Selenite restores Pax 6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters

1Universidad Autónoma de Nuevo León, UANL, Fac. De Biología, San Nicolás delos Garza, Nuevo León, Mexico; 2Grup de Mutagènesi, Departament de Genèticai de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain CIBER Epidemiologíay Salud Pública, ISCIII, Madrid, Spain; 3Tecnológico de Monterrey, Escuela de Medicina, Dirección de Investigación e Innovación. Monterrey, Nuevo León, México; 4Department of Molecular Biology, Centro de Investigación Biomédica del Noreste,Instituto Mexicano del Seguro Social, Monterrey, Nuevo León, Mexico; 5Departamento de Ciencias Básicas, Vicerrectoría de Ciencias de la Salud, Universidad de Monterrey, San Pedro Garza García, Nuevo León, Mexico


INTRODUCTION
Arsenic in drinking water is one of the major threats to public health worldwide.In several countries, the arsenic concentration in water used for human consumption exceeds the maximum permitted limits (WHO, 2010).Furthermore, arsenic exposure has been associated with various types of cancers; dermal effects; cardiovascular, renal, and hepatic diseases; and neurological disorders (Jomova et al., 2011).All the mechanisms of action by which arsenic induces toxicity and cancer have not yet been clarified; however, results of numerous in vitro and in vivo studies attempting to elucidate these mechanisms have been reported.Some authors attribute arsenic toxicity to the induction of oxidative stress in cells, leading to increased production of reactive oxygen species and subsequent DNA damage (Bach et al., 2014).This increase in free radicals generates instability in the whole genome, which is evidenced by deregulation of the expression of various genes, including the Pax6 gene (Bhattacharjee et al., 2013;Tyler & Allan, 2014).
The Pax6 gene is a member of the paired-box family that encodes a series of transcription factors highly conserved among species.PAX6 protein is essential for the development of the central nervous system and pancreatic cells (Blake & Ziman, 2014).PAX6 also has a crucial role in maintaining stability of genes involved in the self-renewal and neurogenesis of stem cells (Zhang et al., 2010).The up-and downregulation of Pax6 expression in neural tissues provokes abnormalities through different mechanisms (Sansom et al., 2009).Reports have shown that the Pax6 gene is a target of arsenic (Tyler & Allan, 2013).Therefore, it is important to understand the effects of arsenic on Pax6 expression.
One possible approach to reverse the toxic effects of arsenic in exposed individuals is the use of antioxidants to control the production of free radicals resulting from arsenic metabolism (Kaur et al., 2009).In particular, α-tocopheryl succinate (α-TOS), a vitamin E analog, has proapoptotic properties as well as antioxidant/anti-inflammatory activities (Neuzil et al., 2006).Other essential nutrients, such as selenium, have the ability to sequester arsenic (Wang et al., 2013) and inhibit its metabolism, thereby preventing its incorporation into the cell (Gailer, 2009).Therefore, the aim of this study was to evaluate the effect of two antioxidants, α-TOS and selenium, on Pax6 expression in neural tissues of hamsters chronically exposed to arsenic.Animals and treatments.Twenty adult male Golden Syrian hamsters (Mesocricetus auratus), 8 weeks old and weighing 90-110 g, were used.Food and water were provided ad libitum.The hamsters were randomly divided into six groups (n=3-5 per group), and each group received the respective treatment regimen for 7 months (Table 1).All treatments were administered in drinking water, contained in 200 mL bottles, every two days.The dose of sodium arsenite (100 ppm/L) used in this study was based on previous studies (Tripathi et al., 1997;Flora, 1999;Flora et al., 2005) as well as our own experience in determining levels of arsenic that cause measurable organ damage.The doses of α-TOS (6 mg/kg/day) and of sodium selenite (8.5 mg/kg/day) were selected based on studies that showed no toxicity in animals (Norppa et al., 1980;Hathcock et al., 2005).Animals were sacrificed in a carbon dioxide chamber.After cessation of breathing, the whole forebrains and cerebellums were removed and immediately placed in 15 mL tubes containing 2-3 mL of RNA later reagent to maintain RNA integrity.The tissues were then refrigerated (4°C) overnight, and on the next day they were frozen at -20°C until use.Forebrains and cerebellums from at least three untreated hamsters (controls) were processed in order to determine baseline Pax6 expression levels.
Sequence alignments and primer/probe design.An in silico analysis was conducted with the UCSC Genome Browser (Genome Bioinformatics Group, University of California at Santa Cruz, USA) for Pax6 primers and probe design.The Basic Local Alignment Search Tool (National Center for Biotechnology Information, Bethesda, MD, USA) was used to find orthologous sequences of the Mus musculus (mouse) Pax6 gene in M. auratus, Crisetulus griseus (Chinese hamster), and Rattus norvegicus (rat).Using the MEGA6 program (Tamura et al., 2013), evolutionarily conserved regions of the M. musculus Pax6 gene among the afore mentioned four species were identified, and multiple alignment of these con-served regions was subsequently performed to delimit the exons.File Builder 3.1 Software (Thermo Fisher Scientific-Applied Biosystems, Grand Island, NY, USA) was used to create the base file for designing the TaqMan probe and primers.The probe was designed to span the exon 2-exon 3 junction in order to accurately evaluate the levels of M. auratus Pax6 gene expression.
RNA extraction.Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific-Invitrogen) according to the manufacturer's instructions.Briefly, a sample of each forebrain and cerebellum was taken and frozen in liquid nitrogen.The frozen tissue was then pulverized avoiding thawing and was added to a microtube containing 1 mL of TRIzol reagent.Chloroform (0.2 mL) was then added and the microtube content stirred for 15 s.Samples were incubated at room temperature for 2-3 min and then centrifuged at 12 000 × g for 15 min at 4°C.The aqueous phase was removed and placed in a new microtube.Total RNA was precipitated with 0.5 mL of absolute isopropanol and centrifuged at 12 000 × g for 10 min at 4°C, followed by an ethanol wash.
Finally, the RNA pellet was resuspended in 30 µL of nuclease-free water (Invitrogen).The purity and concentration of total RNA were estimated spectrophotometrically at 260 and 280 nm.RNA integrity was assessed by electrophoresis in 1% agarose gel stained with GelRed.
Quantitative RT-PCR assays.cDNA synthesis was carried out using M-MLV reverse transcriptase and random primers according to the manufacturer's instructions.Briefly, 1 µg of total RNA plus random primers and deoxynucleosidetriphosphates were used to prepare mix1.Mix1 was incubated at 65°C for 5 min and then cooled on ice.Separately, Mix2 was prepared with firststrand buffer, DTT, and RNaseOUT (Invitrogen).Mix2 was added to Mix1 and incubated at 37°C for 2 min.M-MLV reverse transcriptase was then added to the reaction tube, and the PCR took place under the following conditions: 25°C for 10 min, 37°C for 50 min, and finally enzyme inactivation at 70°C for15 min.The functionality of the cDNA was confirmed by end-point PCR amplification of the constitutive r18S gene, which encodes the ribosomal RNA18S subunit (Table 2), and visualization on 1% agarose gel stained with GelRed.Quantitative PCR was performed using the 7500 Fast Real Time PCR System with primers and the TaqMan probe for the Pax6 gene (Table 2).The PCR reaction was carried out in a 20 μL volume containing 10 μL of TaqMan Universal PCR Master Mix, 1 μL primer/probe, 3 μL of cDNA template and 6 μL of nuclease-free water.Amplification was performed in the standard mode under the following reaction conditions: initial denaturation at 50°C for 2 min; 40 cycles of denaturation at 95°C for 10 min, annealing and extension at 95°C for 15 s, 60°C for 1 min.The dynamic range curve was established using triplicate samples assayed in a 96-well format, and the 1:128 dilution was chosen.Negative template controls were included for all assays.As an endogenous control, analysis of r18S gene expression was performed in par- allel (Table 2).Threshold (Fraga et al., 2005) values were analyzed by the comparative 2 -ΔCt method described by Livak and Schmittgen (Livak Schmittgen, 2001).Statistical analysis.The Kolmogorov-Smirnov test was performed to evaluate the normal distribution of data.Results for quantitative PCR were analyzed using one-way analysis of variance (ANOVA).Dunnett's test and Tukey's test for multiple comparisons were performed using SPSS statistical software package (version 23.0;IBM Corp, Armonk, NY, USA).Student's t-test was used to compare relative expression data between forebrain and cerebellum.In all cases, the criterion for significance was set at P<0.05.Data are shown as the mean ± standard deviation (S.D.).

RESULTS
As depicted in Fig. 1, comparison of Pax6 baseline expression between tissues revealed a mean level of PAX6 mRNA in the forebrain that was 18.5-fold higher than that in the cerebellum (0.74 ± 0.55 vs. 0.04 ± 0.04).When all forebrain and cerebellum samples were compared, the significance of this difference was much greater (P<0.001)regardless of the treatments (Fig. 2).
Similarly, the differences in the magnitude of Pax6 expression between tissues were maintained despite the arsenic exposure (Fig. 2A and 2B).Kolmogorov-Smirnov analysis revealed a normal distribution of the data (P>0.05).Although ANOVA did not show any significant differences, mean PAX6 mRNA levels in both forebrain and cerebellum of arsenic-treated hamsters were decreased by 53 ± 21% and 32 ± 7%, respectively, in comparison to the untreated group.
In addition, the effect of α-TOS and selenite on Pax6 expression revealed unexpected phenomena.
In both forebrain and cerebellum, α-TOS reduced the mean Pax6 expression level to 30% of that in the untreated group.Also, selenite decreased the mean PAX6 mRNA level in the forebrain and cerebellum to 49% and 43% of that in untreated hamsters, respectively (Fig. 2C and 2D).
In arsenic-exposed hamsters treated with α-TOS, the downregulation of Pax6 expression in the forebrain and cerebellum was maintained: mean PAX6 mRNA levels were similar to those observed in the group treated only with α-TOS (Fig. 3).Interestingly, treatment of arsenic-  exposed hamsters with selenite restored PAX6 mRNA to the control levels in the forebrain (P=0.394)but not in the cerebellum.

DISCUSSION
Our gene expression analysis by quantitative PCR revealed that PAX6 mRNA was more abundant in forebrain than in cerebellum, regardless of arsenic exposure or treatment with antioxidants.Although higher expression of Pax6 has been reported in the cerebellum during adulthood (Stoykova & Gruss, 1994;Duan et al., 2013), the granulosa cells, in which Pax6 is expressed, are more susceptible to oxidative stress compared to other neural cells (Fujimura & Usuki, 2014).Considering that in our study we extracted total RNA from the forebrain and cerebellum, the oxidative response may vary in different structures of the brain, which may explain the observed differences in Pax6 expression (Vaz et al., 2011;Medeiros et al., 2012).
Our expression assays further revealed that hamsters exposed to arsenic showed decreased Pax6 expression levels in both forebrain and cerebellum.This finding is consistent with a previous report of Pax6 down regulation in adult mice treated with arsenic during the embryonic stage (Tyler & Allan, 2013).Other studies of nervous tissue have shown altered gene expression in response to arsenic exposure (Wlodraczyk et al., 1996;Luo et al., 2012;Zhang et al., 2014).Although PAX6 protein appears to be crucial for regulating genes involved in self-renewal and stem cell neurogenesis (Sansom et al., 2009), our findings suggest that the downregulation of Pax6 in neural tissues following arsenic exposure may trigger changes in essential cellular processes that lead to neurologic abnormalities.
In our study, the expression of Pax6 was dramatically reduced by α-TOS treatment.This decrease in gene expression triggered by α-TOS has been previously reported by Changand coworkers (2003).Likewise, treatment with selenite resulted in a decrease in Pax6 expression, but the magnitude of this decrease was much smaller compared to that of the α-TOS group.This reduced effect of selenite on decreasing Pax6 expression may be related to increased biosynthesis of the cytosolic selenoprotein, glutathione peroxidase, which is believed to protect cells against peroxide damage (Bermingham et al., 2014).
Treatment of arsenic-exposed hamsters with α-TOS did not show a positive effect on Pax6 regulation neither in the forebrain nor cerebellum.Although other studies have demonstrated a protective effect of α-TOS against oxidizing agents (Prasad et al., 2003;Stankov et al., 2007;Bellezza et al., 2014), it is important to consider that, by oral administration, some characteristic properties (i.e., proapoptotic activity) of α-TOS could be lost (Neuzil & Massa, 2005).In addition, it has been reported that α-tocopheryl is protective against arsenic (Chung etal., 2011); however, high doses of α-tocopheryl may increase the toxic effect of arsenic (Rocha et al., 2011).Therefore, studies using different doses of α-TOS must be done to determine the threshold level of cellular toxicity and to characterize its interactions with oxidizing agents (e.g., arsenic).By contrast, treatment of arsenic-exposed hamsters with selenite resulted in restoration of Pax6 expression to control levels in the forebrain.Similarly, Li and coworkers (2013) demonstrated the capacity of selenite to protect against lead neurotoxicity.These results suggest that selenite has the ability to bind arsenic and form a complex with glutathione, thereby preventing arsenic from entering the cells (Gailer, 2009).
Finally, we observed a large degree of variability in forebrain and cerebellum Pax6 expression levels among the individual animals (as denoted by a markedly increased S.D.) in the control and arsenic-exposed/selenite-treated groups.These expression variations suggest that the genetic background of the animals plays the key role in gene regulation.Another explanation for these Pax6 expression differences in our study is the fact that the treated animals were not from the same litter, and thus there were differences in genetic load.
In conclusion, exposure to arsenic leads to decreased Pax6 gene expression in nervous tissues of the Golden Syrian hamster, and this effect can be reversed by selenite.Although our results suggest that selenite can be used to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population must be evaluated in future studies.

Disclosure of potential conflicts of interest
The authors declare that there are no conflicts of interest.

Figure 1 .
Figure 1.Relative Pax6 expression in forebrain and cerebellum of M. auratus.Each bar represents the mean ± S.D. of 3 independent experiments.Results were normalized to r18S gene expression.*P<0.001.

Figure 2 .
Figure 2. Effect of arsenic, α-TOS, and selenite on Pax6 expression in forebrain and cerebellum of M. auratus.(A) Pax6 expression in forebrain of arsenic-exposed animals.(B) Pax6 expression in cerebellum of arsenic-exposed animals.(C) Pax6 expression in forebrain of α-TOS-and selenite-treated animals.(D) Pax6 expression in cerebellum of α-TOS-and selenite-treated animals.Each bar represents the mean ± S.D. of 3 or 4 replicates from 3 independent experiments.Results were normalized to r18S gene expression.P>0.05.

Figure 3 .
Figure 3.Effect of α-TOS and selenite on Pax6 expression in arsenic-exposed M. auratus.(A) Pax6 expression in forebrain of arsenic-exposed animals treated with α-TOS and with selenite.(B) Pax6 expression in cerebellum of arsenic-exposed animals treated with α-TOS and with selenite.Each bar represents the mean ±S.D. of 3 or 4 replicates from 3 independent experiments.Results were normalized to r18S gene expression.Dashed lines represent the mean level of Pax6 expression in the untreated group.P>0.05.
A. Aguirre-Vázquez and coworkers Local Committee of Research and Ethics in Health No. 1902 at the Instituto Mexicano del Seguro Social and were in full compliance with the Official Mexican Standard NOM-062-ZOO-1999 for the production, care, and use of laboratory animals for scientific purposes.

Table 2 . Primers and probe sequences for PCR assays
(Applied Biosystems)Selenite restores Pax6 expression in neuronal cells of hamsters