This study demonstrated that subchronic exposure of transgenic (Tg.AC) mice to both inorganic and organic arsenicals through drinking water produced various effects on the liver, a major target organ of arsenic toxicity and carcinogenesis (Centeno et al. 2002
; NRC 1999
; Waalkes et al. 2003
). Arsenic-induced toxicity was evidenced by an increase in moribundity and death, a depression in body weight, hepatic pathological changes, and significant changes in gene expression.
An original goal of our research was to examine the effects of inorganic and organic arsenic on TPA-promoted skin papilloma development in Tg.AC mice. Although TPA was administered to all mice (including controls that received no arsenic), the effects of this skin tumor promoter were not deemed critical to our analyses of liver pathology, DNA methylation, and gene expression. Interestingly, topical application of TPA in some experimental models has systemic effects; we recently found that it promoted liver tumors initiated by transplacental arsenic exposure in female mice (Waalkes et al. 2004b
). In this study epidermal TPA treatment resulted in no mortality and did not affect hepatic pathology, indicating that the biological end points/ changes measured are most likely dependent on arsenical treatment alone.
Because the liver is a major target organ of arsenic toxicity and carcinogenesis (Waalkes et al. 2000b
), we examined gene expression as well as pathological changes in the livers of Tg.AC mice to further explore the usefulness of this system as an in vivo
model of arsenic carcinogenesis and toxicity. To detect gene expression changes that may be related to arsenic toxicity, animals treated with the maximal dose of each arsenical were selected for analysis. Generally, 150 ppm As(III) produced more toxicity and more dramatic changes in gene expression than 200 ppm As(V). Organic arsenicals at doses [1,500 and 1,000 ppm as arsenic for MMA(V) and DMA(V), respectively] 5-to 10-fold higher produced toxic effects comparable to those produced by As(III). Although rats are tolerant to 200 ppm MMA(V) in drinking water for 104 weeks (Shen et al. 2003a
), the mice in our study did not tolerate MMA(V) at 1,500 ppm, as 40% mortality (i.e., moribundity and death) occurred in these mice over the 17-week exposure period. The dose of DMA(V) in this study was also higher than the doses (50 and 200 ppm) used to induce urinary bladder tumors in rats (Wei et al. 2002
) and also exceeded the maximum tolerated dose, as it produced 20% mortality.
In our study, promoted and nonpromoted, arsenic-treated Tg.AC mice did not display direct evidence of liver tumor formation. However, preneoplastic lesions (e.g., cell proliferation) occur in the liver after chronic oral arsenic exposures in several strains of mice (Chen et al. 2004
; Shen et al. 2003a
; Waalkes et al. 2000b
) and were also observed in the liver of Tg.AC mice exposed to arsenic in this study. Exposure to arsenic in the drinking water resulted in a dose-dependent accumulation of arsenic in the liver that was independent of chemical form. The highest hepatic content, which was observed in the high-dose (1,500 ppm) MMA(V) group, might contribute to the high degree of mortality (40%) in this group. The hepatic arsenic contents in the Tg.AC mice receiving 150 ppm As(III) and 200 ppm As(V) in this study were 1.2 and 2.0 μg/g tissue, respectively. This was less than the arsenic content in the skin (8.3 μg/g tissue) and much less than that in the hair (170.2 μg/g tissue) of Tg.AC mice exposed to 200 ppm As(III) in the drinking water for 14 weeks in our previous study (Germolec et al. 1998
), indicating that arsenic accumulation in the liver is lower than that in the hair or skin. This may be because liver is the major target organ for arsenic metabolism, and arsenic elimination generally occurs through the bile (Gregus et al. 2000
) or urine.
DNA hypomethylation occurs after chronic arsenic exposure in cells (Zhao et al. 1997
) and also in intact animals (Chen et al. 2004
; Okoji et al. 2002
). In the present study, all arsenicals produced significant DNA hypomethylation in the liver, regardless of dose. Although the doses of MMA(V) (1,500 ppm) and DMA(V) (1,000 ppm) used in our study were much higher than those of As(III) (150 ppm) and As(V) (200 ppm), MMA(V) and DMA(V) induced less hypomethylation of hepatic DNA than As(III) and As(V). This suggests that inorganic arsenicals are more potent stimulators of DNA hypomethylation compared with MMA(V) and DMA(V). It should be noted that global DNA hypomethylation could co-exist with regional or individual gene hypermethylation, as arsenic-induced p53 hypermethylation has been reported (Mass and Wang 1997
). In our recent study, we proposed that arsenic-induced hypomethylation of the estrogen receptor-α gene plays an important role in hepatocellular proliferation (Chen et al. 2004
; Waalkes et al. 2004a
). Efforts are currently being undertaken to examine the methylation status of individual genes after arsenic exposure.
DNA hypomethylation is an important mechanism involved in aberrant gene expression and carcinogenesis (Baylin et al. 1998
; Goodman and Watson 2002
). In particular, it is thought that aberrant DNA methylation is central to the development of liver cancers (Goodman and Watson 2002
) and is an epigenetic mechanism that underlines the aberrant expression of genes involved in mouse liver carcinogenesis (Counts et al. 1997
). In the present study, As(III), As(V), MMA(V), and DMA(V) produced variable gene expression changes, accounting for approximately 10% of genes on the array. We focused primarily on a few categories, for example, glutathione (GSH)-, apoptosis-, and cell proliferation–related genes, and genes important for tumor development, as previous studies have shown these to be related to aberrant cell growth and neoplasia.
Glutathione systems play important roles in arsenic toxicity and carcinogenesis (NRC 1999
; Trouba et al. 2002
; Xie et al. 2004
). In the present study, the expression of GST-μ, GST-π, GST-α, and GST-τ was increased by all arsenicals, although to a variable extent. GSTs are a group of enzymes catalyzing the conjugation and oxidation of GSH with arsenic (Xie et al. 2004
). An increase in GST expression/activity (particularly GST-π) has been reported to play an important role in cellular efflux of arsenic–GSH conjugates and to be a mechanism of arsenic tolerance (Brambila et al. 2002
; Liu et al. 2001a
; Wang et al. 1996
). Increases in GST-π positive foci have been proposed to be a hepatic preneoplastic biomarker in chronic arsenic-exposed populations (Nishikawa et al. 2002
; Shen et al. 2003a
). Changes in GST activity in humans also are associated with altered arsenic metabolism (Chiou et al. 1997
; Marnell et al. 2003
), and GST polymorphisms are thought to be a susceptibility factor for arsenic toxicity in humans (Marnell et al. 2003
). Together, these data indicate that increases in GST gene expression and/or function are consistent events associated with arsenic carcinogenicity and toxicity.
Arsenic induces apoptosis involved in its mechanism of acute toxicity (NRC 1999
). However, after chronic arsenic exposure and the induction of malignant transformation, the development of apoptosis resistance occurs (Brambila et al. 2002
; Qu et al. 2002
) and is associated with the downregulation of apoptosis-related genes (Brambila et al. 2002
; Chen et al. 2001a
). In the present study, arsenical exposure resulted in downregulation of apoptosis-associated genes such as FasL
, tumor necrosis factor receptor–associated factor 3, Bad
, and granzyme A, and also increased the expression of cell proliferation–related genes including c-myc
, proliferating cell nuclear antigen, and fibroblast growth factor 2. These data are interesting in light of evidence that apoptosis tolerance and cell proliferation are important mechanisms involved in chemical carcinogenesis (Waalkes et al. 2000a
), including arsenic. Apoptosis tolerance also is accompanied by cell proliferation, as seen in arsenic-transformed cells (Brambila et al. 2002
; Chen et al. 2001b
; Qu et al. 2002
) in chronic arsenic-exposed animals (Chen et al. 2004
; Xie et al. 2004
), and in liver tumor and nontumor tissues from mice exposed to arsenic in utero
(Liu et al. 2004
; Waalkes et al. 2003
). Thus, the depression of apoptosis genes and the overexpression of cell proliferation genes could be important in arsenic toxicity and carcinogenesis.
Liver is a major target of arsenic carcinogenesis in transplacentally exposed animal models (Waalkes et al., 2003
) and in arsenic-exposed humans (Centeno et al. 2002
; Chen et al. 1997
; Zhou et al. 2002
). The expression of α-fetoprotein (AFP
), a biomarker for hepatocellular carcinogenesis, was increased in transplacental arsenic-induced hepatocellular carcinoma (HCC) and tumor-surrounding tissues (Liu et al. 2004
). In the present study, all the arsenicals tested increased AFP
expression up to 3-fold in MMA(V)-treated mice. The enhanced expression of AFP
lends further support that preneoplastic alterations occur after subchronic arsenic exposure. Other notable alterations in gene expression were the overexpression of IGFBP-1
and suppression of insulin-like growth factor 1 (IGF-1
). Chronic exposure to nongenotoxic chemicals such as oxazepam and Wyeth-14,643 increased the expression of IGFBP-1
in a time-dependent manner (Iida et al. 2003
), and overexpression of IGFBP-1
was also seen in transplacental arsenic-induced HCC and tumor-surrounding tissues (Liu et al. 2004
). Dysregulation of the IGF axis has been implicated in liver tumor formation and progression (Scharf et al. 2001
). Thus, sub-chronic exposure to arsenicals can produce aberrant gene expression related to hepatocarcinogenesis, some of which were confirmed in the present study.
In summary, this study demonstrated that subchronic exposure to As(III), As(V), MMA(V), and DMA(V) in the drinking water resulted in variable toxic effects, accumulation of arsenic in the liver, hepatic global DNA hypomethylation, and alterations in gene expression in Tg.AC mice. These findings indicate that liver is a target organ of subchronic arsenical exposure in this model and support the idea that altered DNA methylation and its effects on gene expression may contribute in an epigenetic manner to arsenic carcinogenesis.