* Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, NCI at NIEHS, Research Triangle Park, NC 27709;
Veterinary and Tumor Pathology Section, Office of Laboratory Animal Science, NCI-Frederick, Frederick, MD 21702; and
Basic Research Program, SAIC-Frederick, Frederick, MD 21702
Received July 22, 2003; accepted October 21, 2003
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ABSTRACT |
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Key Words: inorganic arsenic; transplacental exposure; hepatocellular carcinoma; microarray; real-time RT-PCR.
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INTRODUCTION |
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The development of rodent models of inorganic arsenic carcinogenesis is thus critical for elucidation of molecular mechanisms. We have recently shown that short-term transplacental arsenic exposure during gestation in mice produced a variety of internal tumors in the offspring when they became adults (Waalkes et al., 2003). This includes aggressive epithelial malignancies such as HCC in males. It is noteworthy that HCC has been identified as a tumor type associated with arsenic exposure in humans (Centeno et al., 2002
; Chen et al., 1997
; Zhou et al., 2002
). Arsenic-induced tumors in a transplacental study (Waalkes et al., 2003
) occurred in the absence of any other treatments. Thus, inorganic arsenic can act as a complete transplacental carcinogen. In this regard, gestation is a period of high sensitivity to chemical carcinogenesis (Anderson et al., 2000
). Orally administered inorganic arsenic during gestation can readily cross the placenta and enter the fetal blood and/or tissue (Concha et al., 1998
; Lindgren et al., 1984
; National Research Council, 1999
), making in utero exposure to arsenic a plausibility in humans.
Tumor progression is typically associated with irreversible changes in gene expression, including fetal gene expression and selection of neoplastic cells for optimal growth. There are a variety of ways arsenic can alter gene expression (Simeonova et al., 2000; Kitchin, 2001
), which can be associated with aberrant cellular phenotypes. We have recently used cDNA microarray technology to profile inorganic arsenic-induced malignant transformation in rat liver epithelial cells (Chen et al., 2001
), inorganic arsenic-induced acute stress response in the mouse liver (Liu et al., 2001
), and chronic arsenic-induced liver disorders in a human population of Guizhou, China (Lu et al., 2001
). In this context, the present study was undertaken to examine genetic events associated with transplacental hepatocarcinogenesis induced by inorganic arsenic by using frozen samples of liver tumors and surrounding nontumorous livers collected during our recent study (Waalkes et al., 2003
). Total RNA from liver tumors and peritumor tissue was extracted and subjected to microarray analysis, followed by confirmation of selected genes by real-time RT-PCR. The results showed remarkable expression differences between arsenic-induced HCC and spontaneous liver tumors and differences between nontumorous livers from control and arsenic-exposed mice. This gene profiling should contribute to our understanding of the mechanism(s) of arsenic-induced carcinogenesis in the liver.
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MATERIALS AND METHODS |
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Animal treatment and sample collection.
The current study was performed using liver tumor and nontumorous liver samples collected as a part of the transplacental arsenic carcinogenesis study (Waalkes et al., 2003). Briefly, pregnant C3H mice were given drinking water containing 42.5 or 85 ppm arsenic as sodium arsenite or unaltered water ad libitum from days 8 to 18 of gestation. The dose of arsenic used did not affect water consumption or the body weights of dams (Waalkes et al., 2003
). Dams were allowed to give birth, and litters were culled to no more than eight at 4 days postpartum. Offspring were weaned at 4 weeks and then randomly put into separate groups (n = 25) according to maternal exposure level. The dose of arsenic used did not affect the body weight gain of the offspring. The male offspring were observed up to 74 weeks (Waalkes et al., 2003
). Only samples from male mice were used in the present work as they showed dramatic increases in HCC after in utero arsenic exposure (Waalkes et al., 2003
). Samples were frozen in liquid nitrogen and stored at -80°C until analysis. A total of 24 liver samples was used in the present study as follows: spontaneous tumors (n = 4, three adenomas and one carcinoma) and the surrounding nontumorous liver from control animals (n = 4); arsenic-induced tumors (n = 4, 4 HCC) from the 42.5 ppm group and corresponding nontumorous liver (n = 4); and arsenic-induced HCC (n = 4, 4 HCC) from the 85 ppm group and corresponding nontumorous liver (n = 4). Total RNA was extracted from individual samples and equal amounts of RNA from each sample were pooled for microarray and RT-PCR analysis.
Microarray analysis.
Customer-designed mouse cancer arrays (600 genes, Clontech) were used for cDNA microarray analysis. Customer-designed arrays have several advantages over commercial arrays in that (1) 600 genes of our interest were selected; (2) a large patch of arrays (300 membranes) was made under the same conditions, thus minimizing lot-to-lot variation; and (3) cost was reduced markedly, allowing multiple group comparisons in replicates under the same conditions. Total RNA was isolated from liver samples with TRIzol agent (Invitrogen, Carlsbad, CA), followed by purification and DNase-I digestion with RNeasy columns (Qiagen, Valencia, CA). Approximately 5 µg of total RNA were converted to [-32P]-dATPlabelled cDNA probe using MMLV reverse transcriptase and the Atlas customer array specific cDNA synthesis primer mix and then purified with a NucleoSpin column (Clontech). The membranes were prehybridized with Expresshyb from Clontech for 2 h at 68°C, followed by hybridization with the cDNA probe overnight at 68°C. The membranes were then washed four times in 2X SSC/1% SDS, for 30 min each time, and twice in 0.1X SSC/0.5% SDS, also for 30 min each time. The membranes were then sealed with plastic wrap and exposed to a Molecular Dynamics Phosphoimage Screen. The images were analyzed densitometrically using AtlasImage software (Clontech, Sunnyvale, CA). The gene expression intensities were first corrected with the external background and then globally normalized.
Real-time RT-PCR analysis.
The levels of expression of the selected genes were quantified using real-time RT-PCR analysis. Briefly, total RNA was reverse transcribed with MuLV reverse transcriptase and oligo-dT primers. The forward and reverse primers for selected genes were designed using Primer Express software (Applied Biosystems, Foster City, CA). The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. The relative differences in expression among groups were expressed using cycle time (Ct) values as follows: the Ct values of the interested genes were first normalized with ß-actin of the same sample, and then the relative difference between the control and each treatment group was calculated and expressed as a relative increase, setting the control at 100%. Assuming that the Ct value is reflective of the initial starting copy and that there is 100% efficiency, a difference of one cycle is equivalent to a 2-fold difference in starting copy.
Statistics.
For microarray and real-time RT-PCR analysis, pooled liver samples (n = 4) were used. Means and SEM from three to four replicates were calculated. For the comparisons of gene expression between two groups, Students t-test was performed. For comparisons among three or more groups, data were analyzed using a one-way analysis of variance, followed by Duncans multiple range test. The level of significance was set at p < 0.05 in all cases.
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RESULTS |
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The decreased expression of selected genes in arsenic-induced HCC include the gene encoding for the tumor metastasisreducing protein (syndecan-1) and certain hormone receptors (epidermal growth factor receptor EGFR and interleukin-1 receptor), the gene encoding various enzymes (betaine-homocysteine methyl-transferase [BHMT] and glutathione peroxidase-1), and stress-related protein genes (LPS binding protein, clusterin, HSP70, glucose-regulated protein Grp78, programmed cell death protein-6, and Grb2 adaptor protein).
Similar gene alterations seen in both spontaneous tumors and arsenic-induced HCC include a decrease in insulin-like growth factor-1 (IGF-1) and a marked increase in IGF binding protein-1 (IGFBP1). Similar increases in early growth response protein-1 (EGR1), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor are also observed. On the other hand, the expressions of Mn superoxide dismutase, c-Fms oncogene, CYP2F2, and S100 calcium-binding protein A1 are decreased.
Gene Alterations in Nontumorous Liver from Arsenic-Exposed Mice
Gene alterations in nontumorous livers of mice exposed to 42.5 ppm arsenic (As42N) or 85 ppm arsenic (As85N) in utero are listed in Table 2. Genes showing different expression from in utero arsenic exposure include the following: oncogenes (c-myc, H-ras, and AFP); stress-related genes (soluble superoxide dismutase, glutathione S-transferases, glutathione peroxidase-1, glutathione reductase, TNF receptor superfamily 1a, FK506 binding protein, Bcl-2 binding protein BAG1, caspase 8, and catalase); genes encoding metabolic enzymes (CYP2A4, CYP7B1, and CYP2J5); and genes encoding for growth factors and cell communication such as IGFBP1, estrogen receptor-
, cytokeratin-8, integrin ß2, and gap junction protein connexion 26. Exposure to 85 ppm arsenic in utero displays more significant changes, but clear dose-related effects between the low and high doses of arsenic exposure are not always evident.
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DISCUSSION |
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Dysregulation of the IGF axis, including IGF-1 and IGF-2, their receptors, and their binding proteins, has been implicated in tumor formation, progression, and metastasis in vivo (Scharf et al., 2001). In some human hepatoma tissue, the expression of IGF-1 mRNA is lower compared with adjacent nontumor tissue (Scharf et al., 2001
; Su et al., 1989
), and serum IGF-1 can be lower in some human patients with HCC (Stuver et al., 2000
). In the present study, the expression of IGF-1 in arsenic-induced HCC was decreased by 32%. IGF-1 bioavailability is regulated, in part, by IGF-binding proteins (Iida et al., 2003
), and the marked increases in IGFBP-1 seen in the present study could further decrease the bioavailability of IGF-1. Overexpression of IGFBP-1 has been also observed in HCC in humans (Kondoh et al., 1999
). In nontumorous liver of arsenic-exposed mice, a greater than 2-fold increase in the expression of IGFBP-1 was observed. Dysregulation of the IGF axis seen in this study is in accord with the mouse liver carcinogenesis induced by nongenotoxic carcinogen oxazepam and Wyeth-14,643 reported previously (Iida et al., 2003
).
Cytokeratins are intermediate filament proteins that play an essential "guardian" role in the liver after exposure to environmental stress (Omary et al., 2002). Cytokeratin-8 and cytokeratin-18 are major cytokeratins in the liver. The majority of the cellular cytokeratin-8 is assembled with its partner cytokeratin-18, and their aberrant expression has been implicated in various liver diseases and hepatocellular carcinoma (Gonias et al., 2001
; Li et al., 2003
; Omary et al., 2002
). In the present study, cytokeratin-8 was increased ~1.5-fold in nontumorous livers of arsenic-exposed mice and ~3-fold in arsenic-induced HCC, while cytokeratin-18 was dramatically increased (~10-fold) in arsenic-induced HCC. Arsenic treatment of human liver carcinoma cells also increased the expression of cytokeratin-18 (Ramirez et al., 2000
). Cytokeratin-8 has been proposed to function as a major plasminogen receptor in certain carcinoma cells (Gonias et al., 2001
), and overexpression of cytokeratin-8, PAI-1, and AFP have been used as biomarkers of HCC (Li et al., 2003
; Omary et al., 2002
). In the present study, PAI-1 was increased in arsenic-exposed nontumorous liver and dramatically increased in arsenic-induced HCC. In Taiwanese patients suffering from black foot disease, higher plasma PAI-1 levels have been detected (Wu et al., 1993
). Thus, the aberrant expression of cytokeratins and PAI-1 could be important events for arsenic toxicity and carcinogenesis in the liver.
In arsenic-induced HCC, cyclin D1 was markedly overexpressed. Overexpression of cyclin D1 has been implicated in the development of HCC (Deane et al., 2001). Overexpression of cyclin D1 has been reported in chronic arsenic-exposed/transformed cells (Chen et al., 2001
; Trouba et al., 2000
), in arsenic-induced co-carcinogenecity in mice (Rossman et al., 2002
), and in dimethylarsinic acid-induced bladder hyperplasia and neoplastic lesions in rats (Wei et al., 2002
). Also, we have observed a remarkable upregulation of cyclin D1 in the liver and uterus of adult mice chronically exposed to arsenic, in association with preneoplastic lesions (Waalkes et al., 2000
). Overexpression of cyclin D1 was also detected in arsenic-exposed human skin samples (Hong et al., 2001
). Thus, accumulating evidence suggests that overexpression of cyclin D1 may be a consistent contributing factor in arsenic-induced malignant transformation.
The alterations in the expression of proto-oncogenes and tumor-suppressing genes were also observed following in utero arsenic exposure. In accord with previous observations (Chen et al., 2001; Trouba et al., 2000
), overexpressions of c-myc and H-ras oncogenes were also detected in the liver of arsenic-exposed mice. Inorganic arsenic together with restricted methyl intake produced genome-wide and specific H-ras DNA hypomethylation in the mouse liver (Okoji et al., 2002
). Overexpressions of H-ras and c-myc can occur from hypomethylation in the 5'-CCGC sequence following arsenic exposure in Syrian hamster cells, causing neoplastic transformation (Takahashi et al., 2002
). The disruption of cellular p53 has been proposed as an important event of arsenic carcinogenesis (Hamadeh et al., 2002
; Hsu et al., 1999
; Kitchin, 2001
). In the present study, the expression of p53 gene was not significantly altered in spontaneous liver tumors or nontumorous livers after transplacental arsenic exposure but was markedly increased in arsenic-induced HCC. Thus, a variety of genes that are associated with oncogenesis show significant expression changes after in utero arsenic exposure.
Arsenic-induced oxidative stress has been proposed as a mechanism for arsenic carcinogenesis (National Research Council, 1999). Alterations in stress-related genes seen in transplacental arsenic exposure bear some similarities to the literature. This included increases in glutathione S-transferases, HO-1, EGR1, COX2, and SOD (Chen et al., 2001
; Hamadeh et al., 2002
; Liu et al., 2001
; Lu et al., 2001
; Simeonova et al., 2000
; Wei et al., 2002
) and decreases in glutathione reductase (Thomas et al., 2001
). It should be noted that these changes are not contemporaneous with arsenic exposure since they occurred in adult mice long after arsenic exposure ended. Exactly how arsenic induces these changes that continue even in its absence will require additional study.
Dysregulation of DNA damage/repair has been proposed to be an important mechanism for arsenic carcinogenesis (Hartwig and Schwerdtle, 2002). The expression of growth arrest and DNA damageresponsive gene GADD45 was higher in arsenic-induced HCC. GADD45 is induced following acute arsenic exposure (Liu et al., 2001
) or chronic arsenic treatment (Simeonova et al., 2000
) and could be an important component of cellular defense/DNA repair mechanisms. Also, expression of other DNA damage components, such as DNA mismatch repair protein MSH2, ERCC1, and DNA ligase-1, were also higher in arsenic-induced HCC compared to spontaneous tumors. Implicit in these changes is that DNA damage has occurred in arsenic-induced HCC. Since these DNA damage/repair genes were not altered in arsenic-exposured nontumorous livers, it is impossible to say if they are a cause or a result of malignant conversion.
It is of interest to note that the expression of genes encoding for metabolic enzymes was also altered in arsenic-induced HCC. Consistent with our recent observation of arsenic-induced overexpression of estrogen receptor- (Waalkes et al., 2000
), expressions of CYP2A4, an estrogen receptor-
linked/regulated gene (Sueyoshi et al., 1999
), and CYP2B9, an estrogen-dependent, female-specific gene (Yamada et al., 2002
), were increased in nontumorous livers and HCC from arsenic-exposed male mice. On the other hand, the expressions of CYP7B1, a sexually dimorphic gene (male > female), and CYP2F2 were suppressed by in utero arsenic exposure. The effects of in utero arsenic exposure on these metabolic enzymes point towards hepatic feminization, a phenomenon implicated in hepatocarcinogenesis.
BHMT is a key liver enzyme that is important for homocysteine homeostasis. BHMT catalyzes the synthesis of methionine and S-adenosylmethionine (SAM) from betaine and homocysteine. Both methionine and SAM are important components in arsenic methylation and DNA methylation (Thomas et al., 2001), and depressed expression of BHMT has been seen in human cirrhosis and HCC (Avila et al., 2000
). In the present study, BHMT was significantly decreased in arsenic-induced HCC, implying that the dysregulation of SAM metabolism occurred as a result of in utero arsenic exposure. In human prostate cells malignantly transformed by chronic arsenic exposure, a decrease in BHMT also occurs (unpublished data). Thus, decreased expression of BHMT and/or other metabolic enzymes could lead to the disruption of metabolic homeostasis, an event that might be implicated in arsenic-induced hepatocarcinogenesis.
In summary, we have demonstrated that in utero exposures to inorganic arsenic cause remarkable alterations in gene expression in the mouse liver, which are associated with HCC formation. There is a considerable lag between the end of arsenic exposure and gene expression changes, and expression changes occurred long after exposure had ended, suggesting events that occurred from in utero arsenic exposure could be important and long-lasting. The relative importance of these changes and how they may act as an integrated process will be the focus of additional study. The multitude of expression changes that occurred in the offspring after in utero arsenic exposure emphasizes the importance of protecting pregnant women from arsenic exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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