Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.

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.

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 β-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.

No abstract

Activation of aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in T-cells is required for TCDD-induced suppression of the allogeneic CTL response and for induction of CD25(hi)CD62L(low) adaptive regulatory T-cells. Here, the ability of a constitutively-active AhR (CA-AhR) expressed in T-cells alone to replicate the effects of TCDD was examined. The response of CA-AhR-expressing B6 donor T-cells in B6xD2F1 mice was compared to the response of wild-type B6 donor T-cells in B6xD2F1 mice given a single dose of TCDD. Expression of CA-AhR in donor T-cells enhanced the down-regulation of CD62L on Day 2 after injection, similar to a single oral dose of TCDD, but did not induce up-regulation of CD25 on Day 2 or affect CTL activity on Day 10. This suggests that activation of AhR in T-cells alone may not be sufficient to alter T-cell responses in this acute graft-versus-host (GvH) model. Since host APC are responsible for activating the donor T-cells, we examined the influence of the F1 host's AhR on donor T-cell responses by creating an AhR(-/-) B6xD2F1 host that had a greatly diminished AhR response to TCDD compared to wild-type F1 mice. As in AhR(+/+) B6xD2F1 mice, the CTL response in AhR(-/-) B6xD2F1 mice was completely suppressed by TCDD. This suggests that either CA-AhR dose not fully replicate the function of TCDD-activated AhR in suppression of the CTL response, or that minimal activation of AhR in host cells is required to combine with activation of AhR in T-cells to elicit the immunosuppressive effects of TCDD.