Delayed Temporal Increase of Hepatic Hsp70 in ApoE Knockout Mice After Prenatal Arsenic Exposure

Abstract

Prenatal arsenic exposure accelerates atherosclerosis in ApoE^−/− mice by unknown mechanism. Arsenic is a hepatotoxicant, and liver disease increases atherosclerosis risk. Prenatal arsenic exposure may predispose to liver disease by priming for susceptibility to other environmental insults. Earlier microarray analyses showed prenatal arsenic exposure increased Hsc70 (HspA8) and Hsp70 (HspA1a) mRNAs in livers of 10-week-old mice. We determined effects of prenatal arsenic exposure on hepatic Hsp70 and Hsc70 expression by Western blot and on DNA methylation by methyl acceptance assay during prenatal and postnatal development. Pregnant ApoE^−/− mice were given drinking water containing 85mg/l NaAsO₂ (49 ppm arsenic) from gestation day (GD) 8 to 18. Hsp70 and Hsc70 expression and DNA methylation were determined in GD18 fetuses and 3-, 10-, and 24-week-old mice. Hsc70 expression was unchanged at all ages. Hsp70 induction was observed at 3 and 10 weeks, but was unchanged in GD18 fetuses and 24-week livers of mice. Global DNA methylation increased with age; arsenic had no effects. Bisulfite sequencing of DNA from livers of 10-week-old mice showed Hsp70 promoter region methylation was unchanged, but methylation was increased within the transcribed region. Hsf1 and Nrf2 nuclear translocation were investigated as potential mechanisms of Hsp70 induction and found unaltered. Putative binding sites were identified in HSP70 for in utero arsenic exposure-suppressed microRNAs suggesting a possible mechanism. Thus, prenatal arsenic exposure causes delayed temporal hepatic Hsp70 induction, suggesting a transient state of stress in livers which can predispose the mice to developing liver disease.

Inorganic arsenic is a natural contaminant in drinking water worldwide and a high-priority hazardous substance in the United States. Elevated ground water arsenic is found in large areas of the United States, and very high levels (sometimes > 300 µg/l) occur in Bangladesh, Taiwan, India, Chile, and Argentina (Nordstrom, 2002).

Epidemiological studies indicate chronic arsenic exposure is associated with high risk of cardiovascular disease (CVD) (States et al., 2009). Atherosclerosis underlies most CVD, which is the leading cause of mortality worldwide. Arsenic exposure in drinking water accelerates atherogenesis in experimental animal models (Srivastava et al., 2009) and the induction is dose dependent (Lemaire et al., 2011; Srivastava et al., 2009). However, the mechanism of arsenic-induced atherogenesis is unknown.

Gestation is a critical period of development. Arsenic readily crosses the placenta in humans (Concha et al., 1998) and in rodents (Jin et al., 2006). Thus, developmental arsenic exposures can predispose to adult diseases as indicated by induction of liver cancers in adult male mice (Waalkes et al., 2003). Reports of myocardial infarction in infants whose mothers consumed water with high levels of arsenic in Chile (Rosenberg, 1973, 1974) suggest a role for prenatal arsenic exposure in the development of CVD. Indeed, prenatal arsenic exposure accelerated atherosclerosis in ApoE^−/− mice without high-fat diet, which is usually a requisite for early atherosclerosis in this strain (Srivastava et al., 2007). The mice exposed prenatally to arsenic developed > 2-fold lesions at 10 and 16 weeks of age, compared with unexposed mice. These lesions were located in the aortic roots and aortic arch. Arsenic-exposed mice also had a 20–40% decrease in total triglycerides, but no change in total cholesterol and phospholipids and total abundance of very low-density lipoprotein or high-density lipoprotein particles.

Although atherosclerosis is a vascular disease, the atherogenic stimulus can come from distant sites, including the liver. Epidemiology studies show that liver disease is an independent risk factor for carotid atherosclerosis (Brea et al., 2005), and that elevated liver enzymes in the plasma (e.g., alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) are risk factors for coronary events (Bellentani et al., 2008). Arsenic is toxic to the liver, causing liver diseases in humans (Guha Mazumder, 2001) and animal models (Santra et al., 2000). It is likely that prenatal arsenic exposure affects liver development predisposing to adult chronic disease. We tested the hypothesis that prenatal arsenic exposure altered developmental programming of the liver of ApoE^−/− mice with accelerated atherosclerosis by microarray analyses of both mRNA and microRNA (miRNA) in newborn and 10-week-old mice. The data showed several differentially expressed genes including constitutive (Hsc70) and inducible (Hsp70) heat shock protein (HSP) 70. Age is the major influence on gene expression thus reflecting developmental changes (States et al., 2012).

HSPs are stress proteins which are constitutively expressed at low levels, but upregulated under stress conditions (e.g., heat shock, toxic metals, and oxidative stress) in order to confer protection against such stressors. HSPs function as molecular chaperones under physiological conditions and prevent protein aggregation under stressed conditions (Gething and Sambrook, 1992). Hsc70 and Hsp70 are members of the HSPA family of HSPs. In spite of numerous in vitro data showing Hsp70 induction by arsenic, there are very few in vivo data. Therefore, it is still unclear how Hsp70 expression is regulated postnatally after prenatal arsenic exposure.

One of the mechanisms by which the expression of a gene can be altered is by epigenetic regulation, particularly DNA methylation. Arsenic interferes with genome-wide and site-specific DNA methylation (Reichard and Puga, 2010). Prenatal arsenic exposure causes alterations in DNA methylation leading to altered gene expression, leading to liver carcinogenesis (Waalkes et al., 2004). In addition to DNA methylation, differential miRNA expression also can modulate expression of proteins encoded by miRNA target mRNAs. Prenatal arsenic exposure alters miRNA profiles in fetal and adult mice (States et al., 2012).

In this study, we determined the hepatic expression of Hsp70 and Hsc70 during prenatal and postnatal development in mice prenatally exposed to arsenic. In addition, we also investigated if the observed effect is due to the epigenetic effects of arsenic. This is the first study to determine how the expression of Hsp70 changes during early postnatal development following prenatal arsenic exposure.

MATERIALS AND METHODS

Chemicals.

Sodium arsenite (NaAsO₂) was obtained from Sigma Chemical Co. (St Louis, MO). Adenosyl-l-methionine, S-[methyl-³H] (SAM [³H]) (specific activity 81.9 Ci/mmol), was purchased from PerkinElmer, Inc. (Boston, MA). Protease inhibitors were purchased from Sigma Chemical Co. and Fisher Scientific (Rockford, IL).

Animal treatment and sample collection.

ApoE^−/− mice were housed and bred under pathogen-free conditions in controlled temperature and 12-h light/12-h dark cycle. Animal care was provided following the guidelines of the Association for the Accreditation of Laboratory Animal Care. Prior to treatment, all mice were maintained on standard chow diet and tap water as previously described (Srivastava et al., 2007). Pregnant mice were given drinking water containing 85mg/l NaAsO₂ (49 ppm arsenic) or tap water (for controls) ad libitum from gestation day (GD) 8 to 18. During prenatal arsenic exposures, arsenic-containing water was changed twice weekly. Dams were allowed to give birth (GD18–GD21), and male offspring were maintained on tap water until euthanized at 3, 10, and 24 weeks of age. Liver samples were frozen at −80°C until analysis. GD18 dams were also euthanized and fetal livers were obtained and stored frozen at −80°C. All mice were anesthetized with pentobarbital (150mg/kg) before euthanization. Studies were performed under protocols approved by the University of Louisville Institutional Animal Care and Use Committee.

Isolation of proteins from total liver homogenates.

Frozen livers from GD18 fetuses and 3-, 10-, and 24-week-old mice were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer, or SDS lysis buffer, supplemented with protease inhibitors. Liver homogenates were centrifuged and the supernatants obtained as protein extracts. Protein concentrations were determined by bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL).

Extraction of cytosolic and nuclear fractions.

Livers of 3- and 10-week-old arsenic-exposed and -unexposed mice were subjected to cytosolic and nuclear extractions. Frozen livers (0.1g) were ground in liquid nitrogen and transferred to a Dounce homogenizer. Using pestle B, tissues were homogenized in 700 µl of ice-cold polyamine A buffer (0.34M sucrose, 13.3mM Tris-HCl pH 7.5, 13.3mM NaCl, 0.1% β-mercaptoethanol, 53mM KCl, 2mM EDTA, 0.5mM ethylene glycol tetraacetic acid, 0.5mM spermidine, 0.5mM spermine, 1mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepsatin, and phosphatase inhibitor) about 15 strokes. Dounce homogenizer was rinsed with 300 µl of polyamine A buffer. The suspension was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 4500 × g for 15min, at 4°C. The supernatant, which is the cytosolic fraction, was removed and transferred to a new tube and stored at −80°C.

The nuclear pellet was resuspended in 300 µl of polyamine A buffer + 2.1M sucrose solution (mixed in equal ratios), and the suspension was layered on top of 200 µl of polyamine A buffer + 2.1M sucrose solution (mixed in equal ratios) in centrifuge tubes. Tubes were centrifuged in a Beckman TLA 120.2 rotor at 95,000 × g, for 1h, at 4°C. The supernatant was removed and nuclear pellet was lysed in 200 µl of Buffer B (20mM Hepes, 1M NaCl, 5mM MgCl₂, 12% glycerol, 5mM dithiothreitol [DTT], 2M urea, 1mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepsatin, and phosphatase inhibitor) and incubated shaking for 30min at 4°C. Nuclear lysate was centrifuged at 14,000 × g, for 15min at 4°C and supernatant was collected and stored at −80°C as nuclear extract. Protein concentrations in cytoplasmic and nuclear extracts were measured using Bio-Rad protein assay.

Western blot analysis.

Proteins (20–25 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred into nitrocellulose membranes. Membranes were incubated with primary antibodies including mouse and rabbit monoclonal Hsp70 antibodies (1:1000, overnight at 4°C; Enzo Life Sciences International, Inc., Plymouth Meeting, PA, and Epitomics Inc., Burlingame, CA); rat monoclonal Hsc70 antibody (1:1000, 1h at 4°C; Enzo Life Sciences International, Inc.); rabbit polyclonal Hsf1 antibody (1:500, 24h at 4°C; Cell Signaling Technology Inc., Danvers, MA); and rabbit polyclonal Nrf2 antibody (1:1000 overnight at 4°C; Cell Signaling Technology Inc.). Membranes were incubated with corresponding secondary antibodies bound to HRP. The membranes were incubated with ECL or ECL plus substrate (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). ECL membranes were exposed to Kodak XAR x-ray film. Signals on ECL plus membranes were visualized directly on a Storm Phosphoimager in blue fluorescence mode (Molecular Dynamics, Sunnyvale, CA). Bands were quantitated using Image Quant software.

Global DNA methylation assay.

Genomic DNA was isolated from frozen liver tissues of GD18 fetuses and 3-, 10-, and 24-week-old mice by the salting out method (Bohr et al., 1985) with modification for tissues as follows. Frozen livers (0.1g) were chopped into small pieces and incubated in 1ml lysis buffer (0.5M Tris, pH 8.0, 20mM EDTA, 10mM NaCl, 1% SDS, and 0.5mg/ml proteinase K) overnight at 37°C. Saturated NaCl solution (1/2 volume) was added to the lysate, and the supernatant after precipitation by addition of saturated NaCl was processed as per protocol. Purified DNA was dissolved in TE buffer and quantitated by A₂₆₀. Global DNA methylation was determined by the methyl acceptance assay as described (Xie et al., 2007) with some modifications. Briefly, 2 μg DNA was incubated for 2h at 37°C in a 30-μl reaction mixture containing 1.25μM (3 μCi) SAM [³H], 4U of CpG Methylase (M. ss I) (New England Biolabs, Inc., Beverly, MA), 10mM DTT, Tris-EDTA buffer (100mM Tris, 10mM EDTA, pH 8.0) and 100mM NaCl. The reaction was stopped on ice and split in two aliquots of 15 µl. Each aliquot was transferred onto a Whatman DE81 filter to bind DNA. Bound DNA was washed on a filtration funnel connected to a vacuum source three times with 5ml of 0.5M sodium phosphate buffer (pH 7.0), once with 5ml of 70% ethanol and twice with 5ml of absolute ethanol. The filter was dried and 5ml of liquid scintillation cocktail (Beckman Coulter, Fullerton, CA) was added. Bound radioactivity was measured by scintillation counting on a Tri-Carb 2910TR liquid scintillation analyzer (PerkinElmer, Inc.).

Promoter region and CpG island methylation of Hsp70.

Genomic DNA was isolated from livers of 10-week-old arsenic-exposed and -unexposed mice, and 400ng DNA was subjected to bisulfite treatment using EZ DNA Methylation Direct Kit (Zymo Research Corp., Irvive, CA) according to manufacturer’s protocol. PCR primers were designed using the MethPrimer software (http://www.urogene.org/methprimer/index1.html) which designs oligonucleotide primers for methylation PCR. Primers were designed to amplify five regions of Hsp70 CpG island, which spans the promoter region and 66% of the body of the gene. Primers were also designed to amplify untreated (original) DNA. Bisulfite-treated DNA (50ng) and untreated DNA (100ng) was amplified by PCR using High Fidelity Platinum Taq DNA Polymerase (Invitrogen Corp., Carlsbad, CA) according to manufacturer’s protocol (see Table 1 for PCR primers). PCR products were visualized on a 1.5–2% agarose gel (based on PCR product size) and single distinct bands were observed. PCR products were purified using Multiwell PCR Purification Kit (Qiagen Inc., Valencia, CA) and sequenced using nested oligonucleotides. In this method, sodium bisulfite converts all unmethylated cytosines to uracil residues, whereas 5-methyl cytosine is resistant to conversion. PCR amplification then converts the uracils to thymidines. After sequencing, unmethylated cytosines are detected as thymidines, thus making it possible for determination of site-specific methylation.

TABLE 1 .

Oligonucleotides Used in Amplifying Hsp70 in Bisulfite-Treated DNA

Region		Primer sequence		Tm (°C)
1		Fw: 5ʹ-TAGTATTTTTAGGAGTTGATTTTTAATAGT-3ʹ		50
		Rv: 5ʹ-TTATCTCTAAATAAAACCAAATTTAATTCT-3ʹ
2		Fw: 5ʹ-ATTTAGAATTAAATTTGGTTTTATTTAGAG-3ʹ		58
		Rv: 5ʹ-TATAATTCACCTACACCTTAAACTTATC-3ʹ
3		Fw: 5ʹ-ATGAAGTATTGGTTTTTTTAGGTGGT-3ʹ		58
		Rv: 5ʹ-ATCCTTCTTATACTTCCTCTTAAACTCCT-3ʹ
4		Fw: 5ʹ-GAAGTATAAGAAGGATATTAGTTAGAATAA-3ʹ		55
		Rv: 5ʹ-AAAAAAATCCTACAACAACTTCTACAC-3ʹ
5		Fw: 5ʹ-TGTAGAAGTTGTTGTAGGATTTTTTTAA-3ʹ		60
		Rv: 5ʹ-AATAACCTCCTAACACTTATCCAACAC-3ʹ

Statistical analysis.

Data are expressed as mean ± SEM. Comparisons among groups were performed using one-way ANOVA, whereas comparisons between groups were performed using Student’s t-test.

RESULTS

Hepatic Hsp70 and Hsc70 Expression During Course of Postnatal Development

Hsp70 expression is induced by stress (e.g., heat shock and toxic metals) in many in vitro systems. In spite of numerous in vitro data showing Hsp70 induction by arsenic, there are very few in vivo data. Thus, we determined by Western blot analysis the effect of prenatal arsenic exposure on Hsp70 expression at different stages of development, in GD18 fetuses and 3-, 10-, and 24-week-old mice. The results show that Hsp70 expression was not altered at GD18 at the end of the arsenic exposure (Fig. 1A). However, 3 weeks later, Hsp70 expression was increased ~70% in arsenic-exposed livers (Figs. 1A and B). Hsp70 expression remained increased at 10 weeks, but returned to unexposed levels by 24 weeks. These data indicate that prenatal arsenic exposure caused a delayed temporal induction of inducible Hsp70, thus suggesting a temporal state of stress in the livers of exposed mice.

FIG. 1.

Western blot analyses of hepatic Hsp70 and Hsc70 expression during course of prenatal and postnatal development. Samples of livers of control and arsenic-exposed mice at four stages of development (GD18 and 3, 10, and 24 weeks) were homogenized with either SDS lysis buffer or RIPA buffer as described in Materials and Methods section. Western blot was performed to probe for Hsp70 and Hsc70. 14-3-3 β was used as the loading and normalization control for GD18 samples, whereas GAPDH was used as the loading and normalization control for 3-, 10-, and 24-week samples. Panel A: Representative Western blot images of Hsp70 in livers of arsenic-exposed and -unexposed mice sacrificed on GD18 and 3, 10, and 24 weeks. Panel B: Densitometric quantitation of Hsp70 at each stage of development; *p < 0.003. Panel C: Representative Western blot images of Hsc70 in livers of arsenic-exposed and -unexposed mice sacrificed on GD18 and 3, 10, and 24 weeks. Panel D: Densitometric quantitation of Hsc70 at each stage of development. Western blots of Hsp70 at 10 weeks taken from States et al. (2012).

In contrast to increased expression observed with inducible Hsp70, the expression of constitutive Hsc70 was not altered during any stage of development (Figs. 1C and D). These data are consistent with the constitutive nature of Hsc70.

Global DNA Methylation Analysis

The effect of prenatal arsenic exposure on global DNA methylation at different stages of development (GD18 fetuses and 3, 10, and 24 weeks) was determined by methyl acceptance assay. This assay uses a bacterial DNA methyltransferase that methylates all unmethylated cytosines in DNA. In this assay, S-adenosylmethionine labeled with [³H] at the donated methyl group is the methyl donor. Thus, lower [³H] incorporation corresponds to higher degree of DNA methylation (i.e., hypermethylation) in the genomic DNA. In the DNA of both arsenic-exposed and -unexposed mice, incorporation of [³H] into DNA decreased with increasing age (Fig. 2). Thus, liver DNA becomes hypermethylated globally as mice age. However, prenatal arsenic exposure did not alter the global DNA methylation status at any age.

FIG. 2.

Analysis of global DNA methylation during course of prenatal and postnatal development, measured by methyl acceptance assay. Higher [³H] methyl incorporation by bacterial DNA methyltransferase into unmethylated cytosines corresponds to lower degree of global DNA methylation. *p < 0.05 compared with GD18, †p < 0.05 compared with 3 weeks, and ‡p < 0.05 compared with 10 weeks. Data are mean ± SEM; n = 6–9.

Methylation Status of the Promoter Region and CpG Island of Hsp70

Methylation of DNA in the promoter region or within the body of a gene can alter expression of the gene. We determined if the increased Hsp70 expression observed with prenatal arsenic exposure corresponded to differential DNA methylation induced by arsenic exposure. The Hsp70 CpG island is 2.5kb long and spans through the promoter region and 66% of the body of the gene (Fig. 3). Hsp70 methylation was determined in livers of 10-week-old mice by bisulfite sequencing of five regions spanning 1.9kb of the CpG island (Fig. 3) as described in Materials and Methods section. The methylation status of analyzed regions of hepatic Hsp70 CpG island which includes the promoter region is shown in Figure 4A. The results are expressed as percentage CpG site methylation per region. The data reveal that the promoter region (R1) of Hsp70 is completely unmethylated in DNAs from both arsenic-exposed and -unexposed mice. However, analysis of regions within the body of Hsp70 (R2–R5) shows differential methylation patterns, with region-specific hypo- and hypermethylation. In many instances, a CpG site is completely methylated or unmethylated (Fig. 4B). Methylation of Region 3 (R3 spans +503 to +856) was increased in DNA of arsenic-exposed mice.

FIG. 3.

Schematic representation of Hsp70 showing regions analyzed for methylation studies. Arrows indicate PCR primers. Regions analyzed are represented as R1–R5. Bars represent sequence length of analyzed regions containing high densities of CpG sites.

FIG. 4.

Analysis of Hsp70 promoter region and CpG island methylation. Genomic DNA was isolated from livers of 10-week-old arsenic-exposed and -unexposed mice and Hsp70 methylation was determined by bisulfite sequencing as described in Materials and Methods section. Panel A: Quantitation of percentage CpG site methylation per region. Data are mean ± SEM. *p < 0.004. Panel B: A representative sequences from R3 (n = 3–5) showing methylated and unmethylated CpG sites. Methylated cytosines are represented as “c” in the boxes, whereas unmethylated cytosines appear as “t”.

HSF1 and Nrf2 Nuclear Translocation and Activation

Hsf1 and Nrf2 are transcription factors which are major regulators of Hsp70 expression. The translocation of Hsf1 and Nrf2 from cytoplasm to nucleus is important for their activation of gene transcription. To determine if arsenic exposure was having an upstream effect on transcription factors regulating Hsp70 expression, Western blot analysis of cytosolic and nuclear Hsf1 and Nrf2 proteins was performed. Data revealed that a greater part of Nrf2 is located in the cytosol, with very limited translocation to the nucleus (Fig. 5). Prenatal arsenic exposure did not alter the cytosolic levels of Hsf1 and Nrf2, nor did it increase nuclear translocation. The levels of Hsf1 in both cytosolic and nuclear fractions were very low and there was no significant translocation difference. Absence of nuclear translocation indicates Hsf1 and Nrf2 are not activated. These data suggest that the mechanism of Hsp70 induction by arsenic exposure is not by the activation of Hsf1 and Nrf2 transcription factors.

FIG. 5.

Western blot analysis of Hsf1 and Nrf2 cytosolic and nuclear protein levels. Cytosolic and nuclear fractions were isolated from livers of 3- and 10-week-old arsenic-exposed or -unexposed mice as described in Materials and Methods section. Western blot was performed to probe for Hsf1 and Nrf2. GAPDH was used as the normalization control for cytosolic proteins and to determine nuclear purity, whereas Histone 3 was used as the normalization control for nuclear proteins and to determine cytosolic purity. Panel A: Representative Western blot images of cytosolic and nuclear Hsf1 and Nrf2 in livers of 3-week-old arsenic-exposed and -unexposed mice. Panel B: Representative Western blot images of cytosolic and nuclear Hsf1 and Nrf2 in livers of 10-week-old arsenic-exposed and -unexposed mice. Panel C: Densitometric quantitation of cytosolic and nuclear Hsf1. Panel D: Densitometric quantitation of cytosolic and nuclear Nrf2.

DISCUSSION

Whereas Hsc70 is a constitutively expressed molecular chaperone, Hsp70 is a stress-inducible type which helps cells to resist stress by solubilizing denatured protein aggregates, facilitating the restoration of the function of renatured proteins, and transporting irreversibly damaged proteins to degradative organelles and proteasomes (Kiang and Tsokos, 1998). This study clearly demonstrates that prenatal exposure to inorganic arsenic results in delayed temporal postnatal induction of the stress-inducible Hsp70 protein in livers of ApoE^−/− mice. Hsc70 expression did not change during any stage of postnatal development consistent with it not being stress inducible. The Hsp70 expression increases several weeks after arsenic exposure is stopped and continues for several more weeks before returning to normal levels. The increased Hsp70 expression at ages 3 and 10 weeks following prenatal arsenic exposure indicates a temporal period of stress and thus suggests a critical window during which time the mice are most susceptible to other environmental insults that may lead to chronic adult disease. These data suggest a low-grade injury in which there is no detectable damage at the time of arsenic exposure, but the tissue becomes sensitized or primed for a greater detectable damage upon a second hit which comes later in life. This hypothesis is supported by the enhanced lipopolysaccharide-induced liver damage caused by chronic arsenic exposure (Arteel et al., 2008). Some in vitro and in vivo studies (Del Razo et al., 2001) have proposed that Hsp70 induction can be used as a biomarker of arsenic exposure. However, we did not see an immediate induction of Hsp70 after arsenic exposure but rather a delayed induction, and therefore suggest that Hsp70 expression is not sufficient to be used as a biomarker of arsenic exposure. Absence of GD18 hepatic Hsp70 induction in our study is consistent with the findings of Petrick et al. (2009) who reported that microarray analysis of GD18 embryonic mouse lungs showed no alterations in Hsp70 expression following prenatal arsenic exposure. Other studies have reported increased Hsp70 expression in vivo following arsenic exposure. Hsp70 is reported to be induced in mouse lung by chronic low-dose postnatal arsenic exposures (Andrew et al., 2007), and in mouse liver after acute subcutaneous injections of sodium arsenite and arsenate (Liu et al., 2001). Also, microarray analysis of adult C3H mouse liver tumors revealed decreased Hsp70 mRNA expression by prenatal arsenic exposure (Liu et al., 2004). However, the decreased Hsp70 expression was not confirmed by RT-PCR or Western blot.

There is also growing evidence implicating Hsp70 in atherosclerosis (Lu and Kakkar, 2010). Stressed cells can actively release extracellular Hsp70 (Asea, 2007) which when present in extracellular membranes can activate macrophages to induce TNF-α expression (Vega et al., 2008), leading to increased inflammatory stimulus promoting atherogenesis. Importantly, Hsp70 expression is increased in the plasmas of patients with atherosclerosis (Wright et al., 2000). However, it is still unclear whether circulating Hsp70 has a cytoprotective or cytotoxic role in atherosclerosis (Bielecka-Dabrowa et al., 2009). Taken as a whole, these observations suggest that the stressed hepatic cells in our mouse model may release excess Hsp70 into the circulation, thus contributing to the observed increased atherosclerosis in arsenic-exposed mice.

Arsenic exposure can alter epigenetic marks, especially causing DNA methylation alterations (Reichard and Puga, 2010). In this study, prenatal arsenic exposure did not alter global DNA methylation during the course of development. This finding is consistent with the report of hypomethylation in GC-rich regions but not globally in hepatic DNA of newborn C3H mice exposed to arsenic prenatally (Xie et al., 2007). The novelty of our study is that we determined global DNA methylation during the course of pre- and postnatal development. The findings are that global DNA methylation does not remain constant, but increases (DNA becomes hypermethylated) with age. Chronic postnatal arsenic exposure is reported to cause global DNA hypomethylation in the livers of adult mice and is associated with arsenic-induced hepatocarcinogenesis (Chen et al., 2004). The apparent difference in global DNA methylation between our findings and those of Chen et al. may be attributed to the difference in arsenic exposure; brief prenatal arsenic exposure versus chronic postnatal exposure.

DNA methylation is an important epigenetic mechanism involved in altered gene expression. For example, prenatal arsenic exposure decreased the methylation of estrogen receptor-α promoter region of adult mouse liver leading to increased gene expression which may play a role in arsenic-induced hepatocellular carcinoma (Waalkes et al., 2004). Global DNA methylation does not predict what happens at gene-specific level. Thus, the role of DNA methylation as a potential mechanism underlying increased Hsp70 expression was determined. The results show that prenatal arsenic exposure did not alter Hsp70 promoter region methylation, but significantly increased methylation within the body of the gene, thus, indicating an epigenetic effect. The methylation pattern across the Hsp70 gene was also determined. The data show that Hsp70 promoter region is generally unmethylated and that the methylation density increases in the body of the gene, with some regions having higher methylation densities than others. It is reported that extensive CpG methylation of a 1.2-kb region spanning the Hsp70 transcription start site is associated with transcriptional silencing of Hsp70 genes in several mouse cell lines in which Hsp70 expression is not activated by heat shock (Gorzowski et al., 1995). The Gorzowski report suggests that the methylation status of Hsp70 promoter region can correlate with gene expression, with increased methylation associated with decreased gene expression and vice versa. However, this correlation between Hsp70 promoter region methylation status and gene expression is not seen in our animal model. The lack of methylation difference indicates that the underlying mechanism of Hsp70 induction is not due to decreased Hsp70 promoter region methylation.

Hsf1 and Nrf2 are the major characterized players involved in Hsp70 transcription. During cellular stress, Hsf1 becomes activated and translocates from the cytoplasm to the nucleus where it directly binds to the Hsp70 promoter region and induces transcription (Kiang and Tsokos, 1998). On the other hand, Nrf2 is a transcription factor that transcriptionally activates expression of antioxidant responsive genes. Nrf2 activation also is associated with nuclear translocation (Kobayashi, 2005). Although Nrf2 does not directly target Hsp70, activation of Nrf2 is reported to be associated with Hsp70 induction (Rinaldi Tosi et al., 2011). Hsf1 is reported to be the mechanism underlying increased Hsp70 expression following acute arsenic treatment in vitro (Kato et al., 1997; Khalil et al., 2006). However, the mechanism underlying arsenic-induced Hsp70 expression in vivo is not clearly understood, because the few reported in vivo studies did not determine the mechanism of increased Hsp70 expression. One study (Wijeweera et al., 2001) reports the involvement of AP-1 transcription factor activation and DNA binding in the induction of stress proteins including Hsp70 in precision-cut rat lung slices exposed to arsenic. However, our data showed that induction of Hsp70 expression by prenatal arsenic exposure is not by activation of transcription factors Hsf1 and Nrf2. The mechanisms underlying delayed increased Hsp70 expression following prenatal arsenic exposure might be different from that of immediate gene induction following acute or chronic arsenic exposure. Immediate Hsp70 induction in response to stress is usually by traditional transcription factor activation which is a transient event. We thus speculate that in addition to altered DNA methylation within the body of Hsp70, another potential mechanism of delayed Hsp70 induction in response to stress is the involvement of miRNA regulation. MiRNAs are short RNA molecules that bind to complementary sequences on mRNAs, resulting in translational repression and gene silencing (Bartel, 2009). Prior microarray analyses of miRNA in the livers of 10-week-old mice revealed that arsenic exposure decreased the expression of 15 miRNAs (States et al., 2012). Of the 15 decreased miRNAs (Table 2), 4 have been predicted to have sites on Hsp70 mRNA as predicted by miRWalk and other programs (http://www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). We speculate that these decreased miRNAs might have a regulatory role in Hsp70 expression in livers of arsenic-exposed mice. Future work will investigate this potential role of the miRNAs.

TABLE 2 .

MiRNAs Decreased by Prenatal Arsenic Exposure and Predicted to Have Sites on Hsp70 mRNA as Predicted by miRWalk and Other Programs

MiRNA	Target region
Mmu-miR-130a	3ʹ-UTR
Mmu-miR-218	CDS
Mmu-miR-412	CDS
Mmu-miR-681	CDS

In summary, this study demonstrated that prenatal exposure to arsenic in drinking water results in delayed temporal induction of inducible-type Hsp70 in the liver. Prenatal arsenic exposure did not alter global DNA methylation during pre- and postnatal development. The underlying mechanism of Hsp70 induction is not by transcription factor activation, but may involve altered DNA methylation within the body of the gene. Other potential mechanisms such as decreased miRNA expression resulting in increased mRNA stability might also be involved. Our findings suggest that prenatal arsenic exposure causes a delayed stress response in the liver. This delayed response may signal a critical window during which the liver is most sensitized to increased susceptibility to other environmental insults, thus predisposing to liver disease and accelerated atherosclerosis.

FUNDING

National Institute of Environmental Health Sciences (R21ES015812, R21ES015812-S1, R21ES015812-S2, and P30ES014443); PhD dissertation completion award from the University of Louisville to N.N.O.N.