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Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio) Patrick Carlson a,b , Rebecca J. Van Beneden a,b,c,∗ a b c

Graduate School for Biomedical Sciences and Engineering, University of Maine, Orono, ME 04469-5751, USA Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469-5751, USA School of Marine Sciences, University of Maine, Orono, ME 04469-5751, USA

a r t i c l e

i n f o

Article history: Received 30 June 2013 Received in revised form 27 September 2013 Accepted 2 October 2013 Keywords: Arsenic Zebrafish Liver

a b s t r a c t Adult zebrafish (Danio rerio) were used to investigate mRNA expression in the liver following 7-day and 21-day exposures to 0, 10, 50, or 500 ppb sodium arsenite. Arsenic exposure has been linked to several human disorders including cancers and cardiovascular and metabolic diseases. Quantitative PCR was employed to determine the mRNA expression of genes involved in cell cycle regulation [cyclin E1 (ccne1), WEE1 A kinase (wee1)], DNA damage repair [breast cancer 2 (brca2)] and lipid transport and metabolism [carnitine O-octanoyltransferase (crot), fatty acid binding protein-3 (fabp3) and 3-hydroxy-3methylglutaryl-CoA synthase 1 (hmgcs1)]. Results from the 7-day exposure showed sex- and dose-specific changes in expression of wee1, brca2, crot and hmgcs1. No significant differences from controls were observed in fish exposed for 21 days. Expression of all genes, except ccne1, was significantly different between the 7- and 21-day exposures. The results presented here correlate with prior findings from our lab and others, and offer further insight into potential mechanisms of low-dose arsenic exposure. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Arsenic is found in ground and surface water world-wide, including the United States, Mexico and southeast Asia (NRC, 2001). The arsenic concentration in drinking water in many places far exceeds the EPA and WHO action level of 10 ppb. In the United States, particularly Maine and New Hampshire, some private wells contain arsenic in the high parts per billion (ppb) (Ayotte et al., 2003). Epidemiological studies have linked exposure to high levels of arsenic to increased incidences of lung, skin and bladder cancers (Erraguntla et al., 2012) as well as cardiovascular disease, type 2 diabetes and other metabolic diseases (Del Razo et al., 2011; Guo, 2011). The impact of chronic, low-dose arsenic exposure to both human and ecosystem health has not been well characterized. Understanding the mechanism of arsenic toxicity has been hampered by a number of factors, including nonlinear dose response at low levels, tissue and species specificity, and is further complicated by sex differences. Health effects in humans due to chronic exposure to inorganic arsenic in drinking-water have been shown to be sex-specific (Vahter et al., 2007) and are likely due, in

∗ Corresponding author at: School of Marine Sciences, University of Maine, 5751 Murray Hall, Orono, ME 04469-5751, USA. Tel.: +1 207 581 2602; fax: +1 207 581 2537. E-mail addresses: [email protected] (P. Carlson), [email protected] (R.J. Van Beneden).

part, to differential methylation of inorganic arsenic (Lindberg et al., 2007). Sex-specific effects have also been demonstrated in other species including Fundulus heteroclitus (mummichog), zebrafish, and mouse, and contribute to the complexity of the response (Carlson et al., 2013; Ferrario et al., 2008; Gonzalez et al., 2010). Male and female mice exposed to high doses of arsenic in utero exhibit very different pathologies (Ahlborn et al., 2009; Waalkes et al., 2004a,b, 2003). Male mice had increased incidence of liver and adrenal tumors; females, however, developed urogenital tumors. Microarray studies (Gonzalez et al., 2010) recently showed sex-dependent responses in the mummichog exposed to environmentally relevant levels of arsenic. Serum amyloid precursor was up-regulated in livers from females in the highest exposure group, but down-regulated in males. In each case, sex-specific differences in response to arsenic appear to be a common occurrence across phyla. Fish can serve as models for both human diseases as well as models for the health of the ecosystem. This is especially true in addressing the effects of chronic, low-dose exposures to aquatic contaminants. A number of recent studies using fish models have contributed significantly to our understanding of the molecular mechanisms underlying arsenic toxicity. Acute and chronic exposure can induce different responses, including immune suppression (Datta et al., 2009) and adaptation to changing environment (Shaw et al., 2007). Zebrafish exposed to arsenic during development show impaired reproductive ability and developmental abnormalities such as dorsal curvature, cardiac malformations and

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Please cite this article in press as: Carlson, P., Van Beneden, R.J., Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio). Aquat. Toxicol. (2013), http://dx.doi.org/10.1016/j.aquatox.2013.10.006

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Table 1 Genes investigated and primers used for qPCR analysis. Gene

Expected amplicon

Accession number

Forward primer (5 –3 )

Reverse primer (5 –3 )

brca2 ccne1 crot fabp3 gapdh hmgcs1 wee1

144 125 135 132 140 141 111

NM 001110394 NM 130995 NM 001018151 NM 152961 BC083506 NM 201085 NM 001005770

GGCTTGCTTACAGAGGGACA TTGGAACAACCTGCTCGGAA TGAGGAAAGAACACGCTGGG CGTGGAACTTGAAGGAGAGCA CCGTCTTGAGAAACCTGCCAAG ACACCTCAGCAGTTAGCAGG GGCTCTGTGGATGAGCAAAATG

CAACCATTCTGGAGACACCTCA CCTCACAAACCTCCATTAGCCA GGTGTGGCATAAGGTTTGGC GTGTGAAAACGTCACCCTCCT TGGATGAACGGCAATCCCCA GTCACACAAGCTGGAGACCA CATGTGGTCGTCTTCTGCCC

neuromuscular system defects (Li et al., 2009). These gross morphological abnormalities correlated with differential expression of genes important in cellular and organismal structure (Gonzalez et al., 2006, 2010; Li et al., 2009). The present study is a follow up of our recent work on the effect of arsenic on the zebrafish liver proteome (Carlson et al., 2013) and part of ongoing comparative studies in our laboratory using both fish and mouse models. Here, adult zebrafish were exposed to 0, 10, 50 or 500 ppb sodium arsenite in their water for 7 or 21 days. We examined: (1) dose responsiveness, (2) sex-specific differences and (3) the effect of the duration of the exposure. Based on the response of the mouse to low-dose arsenic exposure, we focused our investigations in zebrafish on three genes involved cell cycle regulation/DNA damage response [breast cancer related 2 (brca2), cyclin E1 (ccne1) and WEE1 homologue (wee1)] and three genes with roles in lipid transport/metabolism [carnitine Ooctanoyltransferase (crot), fatty acid binding protein 3 (fabp3), and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (hmgcs1)]. Overall, following the 7-day exposure, wee1 and brca2 expression changed at 500 ppb, while crot and hmgcs1 were responsive at levels as low as 10 ppb sodium arsenite. Sex-specific and duration-dependent responses were also observed.

whole liver from two fish was pooled; three biological replicates were prepared for each duration, treatment and sex. Each biological replicate was homogenized by micropestle in RLT homogenization buffer and RNA was purified by spin column. RNA concentration was determined by Nanodrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and integrity was determined using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). RNA was reverse transcribed into cDNA using iScript cDNA synthesis reagents (BioRad, Hercules, CA, USA) as per manufacturer’s recommendations. 2.4. Primer design and qPCR Primers for gene targets were identified using BLAST primer design (Table 1); efficiency was determined to be between 90 and 110%. Quantitative PCR, using iTaq Universal Sybr Green reagents (BioRad), was performed using a cDNA template. Amplification protocol: 5 min polymerase activation at 95 ◦ C, followed by 40 cycles of denaturation at 95 ◦ C for 15 s and annealing and amplification at 60 ◦ C for 20 s. A standard melt curve analysis was performed between 65 and 95 ◦ C. All amplifications were completed using a CFX96 Real Time Thermocycler (BioRad). 2.5. Statistical analysis

2. Materials and methods 2.1. Animal treatment AB strain adult zebrafish, between four and eight months old, were maintained at 28 ◦ C on a 14/10 light/dark cycle and used in accordance with standard animal care practices at the University of Maine Zebrafish Facility. They were fed standard lab diet every other day (Hikari, Hayward CA, USA). 10 fish per tank, separated by sex, were exposed to 0, 10, 50 or 500 ppb Na2 AsO (Sigma, St. Louis, MO, USA) for either 7 or 21 days in static treatment tanks with 2 L water. Treatment water was renewed twice daily by siphoning water off, followed by the addition of fresh treatment water. No dead animals were observed during the exposure period. 2.2. Histopathology Following a seven-day arsenic exposure described above, whole zebrafish were fixed in 10% buffered formalin for 24 h, and then transferred to 70% ethanol. Tissues were subsequently dehydrated and paraffin embedded using a Leica TP1020 automatic tissue processor. Sagittal sections were mounted and stained with hemotoxylin and eosin (University of Maine Animal Health Laboratory Diagnostic Center). Slides were analyzed by light microscopy (Zeiss Axio Observer A.1, Carl Zeiss Microscopy, Thornwood, NY, USA) and images were captured with a digital camera (QICam, QImaging, Surrey, BC, Canada). 2.3. RNA extraction and cDNA synthesis RNA was extracted following the manufacturer’s protocol (RNeasy, Qiagen, Valencia, CA, USA). For each biological replicate,

All qPCR data were normalized to gapdh expression. Data homogeneity was determined using Levene’s test (p < 0.05) and normal distribution was measured with Shapiro–Wilk test (p < 0.05), prior to 2-way ANOVA. Significance between doses was determined using Dunnett’s test and confirmed with Tukey’s HSD. Differences between sexes and differences in durations of exposure were determined using Tukey’s HSD. Error is reported as the standard error of the mean (SEM). 3. Results and discussion No histopathological changes were evident following the 7-day exposure to arsenic (Fig. 1). Three genes involved cell cycle regulation and DNA damage response (brca2, ccne1 and wee1) and three genes with a role in lipid transport and metabolism (crot, fabp3 and hmgcs1) were analyzed. The results of this initial investigation showed that 500 ppb arsenic exposure for seven days induced changes in expression in the cell cycle regulatory genes brca2 and wee1, but not ccne1. The level of expression of brca2 and wee1 returned to that of the untreated controls by 21 days. mRNA expression of crot, fabp3 and hmgcs1, involved in lipid metabolism and transport, was altered in a sex-, dose- and duration-specific manner. 3.1. Cell cycle regulation and DNA damage response BRCA2 protein is involved in homologous recombination and DNA double strand break repair. No statistically significant change in brca2 expression was measured in males, relative to either dose or duration of exposure (Fig. 2A). Females exhibited an approximate 12-fold decrease in brca2 expression at the 7-day, 500 ppb

Please cite this article in press as: Carlson, P., Van Beneden, R.J., Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio). Aquat. Toxicol. (2013), http://dx.doi.org/10.1016/j.aquatox.2013.10.006

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Fig. 1. Histopathology (H&E stain) shows no change in liver morphology in either sex following 7-day exposure to 0 or 500 ppb sodium arsenite. No changes were observed at either 10 or 50 ppb (not shown). Magnification is 400×; scale bar is 50 ␮m.

(p = 0.021) exposure; however, by 21 days, there was no significant difference between females exposed to 500 ppb and control fish (Fig. 2A). Decreased expression and activity of the Brca2 gene has been implicated in several cancers, including breast and ovarian (Antoniou et al., 2003). BRCA2-deficient cells treated with 5 ␮M sodium arsenite accumulated DNA adducts (Ying et al., 2009). C3H mice exposed to 85 ppm sodium arsenite in utero had significantly lower Brca2 expression in the liver of hepatocellular carcinoma positive animals (Liu et al., 2006). Overall, lower Brca2 expression is thought predispose cells to accumulate DNA damage. CCNE1 protein is characterized by a dramatic pattern of cyclical expression, increasing through G1 of the cell cycle, accumulating at the G1/S boundary and degrading as cells progress through S phase. CCNE1 forms a complex with, and acts as a regulatory subunit of, CDK2 whose activity is required for the G1/S transition. Over-expression of CCNE1 results in chromosome instability and has been observed in many human tumors (Rajagopalan et al., 2004). Studies in non-fish models have shown Ccne1 to be modulated in response to arsenic exposure; however, no change in mRNA expression was detected in this study (Fig. 2B). Human renal cells exposed to 2.5 ␮M arsenic trioxide for 72 h had depressed CCNE1 protein expression (Hyun Park et al., 2003). Synchronized cultures of mouse embryonic epithelial cells exposed to up to 2 ␮M sodium arsenite had decreased CCNE1 protein expression (Habib, 2010). Mice exposed to 100× higher concentrations of arsenic, which induced hepatocellular carcinoma, exhibited decreased Ccne1 mRNA expression (Liu et al., 2006). Zebrafish in our study were exposed to much lower arsenic levels relative to the above studies, and under these conditions, ccne1 expression was not significantly altered. WEE1 is an essential tyrosine kinase at both the S and G2/M checkpoints. WEE1 phosphorylates and inhibits CDK1 and CDK2 at the conserved Tyr15 residue, delaying entry into mitosis and coordinating proper timing of cell division (Guertin et al., 2012). WEE1 activity is also critical to DNA damage checkpoints and loss of function results in accumulated DNA damage. Our data showed that mRNA expression of wee1 was decreased 6-fold in both males (p = 0.007) and females (p = 0.009) following the 7-day exposure to 500 ppb arsenic, returning to basal expression levels at day 21

(Fig. 2C). Decreased Wee1 expression has been detected in prostate and non-small cell lung carcinomas; conversely, Wee1 expression was increased in Tp53 deficient breast and osteosarcomas (Vriend et al., 2013). In a separate study, arsenite-transformed rat liver epithelial cells showed a slight increase in Wee1 mRNA expression after 24 weeks of 500 nM sodium arsenite exposure, but no change in Tp53 mRNA expression (Chen et al., 2001). These studies suggest that either Tp53 or WEE1 activation is required to prevent cell cycle progression in response to DNA damage. It is possible that decreased wee1 expression observed in this study may be compensated by the functional zebrafish Tp53 gene. 3.2. Lipid metabolism and transport CROT, a member of the carnitine/choline acetyltransferase family, is localized to the peroxisome and plays a role in lipid metabolism and ␤-oxidation of C6–C10 chain fatty acids (Le Borgne et al., 2011). The expression of crot in male zebrafish was not significantly altered relative to control in either the 7- or 21-day exposure (Fig. 3A). There was a significant difference in expression at 10 ppb (p = 0.007) and 50 ppb (p = 0.033) sodium arsenite between the 7and 21-day exposures (Fig. 3A). Females showed an approximate 8fold decrease in mRNA expression at 10 ppb (p = 0.015) and 500 ppb (p = 0.018) sodium arsenite at the 7-day time point relative to control, but expression returned to baseline following the 21-day exposure (Fig. 3A). mRNA expression at the 10 ppb (p = 0.021) and 500 ppb (p = 0.003) exposures was significantly different between the 7- and 21-day exposures in females (Fig. 3A). To the best of our knowledge, altered Crot mRNA expression following arsenic exposure has not been previously reported. DNA methylation increased in human Crot regulatory elements following a 2 year exposure of up to 1.1 ppm arsenic (Smeester et al., 2011); increased methylation can repress gene expression by preventing the binding of RNA polymerase (Mamrut et al., 2013). These studies point to potential mechanisms whereby arsenic may interfere with crot expression. FABP3 transports long chain fatty acids to the mitochondria (Shi et al., 2013). There were no dose-dependent changes in expression detected for fabp3 in either males or females (Fig. 3B). Differences between 7- and 21-day exposures were observed at 50 ppb

Please cite this article in press as: Carlson, P., Van Beneden, R.J., Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio). Aquat. Toxicol. (2013), http://dx.doi.org/10.1016/j.aquatox.2013.10.006

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Fig. 2. Expression of genes involved in cell cycle and DNA damage response in adult zebrafish with respect to dose, duration of exposure and sex in (A) brca2, (B) ccne1 and (C) wee1;  7-day;  21-day. ‡ signifies difference in expression between duration of exposure at the same dose; * denotes significant difference from the control. Expression was normalized to gapdh expression and is presented using Log2 transformation. Each data point is the average of three biological replicates and significance was determined by Dunnett’s test (p < 0.05) or by Tukey’s HSD (p < 0.05).

(p = 0.041) for males and 10 ppb (p = 0.017) for females (Fig. 3B). We have previously shown an increase of 50% in expression of FABP2 protein in zebrafish exposed for 7 days to 50 ppb sodium arsenite (Carlson et al., 2013). The expression of several fabp genes, including fabp2 and fabp3, in zebrafish liver can vary following agonist activation of PPAR␣ (Venkatachalam et al., 2013); PPAR␥ may also preferentially activate transcription of different fabp genes. Correlation between mRNA and protein expression is highly variable, at times less than 50% (Greenbaum et al., 2003; Vogel and

Marcotte, 2012). mRNA expression changes in fabp3 are sex-, doseand duration-specific, exemplifying the complex effects of arsenic exposure. HMGCS1 is located in the cytoplasm and facilitates condensation of acetyl-CoA with acetoacetyl-CoA, a precursor to steroid biosynthesis. mRNA for hmgcs1 in liver from males exposed for 7 days showed 8-fold decreased expression at 10 ppb (p = 0.023), relative to control (Fig. 3C). No change in mRNA expression was observed at 21 days (Fig. 3C). hmgcs1 expression was significantly

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Fig. 3. Expression of genes involved in lipid metabolism and transport in adult zebrafish with respect to dose, duration of exposure and sex in (A) crot, (B) fabp3 and (C) hmgcs1;  7-day;  21-day. ‡ signifies difference in expression between duration of exposure at the same dose; * denotes significant difference from the control. Expression was normalized to gapdh expression and is presented using Log2 transformation. Each data point is the average of three biological replicates and significance was determined by Dunnett’s test (p < 0.05) or by Tukey’s HSD (p < 0.05).

different between the 7- and 21-day, 50 ppb exposures (p = 0.030) in male fish (Fig. 3C). hmgcs1 expression in females was significantly decreased relative to control approximately 14-fold at 7 days with 500 ppb (p = 0.018) sodium arsenite treatment (Fig. 3C). By 21 days, mRNA expression had returned to basal expression at all treatment levels. There was a significant difference in mRNA expression between the 7- and 21-day, 500 ppb exposure (p = 0.042) in females (Fig. 3C). Specific inhibition of

cytoplasmic HMGCS1 has been shown to limit sterol synthesis in mouse liver (Nagashima et al., 1993). Sterols are required for synthesis of cholesterol-derived endocrine hormones including estrogens and androgens. Deregulation of endocrine signaling is one mechanism associated with low-dose arsenic exposure (Davey et al., 2007). The changes seen here suggest a sex-specific response, potentially leading to deregulation of steroid hormone synthesis.

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Fig. 4. Proposed mechanism of arsenic toxicity. Repression of brca2 and wee1 mRNA expression may be linked to aberrant activation of PKB/AKT1. PBK/AKT1 is known to phosphorylate PPAR␥, which could lead to suppression of crot and hmgcs1 mRNA expression; this could deregulate peroxisomal ␤-oxidation and sterol synthesis. Arrows indicate activation, while bars indicate inhibition. Abbreviations: As, arsenic; P, phosphate; RXR, retinoid X receptor; CDK1, cyclin dependent kinase 1; CCNB1, cyclin B1; FA, fatty acid.

An unexpected finding was that we saw a greater number of genes with altered expression in fish exposed for 7 days compared to 21 days. Several studies using rodent models demonstrated a smaller change in the magnitude of mRNA expression at 21-days, relative to 7-day exposures (Liu et al., 2001; MaciaszczykDziubinska et al., 2012; Markowski et al., 2011). Zebrafish are known to possess a membrane bound ion transporter sensitive to arsenic (Abcc1) (Long et al., 2011a). Exposure of zebrafish embryos to 5 ␮M cadmium increased the expression of Abcc2 which conferred resistance to subsequent arsenic exposure (Long et al., 2011b). These studies suggest an inducible mechanism to mitigate arsenic toxicity, and corroborate our findings that duration of exposure may have a significant impact on gene expression.

expression in the cell cycle regulatory gene wee1 and DNA damage response gene brca2. Altered expression of crot, fabp3 and hmgcs1, was also detected; each of these genes has a role in lipid transport and metabolism. Importantly, this study shows that arsenic at environmentally relevant concentrations can induce gene expression changes within multiple cellular pathways. This suggests, within the narrow scope of this study, that cellular pathways controlling lipid metabolism/transport may be more sensitive to perturbation via arsenic exposure than those pathways involved in regulation of the cell cycle. Conflict of interest The authors have nothing to disclose.

4. Conclusions Our findings suggest a common mechanism of toxicity that may be directly or indirectly regulated by PKB/AKT1 (Fig. 4). PKB/AKT1 can directly phosphorylate, and subsequently inhibit, WEE1 (Katayama et al., 2005). In prostate cancer cells, PKB/AKT1 activation has been shown to decrease both protein and mRNA levels of BRCA2 (Moro et al., 2007). These studies suggest that arsenic can disrupt PKB/AKT1 signaling, which may in turn influence BRCA2 and WEE1 gene expression. PKB/AKT1 also has a role in the regulation of PPAR␥. PPAR␥, a member of the ligand-activated PPAR subfamily of nuclear receptors, has several functions including regulation of peroxisomal ␤-oxidation of fatty acids. PPAR␥ function can be modulated by phosphorylation, binding by specific fatty acid ligands, and protein–protein interactions. Mouse adipocytes exposed to 3 ␮M arsenic trioxide showed phosphorylation of PKB/AKT1, which inhibited its ability to interact with PPAR␥ (Wang et al., 2005). Although PPAR␥ is expressed at low levels in the liver, its activation can impact glucose uptake and lipid transport and metabolism through transcriptional regulation (Monsalve et al., 2013; Tan et al., 2002). Arsenic exposure has been shown to deregulate PPAR␥ activity, and these effects may extend downstream through altered signaling pathways (Cheng et al., 2011). How these changes affect downstream gene expression is still not fully understood. This study demonstrated altered mRNA expression of several genes downstream of PKB/AKT1 and PPAR␥. We show that only the highest exposure, 500 ppb sodium arsenite, can depress mRNA

Acknowledgements This work was supported in part by the Maine Agricultural and Forestry Experiment Station (MAFES) [Grant number ME0850910 to RJVB] and the University of Maine Graduate School for Biomedical Sciences and Engineering. The authors would like to acknowledge the University of Maine DNA Sequencing Facility for PCR product sequence verification, Marissa Giroux and Joshua Manning for assistance with zebrafish treatment, and Dawna Beane at the University of Maine Animal Health Laboratory and Lindsay Grumbach for assistance with histology. References Ahlborn, G.J., Nelson, G.M., Grindstaff, R.D., Waalkes, M.P., Diwan, B.A., Allen, J.W., Kitchin, K.T., Preston, R.J., Hernandez-Zavala, A., Adair, B., Thomas, D.J., Delker, D.A., 2009. Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice. Toxicology 262, 106–113. Antoniou, A., Pharoah, P.D.P., Narod, S., Risch, H.A., Eyfjord, J.E., Hopper, J.L., Loman, N., Olsson, H., Johannsson, O., Borg, Å., Pasini, B., Radice, P., Manoukian, S., Eccles, D.M., Tang, N., Olah, E., Anton-Culver, H., Warner, E., Lubinski, J., Gronwald, J., Gorski, B., Tulinius, H., Thorlacius, S., Eerola, H., Nevanlinna, H., Syrjäkoski, K., Kallioniemi, O.P., Thompson, D., Evans, C., Peto, J., Lalloo, F., Evans, D.G., Easton, D.F., 2003. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. American Journal of Human Genetics 72, 1117–1130. Ayotte, J.D., Montgomery, D.L., Flanagan, S.M., Robinson, K.W., 2003. Arsenic in groundwater in eastern New England: occurrence, controls and human health implications. Environmental Science and Technology 37, 2075–2083.

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Carlson, P., Smalley, D.M., Van Beneden, R.J., 2013. Proteomic analysis of arsenicexposed zebrafish (Danio rerio) identifies altered expression in proteins involved in fibrosis and lipid uptake in a gender-specific manner. Toxicological Sciences 134, 83–91. Chen, H., Liu, J., Merrick, B.A., Waalkes, M.P., 2001. Genetic events associated with arsenic-induced malignant transformation: applications of cDNA microarray technology. Molecular Carcinogenesis 30, 79–87. Cheng, H., Qiu, L., Zhang, H., Cheng, M., Li, W., Zhao, X., Liu, K., Lei, L., Ma, J., 2011. Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells. Acta Biochimica et Biophysica Sinica 43, 204–209. Datta, S., Ghosh, D., Saha, D.R., Bhattacharaya, S., Mazumder, S., 2009. Chronic exposure to low concentration of arsenic is immunotoxic to fish: role of head kidney macrophages as biomarkers of arsenic toxicity to Clarias batrachus. Aquatic Toxicology 92, 86–94. Davey, J.C., Bodwell, J.E., Gosse, J.A., Hamilton, J.W., 2007. Arsenic as an endocrine disrupter: effects of arsenic on estrogen receptor-mediated gene expression in vivo and in cell culture. Toxicological Sciences 98, 75–86. Del Razo, L., Garcia-Vargas, G., Valenzuela, O., Castellanos, E., Sanchez-Pena, L., Currier, J., Drobna, Z., Loomis, D., Styblo, M., 2011. Exposure to arsenic in drinking water is associated with increased prevalence of diabetes: a cross-sectional study in the Zimapan and Lagunera regions in Mexico. Environmental Health 10, 73. Erraguntla, N.K., Sielken Jr., R.L., Valdez-Flores, C., Grant, R.L., 2012. An updated inhalation unit risk factor for arsenic and inorganic arsenic compounds based on a combined analysis of epidemiology studies. Regulatory Toxicology and Pharmacology 62, 329–341. Ferrario, D., Croera, C., Brustio, R., Collotta, A., Bowe, G., Vahter, M., Gribaldo, L., 2008. Toxicity of inorganic arsenic and its metabolites on haematopoietic progenitors in vitro: comparison between species and sexes. Toxicology 249, 102–108. Gonzalez, H.O., Hu, J., Gaworecki, K.M., Roling, J.A., Baldwin, W.S., Gardea-Torresdey, J.L., Bain, L.J., 2010. Dose-responsive gene expression changes in juvenile and adult mummichogs (Fundulus heteroclitus) after arsenic exposure. Marine Environmental Research 70, 133–141. Gonzalez, H.O., Roling, J.A., Baldwin, W.S., Bain, L.J., 2006. Physiological changes and differential gene expression in mummichogs (Fundulus heteroclitus) exposed to arsenic. Aquatic Toxicology 77, 43–52. Greenbaum, D., Colangelo, C., Williams, K., Gerstein, M., 2003. Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biology 4, 117. Guertin, A.D., Martin, M.M., Roberts, B., Hurd, M., Qu, X., Miselis, N.R., Liu, Y., Li, J., Feldman, I., Benita, Y., Bloecher, A., Toniatti, C., Shumway, S.D., 2012. Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition. Cancer Cell International 12, 45. Guo, H.-R., 2011. Age adjustment in ecological studies: using a study on arsenic ingestion and bladder cancer as an example. BMC Public Health 11, 820. Habib, G.M., 2010. Arsenite causes down-regulation of Akt and c-Fos, cell cycle dysfunction and apoptosis in glutathione-deficient cells. Journal of Cellular Biochemistry 110, 363–371. Hyun Park, W., Hee Cho, Y., Won Jung, C., Oh Park, J., Kim, K., Hyuck Im, Y., Lee, M.H., Ki Kang, W., Park, K., 2003. Arsenic trioxide inhibits the growth of A498 renal cell carcinoma cells via cell cycle arrest or apoptosis. Biochemical and Biophysical Research Communications 300, 230–235. Katayama, K., Fujita, N., Tsuruo, T., 2005. Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2/M transition. Molecular and Cellular Biology 25, 5725–5737. Le Borgne, F., Ben Mohamed, A., Logerot, M., Garnier, E., Demarquoy, J., 2011. Changes in carnitine octanoyltransferase activity induce alteration in fatty acid metabolism. Biochemical and Biophysical Research Communications 409, 699–704. Li, D., Lu, C., Wang, J., Hu, W., Cao, Z., Sun, D., Xia, H., Ma, X., 2009. Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos. Aquatic Toxicology 91, 229–237. Lindberg, A.-L., Kumar, R., Goessler, W., Thirumaran, R., Gurzau, E., Koppova, K., Rudnai, P., Leonardi, G., Fletcher, T., Vahter, M., 2007. Metabolism of low-dose inorganic arsenic in a central European population: influence of sex and genetic polymorphisms. Environmental Health Perspectives 115, 1081–1086. Liu, J., Chen, H., Miller, D.S., Saavedra, J.E., Keefer, L.K., Johnson, D.R., Klaassen, C.D., Waalkes, M.P., 2001. Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic. Molecular Pharmacology 60, 302–309. Liu, J., Xie, Y., Ducharme, D.M.K., Shen, J., Diwan, B.A., Merrick, B.A., Grissom, S.F., Tucker, C.J., Paules, R.S., Tennant, R., Waalkes, M.P., 2006. Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure. Environmental Health Perspectives 114, 404–411.

7

Long, Y., Li, Q., Cui, Z., 2011a. Molecular analysis and heavy metal detoxification of ABCC1/MRP1 in zebrafish. Molecular Biology Reports 38, 1703–1711. Long, Y., Li, Q., Wang, Y., Cui, Z., 2011b. MRP proteins as potential mediators of heavy metal resistance in zebrafish cells. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 153, 310–317. Maciaszczyk-Dziubinska, E., Wawrzycka, D., Wysocki, R., 2012. Arsenic and antimony transporters in eukaryotes. International Journal of Molecular Sciences 13, 3527–3548. Mamrut, S., Harony, H., Sood, R., Shahar-Gold, H., Gainer, H., Shi, Y.-J., BarkiHarrington, L., Wagner, S., 2013. DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor. PLOS ONE 8, e56869. Markowski, V.P., Currie, D., Reeve, E.A., Thompson, D., Wise Sr., J.P., 2011. Tissuespecific and dose-related accumulation of arsenic in mouse offspring following maternal consumption of arsenic-contaminated water. Basic & Clinical Pharmacology & Toxicology 108, 326–332. Monsalve, F.A., Pyarasani, R.D., Delgado-Lopez, F., Moore-Carrasco, R., 2013. Peroxisome proliferator-activated receptor targets for the treatment of metabolic diseases. Mediators of Inflammation 2013, 18. Moro, L., Arbini, A.A., Marra, E., Greco, M., 2007. Constitutive activation of MAPK/ERK inhibits prostate cancer cell proliferation through upregulation of BRCA2. International Journal of Oncology 30, 217–224. ¯ Nagashima, H., Kumagai, H., Tomoda, H., Omura, S., 1993. Inhibition of hepatic cholesterol biosynthesis by a 3-hydroxy-3-methylglutaryl coenzyme a synthase inhibitor, 1233A, in mice. Life Sciences 52, 1595–1600. NRC, 2001. Arsenic in Drinking Water: 2001 Update. The National Academies Press, Washington, DC. Rajagopalan, H., JAllepalli, P.V., Rago, C., Velculescu, V.E., Kinzler, K.W., Vogelstein, B., Lengauer, C., 2004. Inactivation of hCDC4 can cause chromosomal instability. Nature 428, 77–81. Shaw, J.R., Gabor, K., Hand, E., Lankowski, A., Durant, L., Thibodeau, R., Stanton, C.R., Barnaby, R., Coutermarsh, B., Karlson, K.H., Sato, J.D., Hamilton, J.W., Stanton, B.A., 2007. Role of glucocorticoid receptor in acclimation of killifish (Fundulus heteroclitus) to seawater and effects of arsenic. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 292, R1052–R1060. Shi, H., Luo, J., Zhu, J., Li, J., Sun, Y., Lin, X., Zhang, L., Yao, D., Shi, H., 2013. PPAR␥ regulates genes involved in triacylglycerol synthesis and secretion in mammary gland epithelial cells of dairy goats. PPAR Research 2013, 10. Smeester, L., Rager, J.E., Bailey, K.A., Guan, X., Smith, N., Garcia-Vargas, G., Del Razo, L.-M., Brobna, Z., Kelkar, H., Styblo, M., Fry, R.C., 2011. Epigenetic changes in individuals with arsenicosis. Chemical Research in Toxicology 24, 165–167. Tan, N.-S., Shaw, N.S., Vinckenbosch, N., Liu, P., Yasmin, R., Desvergne, B., Wahli, W., Noy, N., 2002. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. Molecular and Cellular Biology 22, 5114–5127. Vahter, M., Åkesson, A., Lidén, C., Ceccatelli, S., Berglund, M., 2007. Gender differences in the disposition and toxicity of metals. Environmental Research 104, 85–95. Venkatachalam, A.B., Sawler, D.L., Wright, J.M., 2013. Tissue-specific transcriptional modulation of fatty acid-binding protein genes, fabp2, fabp3 and fabp6, by fatty acids and the peroxisome proliferator, clofibrate, in zebrafish (Danio rerio). Gene 520, 14–21. Vogel, C., Marcotte, E.M., 2012. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics 13, 227–232. Vriend, L.E.M., De Witt Hamer, P.C., Van Noorden, C.J.F., Würdinger, T., 2013. WEE1 inhibition and genomic instability in cancer. Biochimica et Biophysica Acta (BBA) – Reviews on Cancer 1836, 227–235. Waalkes, M.P., Liu, J., Ward, J.M., Diwan, B.A., 2004a. Animal models for arsenic carcinogenesis: inorganic arsenic is a transplacental carcinogen in mice. Toxicology and Applied Pharmacology 198, 377–384. Waalkes, M.P., Ward, J.M., Diwan, B.A., 2004b. Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers. Carcinogenesis 25, 133–141. Waalkes, M.P., Ward, J.M., Liu, J., Diwan, B.A., 2003. Transplacental carcinogenicity of inorganic arsenic in the drinking water: induction of hepatic, ovarian, pulmonary, and adrenal tumors in mice. Toxicology and Applied Pharmacology 186, 7–17. Wang, Z.X., Jiang, C.S., Liu, L., Wang, X.H., Jin, H.J., Wu, Q., Quan, C., 2005. The role of Akt on arsenic trioxide suppression of 3T3-L1 preadipocyte differentiation. Cell Research 15, 379–386. Ying, S., Myers, K., Bottomley, S., Helleday, T., Bryant, H.E., 2009. BRCA2-dependent homologous recombination is required for repair of arsenite-induced replication lesions in mammalian cells. Nucleic Acids Research 37, 5105–5113.

Please cite this article in press as: Carlson, P., Van Beneden, R.J., Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio). Aquat. Toxicol. (2013), http://dx.doi.org/10.1016/j.aquatox.2013.10.006

Arsenic exposure alters expression of cell cycle and lipid metabolism genes in the liver of adult zebrafish (Danio rerio).

Adult zebrafish (Danio rerio) were used to investigate mRNA expression in the liver following 7-day and 21-day exposures to 0, 10, 50, or 500 ppb sodi...
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