Transplacental exposure to inorganic arsenic at a hepatocarcinogenic dose induces fetal gene expression changes in mice indicative of aberrant estrogen signaling and disrupted steroid metabolism
Introduction
Inorganic arsenic is a human carcinogen, associated with tumors of the skin, urinary bladder, lung, liver, prostate, kidney, and possibly other sites (NRC, 2001, Morales et al., 2000, Centeno et al., 2002, IARC, 2004). We have shown that short-term exposure in mice to inorganic arsenic in utero produces a variety of internal tumors in the offspring when they reach adulthood (Waalkes et al., 2003, Waalkes et al., 2004a, Waalkes et al., 2006a, Waalkes et al., 2006b). Gestation is a period of high sensitivity to chemical carcinogenesis in rodents and probably in humans (Anderson et al., 2000). Inorganic arsenic can readily cross the rodent and human placenta and enter the fetus (Concha et al., 1998, NRC, 2001). After in utero exposure to inorganic arsenic at carcinogenic doses, significant amounts of inorganic arsenic and its methylated metabolites (DMA and MMA) are detected in various mouse fetal tissues including the liver (Devesa et al., 2006). In arsenic-exposed human populations all life stages of exposure are involved (IARC, 2004). Thus, it is likely that significant in utero arsenic exposure occurs in human populations, and it is prudent to assume that the transplacental carcinogenic risks defined in rodents may predict similar effects in humans.
The liver is a major target organ of arsenic toxicity (Lu et al., 2001, Mazumder, 2005) and carcinogenesis in humans (Chen et al., 1997, Zhou et al., 2002, Centeno et al., 2002, Chen and Ahsan, 2004). In accord with human data, transplacental exposure to inorganic arsenic induced a marked, dose-related increase in hepatocellular tumors, including carcinoma, in adult male mice (Waalkes et al., 2003, Waalkes et al., 2004a, Waalkes et al., 2006b). Genomic analysis of liver samples taken at necropsy 1–2 years after gestational arsenic exposure alone or combined with postnatal exposure to 12-O-teradecanoyl phorbol-13-acetate (TPA) revealed a variety of hepatic genes to be aberrantly expressed in adulthood, including genes critical to the carcinogenic process (Liu et al., 2004, Liu et al., 2006a, Liu et al., 2006b). Although the expression changes in adult mouse liver are clearly associated with liver tumors, whether they are involved in cancer causation specifically by arsenic cannot be defined in fully developed tumors. Thus, genomic analysis of early molecular events following gestational arsenic exposure is clearly warranted.
The spectrum of tumors and/or proliferative lesions induced by in utero arsenic exposure, including tumors of liver, ovary, adrenal, uterus and oviduct, resembles the potential targets of carcinogenic estrogens (Waalkes et al., 2003, Waalkes et al., 2004a, Waalkes et al., 2006a, Waalkes et al., 2006b). This has led us to the hypothesis that arsenic could somehow produce estrogen-like effects, possibly through estrogen receptor-alpha (ER-α), as part of the mechanisms causing tumor formation (Waalkes et al., 2004b). Aberrant over-expression of ER-α is associated with a variety of human and rodent tumors (Fishman et al., 1995). Indeed, in livers and liver tumors from male mice exposed to arsenic in utero, the over-expression of the ER-α and estrogen-linked cyclin D1 is a prominent feature, and a feminized pattern of hepatic metabolic enzyme genes is evident (Waalkes et al., 2004b, Liu et al., 2004, Liu et al., 2006b). Samples of human livers from populations highly exposed to inorganic arsenic also show ER-α over-expression (Waalkes et al., 2004b).
Thus, this study investigated aberrant gene expression in the fetal male livers following in utero exposure to a hepatocarcinogenic dose of arsenic. Global genomic analysis was performed through the National Center for Toxicogenomics, using the Agilent 22K chip array. Expression of key genes was followed up by real-time RT-PCR analysis. This study clearly showed that in utero arsenic exposure produced dramatic alterations in gene expression in fetal liver, providing evidence for enhanced estrogen signaling and aberrant steroid metabolism in the developing fetus as a result of transplacental arsenic exposure. This arsenic-induced early life stage disruption of genetic programming could potentially lead to tumor formation much later in adulthood.
Section snippets
Chemicals
Sodium arsenite (NaAsO2) was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in the drinking water at 85 mg arsenic/L (85 ppm). The Agilent 22K mouse oligo array was obtained from Agilent Technologies (Palo Alto, CA).
Animal treatment and sample collection
Timed pregnant C3H mice were given drinking water containing 85 ppm arsenic or unaltered water ad libitum from day 8 to day 18 of gestation. At day 18 of gestation, mice were killed by CO2 asphyxiation and fetuses removed. Only male fetal livers were used for the
Microarray analysis of aberrantly expressed genes
Total RNA from male fetal liver samples of control and arsenic-exposed mice was subjected to microarray analysis. Under the criteria of p < 0.001 by the Rosetta Resolver (v4.0) system, the transcript levels of 187 genes among 22,000 contained on the array were significantly altered by arsenic exposure compared to control. Clustering analysis of these altered genes is shown in Fig. 1. A cluster of increased genes (shown in red) included various genes related to estrogen signaling, and a cluster of
Discussion
The hypothesis that inorganic arsenic might somehow act through aberrant activation of estrogen signaling pathways (Waalkes et al., 2004b) comes from several lines of evidence. This includes the fact that the transplacental arsenic carcinogenesis shows consistent targets (i.e., liver, ovary, adrenal, uterus) which are also targets of broad range or tissue-selective carcinogenic estrogens (Birnbaum and Fenton, 2003, Newbold, 2004). In addition, estrogen-linked gene/protein over-expressions are
Acknowledgments
The authors thank Drs. Erik Tokar, Ronald Cannon and Larry Keefer for their critical review of the manuscript. Research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, the Federal funds from the National Cancer Institute, National Institutes of Health, under contract No. NO1-CO-12400, and the National Center for Toxicogenomics at NIEHS. The content of this publication does not necessarily reflect the views or policies
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