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Transgenerational reproductive effects of arsenite are associated with H3K4 di-methylation and SPR-5 downregulation in Caenorhabditis elegans Chan-Wei Yu, and Vivian Hsiu-Chuan Liao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02173 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 4, 2016
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Manuscript submitted to: Environmental Science & Technology
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es-2016-02173j-R2
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Transgenerational reproductive effects of arsenite are associated
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with H3K4 di-methylation and SPR-5 downregulation in Caenorhabditis
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elegans
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Chan-Wei Yu, Vivian Hsiu-Chuan Liao*
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Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan
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ABSTRACT
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Arsenic is a prevalent environmental toxin. Arsenic is associated with a wide variety of
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adverse effects, however, studies on whether As-induced toxicities can be transferred from
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parents to offspring have received little attention. Caenorhabditis elegans has become an
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important animal model in biomedical and environmental toxicology research. In this study,
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the transgenerational reproductive toxicity by arsenite exposure and the underlying
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mechanisms in C. elegans were investigated over six generations (F0 to F5). Following
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arsenite maternal exposure to the F0 generation, subsequent generations (F1–F5) were
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cultured under arsenite-free conditions. We found that the brood size of C. elegans was
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significantly reduced by arsenite exposure in the F0, and that this reduction in brood size was
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also observed in the offspring generations (F1–F5), after the toxicant had been removed from
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the diet. In addition, adult worms from F0 and F1 generations accumulated arsenite and
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arsenate when F0 L4 larvae were exposed to arsenite for 24 h. We found that the mRNA level
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of the H3K4me2 demethylase LSD/KDM1, spr-5, was significantly reduced in the F0
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exposed generation and subsequent unexposed generations (F1–F3). Likewise, the mRNA
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levels of spr-5 were also significantly decreased in the F1 to F3 generations. Moreover,
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di-methylation of global H3K4 was increased in the F0 to F3 generations. Our study
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demonstrates that maternal arsenite exposure causes transgenerational reproductive effects in
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C. elegans, which might be associated with H3K4 di-methylation and SPR-5 downregulation.
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Keywords: arsenic, transgeneration, reproductive toxicity, Caenorhabditis elegans, epigenetic, histone H3K4di-methylation, SPR-5
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INTRODUCTION
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Arsenic is a naturally occurring chemical found widely in the environment. Arsenic is
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released into the environment from both natural and human activities.1 Arsenic is a
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well-known human carcinogen and was ranked first on the Agency for Toxic Substances and
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Disease Registry (ATSDR) priority list of hazardous substances in USA for several decades.2
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High concentrations of arsenic in groundwater have been found in many countries,3,4 making
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exposure to arsenic in drinking water a significant health problem for people around the world.
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Studies have reported that arsenic is associated with a wide variety of adverse effects, such as
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skin lesions, peripheral vascular diseases, reproduction toxicity and neurological effects.5
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Arsenic is a recognized reproductive toxicant in laboratory animals.6-9 It has been
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reported that male mice exposed to 0.15 or 0.3 mM arsenite in drinking water for 5 weeks
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have decreased epididymal sperm counts and testicular weights compared to non-exposed
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mice.9 In addition, exposure to high levels of arsenic in drinking water produces a high risk of
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fetal loss and premature delivery in pregnant woman.10 Pregnancy has long been recognized
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as a potential critical period of vulnerability for exposure to toxins and questions have been
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raised about the long-term impact of toxicant exposure during pregnancy. Therefore, the early
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developmental phase of pregnancy is thought to be very important in determining long-term
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growth and overall health. Several studies have shown positive associations of arsenic
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exposure with spontaneous abortion, stillbirth, preterm birth, low birth-weight and infant
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mortality but the underlying mechanisms are unclear.11-15
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The evaluation of arsenic toxicology has been carried out in many organisms, including
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fish, invertebrates and mammals.16-18 However, most of these studies have focused their
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attention on the observed toxic effects of arsenic, rather than its transferable property. Arsenic
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permeates the environment and human beings are continuously exposed to it. Although
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humans have a high risk for long-term exposure to arsenic, studies on arsenic-induced
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transgenerational toxicity has received little attention. Many environmental factors are
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recognized as causing epigenetic changes.19,20 For example, exposure to environmental
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toxicants, such as endocrine disruptors, during development alters DNA methylation,
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affecting the germline methylation and causing disease in adults for multiple generations.21-23
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Furthermore, endocrine disruptors have been reported to induce a transgenerational disease
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phenotype.24 It has been reported that arsenic acts as a potent endocrine disruptor by
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interfering with hormone systems and affecting DNA methylation in offspring,25,26 raising the
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possibility of transgenerational effects for arsenic exposure. Several studies have
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demonstrated the impact of environmental toxins, such as toxic metals, on the epigenome.27 It
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has been reported that exposure to arsenic is associated with changes in DNA methylation,
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histone modification and miRNA expression.27-29 Recently, Zhang et al. (2016) reported that
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arsenic causes aberrant repressive histone modification to silence Pyruvate dehydrogenase
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kinase 4 (PDK4) in both HCC cells and in mouse liver.30 Arsenic exposure causes the levels
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of H3K9me3 and H3K9Ac to change in lymphocytes.31 Despite the substantial data on the
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epigenetic consequences of arsenic exposure,32 little is known about the potential multi- and
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transgenerational phenotypes associated with these epigenetic effects. Specifically, there are
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important outstanding questions about the long-term impact of arsenic exposure and whether
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the toxic effects persist even after removal of this toxin. Recently, it has been reported that in
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utero and continuous early-life exposure to arsenite causes metabolic vulnerability later in life
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in a rodent model.33
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The nematode C. elegans has become an important animal model in biomedical and
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environmental toxicology research.34 Many basic physiological processes, stress responses
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and signal transduction pathways in higher organisms (e.g., humans) are conserved in C.
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elegans.34 C. elegans also contains 60–80% of human homologues.35 In C. elegans, the
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germinal cells mainly divide during the L2 stage and oocytes are matured during the end of
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L3–L4 stage. The sperm cells are matured during the L3 stage and have produced 200–300
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sperm around L4–adult molt.36,37
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Di-methylation of histone H3 on lysine 4 (H3K4me2) has been identified as a form of
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epigenetic memory and maintains transcription patterns during development in Drosophila.
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epigenetic reprogramming mechanisms.40 It has been reported that, in C. elegans, deficiency
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of spr-5 leads to high levels of sterility for many generations, resulting in global accumulation
The C. elegans ortholog of H3K4me2 demethylase LSD1/KMD1, SPR-5, regulates
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of H3K4me2.40,41 We hypothesized that arsenite exposure may lead to altered levels of SPR-5,
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which is involved in maintenance of epigenetic marks, and that the resulting epigenetic
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abnormalities may be transmitted to the offspring.
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Few studies have addressed the transgenerational effects of arsenic exposure. Taking
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advantage of the relatively short lifecycle of C. elegans, we investigated the transgenerational
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reproductive toxicity of arsenite exposure in C. elegans, where only the parental generation
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(F0) was exposed to arsenite. In addition, the potential underlying mechanisms of the
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transgenerational reproductive toxicity in C. elegans was investigated.
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MATERIALS AND METHODS
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C. elegans Strains and Handling Procedures.
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All chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK), unless
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otherwise described. The nematodes used in this study were wild-type N2. C. elegans strains
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and Escherichia coli OP50 strain were obtained from the Caenorhabditis Genetics Center
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(CGC) (University of Minnesota, MN, USA), which is funded by the NIH National Center for
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Research Resources. C. elegans was maintained and assayed at 20°C on nematode growth
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medium (NGM) agar plates carrying a lawn of E. coli OP50 as a food source.42
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Synchronization of C. elegans cultures were obtained by hypochlorite treatment of gravid
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hermaphrodites.43 Gravid nematodes were washed from the NGM plates and lysed with the
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bleaching solution (0.45M NaOH, 2% HOCl). An aged synchronous population of L1 larvae
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was obtained and cultured to L4 larval stage.
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Arsenite-contaminated NGM plates were prepared by adding the stock solution (1 M
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arsenite) into liquid NGM medium at 55°C to give final concentrations of arsenite at 0.1, 0.25,
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0.5, and 1 mM (nominal concentration). In NGM medium, 1 mM arsenite did not influence
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the pH and Eh values with pH and Eh at 6.4 and 17 (mV), respectively, and these values
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remained unchanged after 24 h.
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Measurement of C. elegans Growth Endpoint.
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C. elegans growth was examined by measurement of change in body length.
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Synchronized wild-type L4 nematodes were exposed to various concentrations (0, 0.1, 0.25,
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0.5, and 1 mM) of arsenite for 24 h on NGM plates with E. coli OP50. After 24 h exposure at
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20°C, nematodes were immediately photographed on plates and images from individual
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animals were captured from a dissecting microscope (Leica, Wetzlar, Germany) equipped
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with a cooled charge coupled device (CCD) camera. Nematodes lengths were measured using
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Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). At least 20 worms
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were measured for each treatment and at least three independent experiments were performed.
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Measurement of C. elegans Reproduction Endpoint.
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The brood size was assayed as previously described.44 Brood size was measured by
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randomly placing single synchronized wild-type L4 nematode onto individual NGM plates
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containing various concentrations (0, 0.1, 0.25, 0.5 and 1 mM) of arsenite for 24 h at 20°C.
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Subsequently, animal was transferred to a new plate (normal diets, arsenite-free) every day
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and the offspring of each animal was counted at the L2 or L3 stage following transfer. At least
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20 worms were assayed for each treatment and at least three biological independent trials
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were performed.
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Transgenerational Assays.
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Figure 1 illustrates the experimental design of the transgenerational assays. Briefly,
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around 5000 synchronized wild-type L4 nematodes of F0 generation were placed on control
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(arsenite- free) or 1 mM arsenite-containing 10-cm NGM plates (10 plates for each treatment)
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for 24 h at 20°C. Nematodes from F0 were then collected from each plate and washed at least
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3 times with K-medium (0.032 M KCL, 0.051 M NaCl) to remove arsenite. The offspring
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embryos (F1) were then obtained by hypochlorite treatment.43 The egg pellet was resuspended
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in K-medium without E. coli OP50 and let the eggs hatch overnight at 20°C with gentle
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rocking. Since there is no food source, the larvae should be halted at the L1 larval stage.
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Subsequently, a portion of F1s (~ 5000 L1) was placed onto 10 individual 10-cm NGM plates
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(arsenite- free) at 20°C. For reproduction assay, brood size of F1 generation was measured by
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randomly placing single synchronized wild-type L4 nematode from F1s onto individual NGM
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plates (normal diet, arsenite-free) as described above. For RNA and histone assays, F1 adult
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nematodes from 7 individual 10-cm NGM plates were pooled and divided into 2 portions for
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subsequent RNA and histone assays, respectively. F1 adult nematodes from the remaining 3
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individual 10-cm NGM plates were pooled and performed bleaching procedures43 for the next
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generation assays. These procedures were repeated in subsequent generations (F2–F5). The
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non-exposed control nematodes were also followed over the six generations. There were no
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significant differences between the control generations allowing us to pool the controls. The
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exposed individuals (F1–F5) were compared to the pool of the non-exposed control of six
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generations. At least three biological independent trials were performed.
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Measurement of Arsenic Species Accumulation in C. elegans.
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Nematodes from the control, F0, F1 and F2 generations were obtained in the same
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manner as above, described in the transgenerational assays. Total worms of F0, F1, and F2
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generations were counted by the “dilution method” (Supporting Information). Around 50,000
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worms of each condition were used.
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To analyze arsenic species, the worm pellets (F0, F1 and F2) were washed with
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phosphate-buffered saline (PBS) at least six times to remove extraneous material. F0, F1 and
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F2 generation worms were broken up by sonication and centrifuged to isolate the pellets from
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the supernatants. Subsequently, supernatants containing the worms lysates were collected and
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the presence of arsenic species (arsenite[As(III)], arsenate[As(V)], monomethylarsonic
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acid[MMA], and dimethylarsinic acid[DMA]) and arsenic concentrations were analyzed by
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High Performance Liquid Chromatography / Inductive Coupled Plasma / Mass Spectrometry
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(HPLC-ICP-MS) (Agilent 1260 Infinity Quaternary LC System; Agilent 7700x, Santa Clara,
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CA, USA). Limit of detection (LOD) using HPLC-ICP-MS for the four species:
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arsenite[As(III)], arsenate[As(V)], monomethylarsonic acid[MMA], and dimethylarsinic
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acid[DMA], is 0.15 µg/L (0.0011 µM), 0.23 µg/L (0.0014 µM), 0.12 µg/L (0.0004 µM), and
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0.07 µg/L (0.0005 µM), respectively, which was in agreement with the previous report.45 At
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least three biological independent trials were performed.
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Histone Extraction and Di-methyl Histone H3K4 Quantification.
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F0, F1, F2, F3, F4 and F5 generation nematodes were cultured as described above. Each
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generation of adult nematodes were collected and homogenized by sonication. Total histones
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were extracted using the EpiQuick total histone extraction kit (Epigentek, Farmingdale, NY,
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USA). Di-methyl histone H3K4 level was analyzed using EoiQuilk global di-methyk histone
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H3K4 quantification kit (colorimetric) (Epigentek). Normalization was ensured by adding 500
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ng of total histone to each sample. At least three biological independent trials were performed.
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Real-time Quantitative RT-PCR Analysis.
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Nematodes were cultured as described above. Total RNAs of F0, F1, F2, F3, F4 and F5
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generation adult worms were isolated using TRIzol, according to the manufacturer’s
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instructions (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using Super-Script
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III First-strand synthesis super-mix for qRT-PCR (Invitrogen). qRT-PCR was performed on a
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Step One real-time cycler (Applied Biosystems, Carlsbad, CA, USA), using a SYBR Green
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qPCR kit (Affymetrix, Inc., Cleveland, Ohio, USA). Primers used in qRT-PCR are presented
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in Supplementary Table 1 (Supporting Information). The relative quantities of mRNAs were
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determined using comparative cycle threshold methods, and were normalized against the
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mRNA of mlc-2 or β-actin. The fold change was normalized to non-exposed control worms.
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At least three independent experiments were performed.
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Data Analysis.
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Statistical analyses were performed using SPSS Statistics 19.0 Software (SPSS Inc.,
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Chicago, IL, USA, 2010). The results are presented as the mean ± standard deviations (SD),
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or the mean ± standard errors of the mean (SEM). The statistical significance of differences
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between C. elegans populations were determined using one-way ANOVA and Tukey’s test as
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a post-hoc test. For transgenerational assays, two-way ANOVA was also employed.
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Differences were considered statistically significant at p