Altered estrogen signaling may play a role in induction of HCC by arsenic exposure in utero. Specifically, overexpression of ER-alpha, potentially through promoter region hypomethylation, in livers of such mice may be linked to the hepatocarcinogenicity of arsenic.

Our prior work showed that brief exposure of pregnant C3H mice to inorganic arsenic-induced hepatocellular carcinoma (HCC) formation in adult male offspring. The current study examined the early hepatic events associated with this oncogenic transformation. Pregnant mice were exposed to a known carcinogenic dose of arsenic (85 ppm) in the drinking water from gestation days 8 to 18. The dams were allowed to give birth and liver samples from newborn males were analyzed for arsenic content, global DNA methylation and aberrant expression of genes relevant to the carcinogenic process. Arsenic content in newborn liver reached 57 ng/g wet weight, indicating arsenic had crossed the placenta, reached the fetal liver and that significant amounts remained after birth. Global methylation status of hepatic DNA was not altered by arsenic in the newborn. However, a significant reduction in methylation occurred globally in GC-rich regions. Microarray and real-time RT-PCR analysis showed that arsenic exposure enhanced expression of genes encoding for glutathione production and caused aberrant expression of genes related to insulin growth factor signaling pathways and cytochrome P450 enzymes. Other expression alterations observed in the arsenictreated male mouse newborn liver included the overexpression of cdk-inhibitors and stress response genes including increased expression of metallothionein-1 and decreased expression of betainehomocysteine methyltransferase and thioether S-methyltransferase. Thus, transplacental exposure to arsenic at a hepatocarcinogenic dose induces alterations in DNA methylation and a complex set of aberrant gene expressions in the newborn liver, a target of arsenic carcinogenesis.

Our previous work has shown that exposure to inorganic arsenic in utero produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation. The incidence of HCC in adult male offspring was increased 4-fold and tumor multiplicity 3-fold after transplacental arsenic exposure. Samples of normal liver and liver tumors were taken at autopsy for genomic analysis. Arsenic exposure in utero resulted in significant alterations (p < 0.001) in the expression of 2,010 genes in arsenic-exposed liver samples and in the expression of 2,540 genes in arsenic-induced HCC. Ingenuity Pathway Analysis revealed that significant alterations in gene expression occurred in a number of biological networks, and Myc plays a critical role in one of the primary networks. Real-time reverse transcriptase–polymerase chain reaction and Western blot analysis of selected genes/proteins showed > 90% concordance. Arsenic-altered gene expression included activation of oncogenes and HCC biomarkers, and increased expression of cell proliferation–related genes, stress proteins, and insulin-like growth factors and genes involved in cell–cell communications. Liver feminization was evidenced by increased expression of estrogen-linked genes and altered expression of genes that encode gender-related metabolic enzymes. These novel findings are in agreement with the biology and histology of arsenic-induced HCC, thereby indicating that multiple genetic events are associated with transplacental arsenic hepatocarcinogenesis.

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

Previous research demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment increased the number of skin papillomas in v-Ha-ras transgenic (Tg.AC) mice that had received sodium arsenite [(As(III)] in drinking water, indicating that this model is useful for studying the toxic effects of arsenic in vivo. Because the liver is a known target of arsenic, we examined the pathophysiologic and molecular effects of inorganic and organic arsenical exposure on Tg.AC mouse liver in this study. Tg.AC mice were provided drinking water containing As(III), sodium arsenate [As (

Exposure to inorganic arsenic in utero in C3H mice produces hepatocellular carcinoma in male offspring when they reach adulthood. To help define the molecular events associated with the fetal onset of arsenic hepatocarcinogenesis, pregnant C3H mice were given drinking water containing 0 (control) or 85 ppm arsenic from day 8 to 18 of gestation. At the end of the arsenic exposure period, male fetal livers were removed and RNA isolated for microarray analysis using 22K oligo chips. Arsenic exposure in utero produced significant (p<0.001) alterations in expression of 187 genes, with approximately 25% of aberrantly expressed genes related to either estrogen signaling or steroid metabolism. Real-time RT-PCR on selected genes confirmed these changes. Various genes controlled by estrogen, including X-inactive-specific transcript, anterior gradient-2, trefoil factor-1, CRP-ductin, ghrelin, and small proline-rich protein-2A, were dramatically over-expressed. Estrogen-regulated genes including cytokeratin 1-19 and Cyp2a4 were over-expressed, although Cyp3a25 was suppressed. Several genes involved with steroid metabolism also showed remarkable expression changes, including increased expression of 17beta-hydroxysteroid dehydrogenase-7 (HSD17beta7; involved in estradiol production) and decreased expression of HSD17beta5 (involved in testosterone production). The expression of key genes important in methionine metabolism, such as methionine adenosyltransferase-1a, betaine-homocysteine methyltransferase and thioether S-methyltransferase, were suppressed. Thus, exposure of mouse fetus to inorganic arsenic during a critical period in development significantly alters the expression of various genes encoding estrogen signaling and steroid or methionine metabolism. These alterations could disrupt genetic programming at the very early life stage, which could impact tumor formation much later in adulthood.

Multidrug-resistance gene knockout mdr1a/1b(-/-) mice, which are deficient in P-glycoproteins, are more sensitive than wild-type (WT) mice to acute arsenic toxicity. This study assessed toxic manifestations of chronic oral arsenic in mdr1a/1b(-/-) mice, including oxidative stress and altered gene expression, and investigated altered toxicokinetics as a potential basis of enhanced arsenic toxicity. Thus, mdr1a/1b(-/-) and WT mice were exposed to sodium arsenite (0-80 ppm as arsenic) in the drinking water for 10 weeks at which time hepatic arsenic accumulation, lipid peroxidation (LPO), redox status and change in gene expression level were assessed. All mice survived the arsenic exposure, but body weight gain in the highest dose group was reduced in both mdr1a/1b(-/-) and WT mice. Arsenic induced pathological changes, elevated LPO levels and enhanced glutathione S-transferase (GST) activity, in the liver to a greater extent in mdr1a/1b(-/-) than in WT mice. Arsenic also decreased Cu/Zn superoxide dismutase activity in both mdr1a/1b(-/-) and WT mice. The expressions of certain genes, such as those encoding cell proliferation, GST, acute-phase proteins and metabolic enzymes, were modestly altered in arsenic-exposed mice. The expression of cyclin D1, a potential hepatic oncogene, was enhanced in arsenic-exposed mdr1a/1b(-/-) mice only. At the highest level of exposure, hepatic arsenic content was higher in mdr1a/1b(-/-) than in WT mice, suggesting that enhanced accumulation due to transport deficiency may, in part, account for the enhanced toxicity in these mice. In summary, this study shows that chronic arsenic toxicity, including liver pathology and oxidative stress, is enhanced in mdr1a/1b(-/-) mice, possibly due to enhanced accumulation of arsenic as a result of transport system deficiency.

BackgroundInorganic arsenic (iAs) is a well-known human carcinogen recognized by the World Health Organization and the International Agency for Research on Cancer. Currently, most iAs studies in populations are concerned with drinking water and occupational arsenicosis. In Guizhou province, arsenicosis caused by the burning of coal in unventilated indoor stoves is an unusual type of exposure. Because the poisoning mechanism involved in arsenicosis is as yet unknown and no effective therapy exists, progress has been slow on the prevention and therapy of arsenicosis.ObjectivesWe examined the relationship between arsenic (As) exposure from the burning of coal in unventilated indoor stoves and genetic damage in humans, using cellular and molecular indices. We selected villagers from Jiaole township, Guizhou province, China, who had been exposed to milligram levels of As daily via food and air contaminated by the burning of As-containing coal in unventilated indoor stoves.ResultsThe As-exposed subjects from Jiaole were divided into four groups according to skin lesion symptoms: nonpatients, mild, intermediate, and severe arsenicosis. Another 53 villagers from a town 12 km from Jiaole were recruited as the external control group. In the four groups of exposed subjects, As concentrations in urine and hair were 76–145 μg/L and 5.4–7.9 μg/g, respectively. These values were higher than those in the external control group, which had As concentrations of 46 μg/L for urine and 1.6 μg/g for hair. We measured sister chromatid exchange and chromosomal aberrations to determine human chromosome damage, and for DNA damage, we measured DNA single-strand breaks and DNA–protein cross-links. All measurements were higher in the four exposed groups compared with the external control group. DNA repair was impaired by As exposure, as indicated by the mRNA of O-6-methylguanine-DNA methyltransferase (MGMT), X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1), and, to a lesser extent, by the mismatch repair gene hMSH2 mRNA. The expression of mutant-type p53 increased with aggravation of arsenicosis symptoms, whereas the expression of p16-INK4(p16) decreased. p53 mutated at a frequency of 30–17% in the carcinoma (n = 10) and precarcinoma (n = 12) groups. No mutation was found in p16, although deletion was evident. Deletion rates were 8.7% (n = 23) and 38.9% (n = 18) in noncarcinoma and carcinoma groups, respectively.ConclusionsThe results showed that long-term As exposure may be associated with damage of chromosomes and DNA, gene mutations, gene deletions, and alterations of DNA synthesis and repair ability.