is responsible for arsenite-induced G0/G1 arrest. The accumulated p130 was shown to increase the p130 complex with E2F4, a transcription-suppressing E2F. Comparison by Western blotting of arsenite-induced p130 and p130 accumulated by a proteasome inhibitor suggested that arsenite-induced p130 is hypophosphorylated and hypoubiquitinated and refractory to proteasome-dependent degradation. We also showed that arsenite increases mRNA and protein of p16(INK4a), an inhibitor of CDK4/6 that phosphorylates p130. Down-regulation of arsenite-induced p16(INK4a) by siRNA suppressed the p130 accumulation. We propose a novel mechanism in which arsenite inhibits phosphorylation/ubiquitin-dependent proteasome degradation of p130 by inducing p16(INK4a) and the accumulated p130 causes cell cycle arrest with E2F4.

Potential differences in chitin synthesis ability cause different sensitivities to diflubenzuron among three strains of Daphnia magna.

Ecotoxicity testing of crustaceans using Daphnia magna has been implemented in the chemical management systems of various countries. While the chemical sensitivity of D. magna varies depending on genetically different clonal lineages, the strain used in ecotoxicity tests, including the acute immobilization test (OECD TG202), has not been specified. We hypothesized that comprehensive gene expression profiles could provide useful information on phenotypic differences among strains, including chemical sensitivity. To test this hypothesis, we performed mRNA sequencing on three different strains (NIES, England, and Clone 5) of D. magna under culture conditions. The resulting expression profile of the NIES strain was clearly different compared to the profiles of the other two strains. Gene ontology (GO) enrichment analysis suggested that chitin metabolism was significantly enriched in the NIES strain compared to that in the England strain. Consistent with the GO analysis, evidence of high levels of chitin metabolism in the NIES strain were observed across multiple levels of biological organization, such as expression of chitin synthase genes, chitin content, and chitinase activity, which suggested that the different strains would exhibit different sensitivities to chemicals used to inhibit chitin synthesis. We found that among all strains, the NIES strain was more tolerant to diflubenzuron, a chitin synthesis inhibitor, with a 14-fold difference in the 48 h-EC50 value for the acute immobilization test compared to the England strain. The present study demonstrates that the differences among strains in chitin metabolism may lead to sensitivity difference to diflubenzuron, and serves as a case study of the usefulness of comprehensive gene expression profiles in finding sensitivity differences.

Gestational arsenic exposure induces site-specific DNA hypomethylation in active retrotransposon subfamilies in offspring sperm in mice.

BACKGROUND: Environmental impacts on a fetus can disrupt germ cell development leading to epimutations in mature germ cells. Paternal inheritance of adverse health effects through sperm epigenomes, including DNA methylomes, has been recognized in human and animal studies. However, the impacts of gestational exposure to a variety of environmental factors on the germ cell epigenomes are not fully investigated. Arsenic, a naturally occurring contaminant, is one of the most concerning environmental chemicals, that is causing serious health problems, including an increase in cancer, in highly contaminated areas worldwide. We previously showed that gestational arsenic exposure of pregnant C3H mice paternally induces hepatic tumor increase in the second generation (F2). In the present study, we have investigated the F1 sperm DNA methylomes genome-widely by one-base resolution analysis using a reduced representation bisulfite sequencing (RRBS) method. RESULTS: We have clarified that gestational arsenic exposure increases hypomethylated cytosines in all the chromosomes and they are significantly overrepresented in the retrotransposon LINEs and LTRs, predominantly in the intergenic regions. Closer analyses of detailed annotated DNA sequences showed that hypomethylated cytosines are especially accumulated in the promoter regions of the active full-length L1MdA subfamily in LINEs, and 5'LTRs of the active IAPE subfamily in LTRs. This is the first report that has identified the specific positions of methylomes altered in the retrotransposon elements by environmental exposure, by genome-wide methylome analysis. CONCLUSION: Lowered DNA methylation potentially enhances L1MdA retrotransposition and cryptic promoter activity of 5'LTR for coding genes and non-coding RNAs. The present study has illuminated the environmental impacts on sperm DNA methylome establishment that can lead to augmented retrotransposon activities in germ cells and can cause harmful effects in the following generation.

Tumor-augmenting effects of gestational arsenic exposure on F1 and F2 in mice.

The consequences of early-life exposure to chemicals in the environment are emerging concerns. Chronic exposure to naturally occurring inorganic arsenic has been known to cause various adverse health effects, including cancers, in humans. On the other hand, animal studies by Dr. M. Waalkes' group reported that arsenite exposure of pregnant F0 females, only from gestational day 8 to 18, increased hepatic tumors in the F1 (arsenite-F1) males of C3H mice, whose males tend to develop spontaneous hepatic tumors later in life. Since this mice model illuminated novel unidentified consequences of arsenic exposure, we wished to further investigate the background mechanisms. In the same experimental model, we identified a variety of factors that were affected by gestational arsenic exposure, including epigenetic and genetic changes, as possible constituents of multiple steps of late-onset hepatic tumor augmentation in arsenite-F1 males. Furthermore, our study discovered that the F2 males born to arsenite-F1 males developed hepatic tumors at a significantly higher rate than the control F2 males. The results imply that the tumor augmenting effect is inherited by arsenite-F2 males through the sperm of arsenite-F1. In this article, we summarized our studies on the consequences of gestational arsenite exposure in F1 and F2 mice to discuss novel aspects of biological effects of gestational arsenic exposure.

Increase in incidence of hepatic tumors caused by oncogenic somatic mutation in mice maternally exposed to inorganic arsenic and the multigenerational and transgenerational effects of inorganic arsenic.

Inorganic arsenic is a natural environmental contaminant and known to be a human carcinogen. Although rodent models are pivotal in elucidating the mode of action of arsenic, it has been difficult to verify the carcinogenicity of arsenic in rodents until recently. Waalkes et al. (Toxicol Appl Pharmacol 2003;186:7-17) reported that maternal exposure to arsenite increases the incidence of hepatic tumors in the male pups of C3H mice in adulthood. This finding indicated that the gestational period is vulnerable to arsenic. Using the same experimental model, we found that maternal arsenite exposure increases the incidence of hepatic tumors caused by a somatic mutation of the C61A Ha-ras gene, which encodes an activated oncogenic Ha-ras protein. The G:C to T:A transversion is attributable to oxidative stress. Our further studies of gpt delta transgenic mice, which enable detection of in vivo mutation, and genome-wide analysis of DNA methylation levels using the methylated DNA immunoprecipitation-CpG island microarray method suggest that oxidative-stress-induced mutation and DNA methylation changes are involved in the tumor augmentation in the pups maternally exposed to arsenic. Our recent study has also suggested that maternal arsenic exposure increases the incidence of hepatic tumors even in the grandchildren (the F2 generation). Consideration should be given to multigenerational and transgenerational effects of maternal exposure in future studies.

Late-onset increases in oxidative stress and other tumorigenic activities and tumors with a Ha-ras mutation in the liver of adult male C3H mice gestationally exposed to arsenic.

Tumorigenesis is a complex process involving genetic, epigenetic, and metabolic alterations. Gestational arsenic exposure has been shown to increase hepatic tumors in adult male offspring of C3H mice, which spontaneously develop hepatic tumors often harboring activating Ha-ras mutation. We explored tumor-promoting changes by gestational arsenic exposure with a focus on Ha-ras mutation and gene expression changes. The results of this study demonstrated that gestational arsenic exposure particularly increased hepatic tumors with a C61A Ha-ras mutation. Real-time PCR analyses on the adult normal livers showed that two genes (Creld2, Slc25a30), whose expression are induced by endoplasmic reticulum stress and cellular oxidative stress, respectively, were significantly upregulated and two genes (Fabp4, Ell3), whose products are involved in lipid efflux and apoptosis, respectively, were significantly downregulated more than twofold by gestational arsenic exposure compared with control mice. The expression changes in the four genes were shown to be late-onset events and to some extent to be associated with corresponding histone modifications, and not with DNA methylation changes. The gene expression changes suggested alterations in lipid metabolism and associated oxidative stress augmentation. Consistently, expression of an oxidative-stress-inducible gene heme oxygenase-1 (HO-1) was upregulated in the livers of the arsenic group. We also found increased expression of retrotransposon L1 mRNA in the tumor-bearing livers of the arsenic group in comparison with control mice. These results suggested that gestational arsenic exposure induces tumor-augmenting changes, including oxidative stress and L1 activation, in a late-onset manner, which would particularly promote tumorigenic expansion of cells with a C61A Ha-ras mutation.