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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Environ Int. 2020 Feb 18;137:105560. doi: 10.1016/j.envint.2020.105560

Arsenic and benzo[a]pyrene co-exposure acts synergistically in inducing cancer stem cell-like property and tumorigenesis by epigenetically down-regulating SOCS3 expression

Zhishan Wang a,*, Ping Yang a,b, Jie Xie a,c, Hsuan-Pei Lin a, Kazuyoshi Kumagai d, Jack Harkema d, Chengfeng Yang a
PMCID: PMC7099608  NIHMSID: NIHMS1564862  PMID: 32062438

Abstract

Arsenic and benzo[a]pyrene (BaP) are among the most common environmental carcinogens causing lung cancer. Millions of people are exposed to arsenic through consuming arsenic-contaminated drinking water. High levels of BaP are found in well-done barbecued meat and other food in addition to cigarette smoke. Hence, arsenic and BaP co-exposure in humans is common. However, the combined health effect and the underlying mechanism of arsenic and BaP co-exposure have not been well-understood. In this study we investigate the combined tumorigenic effect of arsenic and BaP co-exposure and the mechanism using both cell culture and mouse models. It was found that arsenic (sodium arsenite, 1.0 μM) and BaP (2.5 μM) co-exposure for 30 weeks synergizes in inducing malignant transformation of immortalized non-tumorigenic human bronchial epithelial cells and cancer stem cell (CSC)-like property to enhance their tumorigenicity. In animal studies, A/J mice were exposed to arsenic in drinking water (sodium arsenite, 20 ppm) starting from gestation day 18. After birth, the dams continuously received arsenic water throughout lactation. At weaning (3 weeks of age), male offspring were exposed to either arsenic alone via drinking the same arsenic water or exposed to arsenic plus BaP. BaP was administered via oral gavage (3 μmol per mouse per week) once a week starting from 3 weeks of age for 8 weeks. All mice were euthanized 34-weeks after the first BaP exposure. It was found that mice in control and arsenic exposure alone group did not develop lung tumors. All mice in BaP exposure alone group developed lung adenomas. However, arsenic and BaP co-exposure synergized in increasing lung tumor multiplicity and tumor burden. Furthermore, 30% of mice in arsenic and BaP co-exposure group also developed lung adenocarcinomas. Mechanistic studies revealed that arsenic and BaP co-exposure does not produce more BPDE-DNA adducts than BaP exposure alone; but acts synergistically in activating aryl hydrocarbon receptor (AhR) to up-regulate the expression of a histone H3 lysine 9 methyltransferase SUV39H1 and increase the level of suppressive H3 lysine 9 dimethylation (H3K9me2), which down-regulates the expression of tumor suppressive SOCS3 leading to enhanced activation of Akt and Erk1/2 to promote cell transformation, CSC-like property and tumorigenesis. Together, these findings suggest that arsenic and BaP co-exposure synergizes in causing epigenetic dysregulation to enhance cell transformation, CSC-like property and tumorigenesis.

Keywords: Arsenic and benzo[a]pyrene co-exposure, mixture exposure, aryl hydrocarbon receptor (AhR), suppressor Of Variegation 3-9 Homolog 1 (SUV39H1), suppressor of cytokine signaling 3 (SOCS3), cancer stem cell (CSC)-like property, tumorigenesis

1. Introduction

Humans and other organisms are consistently exposed to a mixture of environmental pollutants; however, current research mainly focuses on the effects of single pollutants representing a significant knowledge gap in understanding the health impact of environmental exposure (Bellavia et al., 2019; Bopp et al., 2018; Martin and Fry, 2018). Indeed, some previous studies showed that the effects of exposure to a mixture of environmental pollutants could be significantly different from that of exposure to individual chemicals (Tsiaoussis et al., 2019; Andrade et al., 2017; Karri et al., 2016). However, the underlying mechanisms of how mixtures of pollutants act significantly different from the single chemicals remain largely unknown.

Arsenic and benzo[a]pyrene (BaP) are among the most common environmental pollutants that humans are exposed to. Arsenic is a naturally occurring and widely distributing chemical; and arsenic-contaminated drinking water is the main source of general population arsenic exposure (IARC, 2004). BaP, a member of the polycyclic aromatic hydrocarbon (PAH) family, is produced usually when organic matters are incompletely burned (IARC, 1983). Cigarette smoke and well-done barbecued-meat contain high levels of BaP and are the common sources of human BaP exposure (Hecht, 2003; Kazerouni et al., 2001). Both arsenic and BaP are well-recognized human carcinogens causing lung cancer and other types of cancer (Tapio and Grosche, 2006; IARC, 2004; Hecht, 2003; Yang and Frenkel, 2002; IARC, 1983). Since millions of people are exposed to arsenic through arsenic-contaminated drinking water and high levels of BaP are found in cigarette smoke and well done-cooked meat, it is likely that arsenic and BaP co-exposure is common in humans (Chen et al., 2004). However, the combined effects and the underlying mechanism of arsenic and BaP co-exposure have not been well-understood.

Epidemiology studies showed that human exposure to arsenic through drinking water is associated with increased risk of lung cancer (Celik et al. 2008; Tapio and Grosche, 2006; IARC, 2004). Moreover, epidemiology studies also showed that arsenic-exposed people who were cigarette smokers had a significantly higher lung cancer risk than those who were non-smokers, revealing that arsenic exposure and cigarette smoking act synergistically in increasing the risk of lung cancer (Celik et al. 2008; Mostafa et al., 2008; Tapio and Grosche, 2006; Chen et al., 2004). Since BaP is one of the major carcinogens in cigarette smoke, it was speculated that the synergistic effect of arsenic exposure and cigarette smoking on lung cancer risk may be due to the combined effect of arsenic and BaP co-exposure. Indeed, some studies showed that co-exposure of arsenic (arsenic trioxide) and BaP via intratracheal instillation significantly increased the incidence of lung squamous cell carcinoma in Wistar-King rats and lung adenocarcinoma in Hamsters (Pershagen et al., 1984; Ishinishi et al., 1977). However, it has not been studied whether arsenic exposure through drinking water together with BaP exposure via oral ingestion exhibit a synergistic effect on lung cancer risk. Furthermore, the mechanism of the synergistic effect of arsenic and BaP co-exposure on lung cancer risk remains largely unknown.

Cancer stem cells (CSCs) or CSC-like cells refer to a small population of cancer cells exhibiting characteristics associated with normal stem cells (Nguyen et al., 2012). It has been proposed that cancers are originated from CSCs, which means that CSCs or CSC-like cells are cancer initiating and maintaining cells (Nguyen et al., 2012). Ours and other recent studies showed that chronic exposure to arsenic or other metal carcinogens produce CSC-like cells; and that the capability of chronic metal carcinogen exposure to induce CSC-like cells is considered a novel mechanism of metal carcinogenesis (Wang and Yang, 2019). However, it remains to be determined whether arsenic and BaP co-exposure is capable of producing more CSC-like cells.

The objective of this study was to use cell culture and mouse models to investigate the combined effect and the mechanism of arsenic and BaP co-exposure via contaminated-drinking water and oral ingestion, respectively, the common routes of general population arsenic and BaP exposure. The findings of this study not only reveal a novel mechanism of the combined effect of arsenic and BaP co-exposure in increasing lung cancer risk, also provide a foundation for future developing strategies to prevent or reduce the risk of lung cancer resulting from arsenic and BaP co-exposure.

2. Materials and Methods

2.1. Cell culture and cell transformation by chronic arsenic and BaP exposure

Immortalized human bronchial epithelial BEAS-2B cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cell transformation by chronic arsenic and BaP exposure was performed following the protocol described in our previous publications (Wang et al., 2018; 2011). Briefly, BEAS-2B cells were continuously exposed to vehicle control (dimethyl sulfoxide, DMSO), arsenic (NaAsO2, 1 μM, Sigma), BaP (2.5 μM, Sigma) or arsenic (NaAsO2, 1 μM) plus BaP (2.5 μM). When reaching about 80-90% confluence after 48-72 h arsenic and BaP exposure, cells were sub-cultured. Arsenic and BaP were then freshly added to cells each time after overnight cell attachment. Soft agar colony formation assay was performed after every 5-week arsenic and BaP exposure to assess cell transformation, which was stopped after arsenic and BaP continuous exposure for 30 weeks. Both parental BEAS-2B cells, chronic arsenic and BaP exposure-transformed BEAS-2B cells, and passage-matched control BEAS-2B cell were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 5% fetal bovine serum (FBS, Sigma).

2.2. Generation of SUV39H1stable knockdown and SOCS3 stable overexpression cells.

To generate vector control and SUV39H1 stable knockdown cells, arsenic and BaP co-exposure-transformed BEAS-2B cells (BEAS-2B-As+BaP) were transduced with non-targeting control small hairpin RNA (shRNA) lentiviral (pLKO.1-Control shRNA) or SUV39H1 targeting shRNA lentiviral (pLKO .1-SUV39H1 shRNA, Catalogue #: TRCN0000275372 ) particles (Sigma). Forty-eight h after the lentiviral particle transduction, cells were selected with puromycin (1 μg/ml) following the procedure described in our previous studies (Humphries et al., 2014; Zhao et al., 2011). The knockdown of SUV39H1 expression was confirmed by Western blot analysis. To generate SOCS3 stable overexpression cells, parental BEAS-2B cells and arsenic and BaP co-exposure-transformed BEAS-2B cells (BEAS-2B-As+BaP) were transduced with vector control (pLenti6.3) or SOCS3 overexpressing (pLenti6.3-SOCS3) lentiviral particles (Invitrogen) and selected with blasticidin (10 μg/ml) following the procedures described in our previous studies (Wang et al., 2019a; 2014; Humphries et al., 2017). The overexpression of SOCS3 was confirmed by Western blot.

2.3. Soft agar colony formation assay.

The soft agar colony formation assay was performed following our previous protocol (Li et al., 2019; Yang et al., 2005). Briefly, passage-matched control BEAS-2B cells and arsenic-, BaP-exposed BEAS-2B cells were harvested and suspended in DMEM containing 10% FBS at a concentration of 0.5 × 104 cells/ml. Normal melting point agar (5 ml of 0.6% agar in DMEM) was placed into each 60-mm cell culture dish as the bottom agar. After solidification of the bottom agar, 4 ml of cell mixture consisting of 2 ml of cell suspension (0.5 × 104 cells/ml) and 2 ml of 0.8% lower melting point agar in DMEM containing 10% FBS were poured over the bottom agar. After solidification of the upper agar, 3 ml of DMEM containing 10% FBS were added, and dishes were incubated at 37°C in a humidified 5% CO2 atmosphere. For testing the effect of pharmacological inhibitors treatment on soft agar colony formation of arsenic and BaP co-exposure-transformed cells, the inhibitors were added to upper agar and culture media. Soft agar colonies were stained with 0.003% crystal violet, photographed and counted (if > 100 μm) after 4-week incubation.

2.4. Serum-free suspension culture sphere formation assay.

The sphere formation under serum-free suspension culture conditions reflecting the stem cell property was determined following the published protocol (Wang et al., 2019b; Xiao et al., 2018; Dontu et al., 2003) with minor modifications. Briefly, single cells from passage-matched control group, arsenic and BaP exposure groups were plated in ultralow attachment 24-well culture plates (Corning, Corning, NY) at a density of 2.5 × 103 cells per well suspended in serum-free DMEM containing human recombinant basic fibroblast growth factor (bFGF, 20 ng/ml), human recombinant epidermal growth factor (EGF, 20 ng/ml) (R&D, Minneapolis, MN), B27 (50 times diluted from the original 50× stock solution, Invitrogen, Carlsbad, CA) and heparin (4 μg/ml, Sigma). Plates were incubated at 37°C in a humidified 5% CO2 atmosphere. Spheres were viewed, photographed and counted (if > 100 μm) under a phase-contrast microscope after 10-day culture.

2.5. ALDEFLUOR assay

The ALDEFLUOR assay is another assay used to assess CSC property based on the fact that CSCs have high aldehyde dehydrogenase (ALDH) activity (Sugihara and Saya, 2013). The ALDEFLUOR Kit from Stem Cell Technologies (Vancouver, BC, Canada) was used for performing ALDEFLUOR assay following instructions from the manufacturer. The ALDH inhibitor DEAB treatment group was included to verify that the gated ALDEFLUOR positive cells are diminished upon DEAB treatment and thus indeed ALDEFLUOR positive cells.

2.6. Western blot and quantitative PCR (q-PCR) analysis.

For Western blot analysis, cultured-cells were harvested and washed with PBS and lysed using cell lysis buffer following our published protocol (Wang et al., 2014; 2013; 2012) and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10 to 30 μg of protein/lane). Western blots were performed three times using cell pellets collected at different time. For q-PCR analysis, cellular total RNAs were extracted using TRIzol Reagent and used for q-PCR analysis of specific gene mRNA level carried out in ABI QuantStudio 3 q-PCR System using ABI TaqMan gene expression assay and β-actin or 18S rRNA was used to normalize gene mRNA expression levels as described in our recent publications (Wang et al., 2019; 2014).

2.7. Chromatin immunoprecipitation (ChIP)-q-PCR analysis

The ChIP-q-PCR analysis was used to analyze the enrichment of the transcription factor aryl hydrocarbon receptor (AhR) in the promoter region of SUV39H1 (suppressor of variegation 3-9 homolog 1) or the enrichment of dimethylated lysine residue 9 of histone H3 (H3K9me2) in the promoter region of SOCS3 (suppressor of cytokine signaling 3). The ChIP was performed using the Pierce™ Agarose ChIP Kit (Thermo Scientific) following manufacturer’s instructions. Briefly, cells were cross-linked with 1% formaldehyde, washed and digested with Micrococcal Nuclease (ChIP grade) to obtain 200-500 bp DNA fragments for immunoprecipitation (IP) followed by q-PCR analysis. ChIP grade antibodies against AhR (Catlogue #: 83200) and H3K9me2 (Catlogue #: 4658) (Cell Signaling Technology) were used for IP. AhR and H3K9me2 ChIP DNA were analyzed by q-PCR using ABI TaqMan gene expression assay as described above. The ChIP-qPCR results are expressed using the Percent Input Method.

2.8. BPDE-DNA adduct level analysis

The BPDE-DNA adduct levels in cultured cells were analyzed using an assay kit (OxiSelect™ BPDE DNA Adduct ELISA Kit, Catalog Number: STA-357) from Cell Biolabs, Inc. (San Diego, CA) following the protocol from the manufacturer. The measured-BPDE-DNA adduct levels in cultured cells were calculated using the BPDE-DNA adduct standard curve and expressed as BPDE-DNA adduct (ng)/cellular DNA (mg).

2.9. H&E, immunohistochemistry (IHC) and immunofluorescence (IF) staining

The H&E and IHC staining of mouse lung tissue sections were carried out following our previous procedures (Zhao et al., 2010). The IF staining of AhR in cultured cells were performed as described in our recent publication (Wang et al., 2018). The presented IF staining pictures are the overlaid images of AhR staining in red fluorescence with nuclear 4′6-diamidino-2-phenylindole (DAPI) staining in blue fluorescence. The IF staining images were taken and overlaid using Nikon NIS-Elements software.

2.10. Nude mouse xenograft tumorigenesis study

The nude mouse xenograft tumorigenesis study protocol was reviewed and approved by Michigan State University Institutional Animal Care and Use Committee. Six-week old female nude mice (Nu/Nu) were purchased from Charles River Laboratory. After one week acclimation, mice were randomly divided into 4 groups and 5 mice in each group. Passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells, or arsenic plus BaP co-exposure-transformed cells (0.25 × 106 cells in 0.1 ml of 1:1 growth factor-reduced matrigel and PBS) were injected subcutaneously into the right flank of female nude mice (Nu/Nu, Charles River laboratories, five mice in each group). After cell injection, nude mice were maintained under specific pathogen-free conditions. All animals were euthanized 12 weeks after cell injection, and the xenograft tissues were harvested and fixed with 10% formalin solution.

2.11. Arsenic and BaP co-exposure mouse lung tumorigenesis study

The arsenic and BaP co-exposure animal study was carried out in A/J mice (The Jackson Laboratory) and the animal study protocol was reviewed and approved by Michigan State University Institutional Animal Care and Use Committee. Since previous studies showed that only gestational or whole life arsenic exposure through drinking water produces lung tumors in mice (Waalkes et al., 2003, 2007; Tokar et al., 2011), our A/J mice arsenic exposure started from gestation day 18 and the dose of arsenic (NaAsO2) in drinking water in this study was 20 ppm, which captures the upper level reported for human drinking water arsenic exposure (IARC, 2004; Waalkes et al., 2007; Tokar et al., 2011) and has been reported to be well-tolerated by the pregnant mice and their offspring in an arsenic whole life exposure study (Tokar et al., 2011). Briefly, pregnant A/J mice received normal drinking water or drinking water with arsenic starting from gestation day 18. After birth, the dams continuously received the same normal drinking water or arsenic water throughout lactation. At weaning (3 weeks of age), male offspring from maternal mice received normal drinking water were randomly divided into two groups: Control group and BaP exposure alone group (n=10); male offspring from maternal mice received arsenic water were also randomly divided into two groups: arsenic exposure alone group and arsenic plus BaP co-exposure group (n=10) and these two groups of mice continuously received the same arsenic water untill the end of the experiment. Mice in BaP exposure alone group and arsenic plus BaP co-exposure group received BaP via oral gavage (3 gmol of BaP in 0.1 ml of corn oil (Mazola cholesterol free corn oil) per mouse per week) once a week starting from 3 weeks of age for 8 weeks, a well-established and widely used BaP exposure protocol for mouse lung tumorigenesis study (Hecht et al., 1994). Meanwhile, mice in control and arsenic exposure alone group received 0.1 ml of corn oil via oral gavage once a week starting from 3 weeks of age for 8 weeks. All mice were sacrificed 34 weeks after the first BaP exposure following the protocol from the previous study (Hecht et al., 1994). During mouse sacrifice, lungs, liver, stomach, and intestines were examined to detect tumor formation. Lungs were then fixed in 10% formalin solution and processed for further histology and pathology examination and IHC staining. Mouse lung tumors were analyzed by board-certified pathologists. The lung tumor burden % was determined by calculating volumes of tumors (bronchioloalveolar adenomas/carcinomas) and volumes of reference lung tissues using point counting technique as described in STEPanizer (ver.1-8: http://www.stepanizer.com/). The lung tumor multiplicity refers to the average number of lung tumors of each mouse.

2.12. Statistical analysis

The statistical analyses for the significance of differences between different groups (mean ± SD) were carried out by testing different treatment effects using two-tailed t-tests for a comparison of two data sets or one-way analysis of variance (ANOVA) for multiple data sets. A p-value of <0.05 was considered statistically significant. The significance of difference in nude mouse xenograft tumor incidence and A/J mouse lung tumor incidence was tested using Fisher’s exact test. The significance of difference in lung tumor burden and tumor multiplicity was analyzed using Student’s t-test. A p-value of <0.05 was considered statistically significant.

3. Results

3.1. Arsenic and BaP co-exposure acts synergistically in inducing cell transformation, cancer stem cell (CSC)-like property and tumorigenesis

To investigate the combined tumorigenic effect of arsenic and BaP co-exposure and the underlying mechanism, we first performed chronic cell transformation experiment by continuously exposing immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells to low levels of arsenic (1.0 μM of sodium arsenite) and BaP (2.5 μM) for 30 weeks. It was found that arsenic and BaP co-exposed cells (BEAS-2B-As+BaP) form significantly more colonies in soft agar than cells exposed to arsenic or BaP alone (Fig. 1A), suggesting that arsenic and BaP co-exposure synergizes in inducing cell transformation.

Figure 1.

Figure 1.

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Arsenic and BaP co-exposure acts synergistically in inducing cell transformation, CSC-like property and tumorigenesis. A-C. Arsenic and BaP co-exposure synergizes in inducing cell transformation (A) and cancer stem cell (CSC)-like property (B-C) (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells (BEAS-2B-Control); # p<0.05, compared to arsenic exposure alone-transformed cells (BEAS-2B-As); $ p<0.05, compared to BaP exposure alone-transformed cells (BEAS-2B-BaP). Panel B shows the representative images of suspension culture spheres. Scale bar: 100 μm. D. Arsenic and BaP co-exposure-transformed cells (BEAS-2B-As+BaP) display a significantly stronger tumorigenicity in nude mice. E-H. Arsenic and BaP co-exposure via oral ingestion synergizes in inducing lung tumorigenesis and tumor malignancy in A/J mice (mean ± SD, n=10). *p<0.05, compared to BaP exposure alone group. Panel F shows the representative images of mouse lung section H&E staining from control, arsenic exposure alone (As), BaP exposure alone (BaP) and arsenic and BaP co-exposure (As+BaP) group.

A serum-free suspension culture sphere formation assay was carried out to determine whether arsenic and BaP co-exposure acts synergistically in producing cancer stem cell (CSC)-like cells. As shown in Fig. 1B–C, arsenic and BaP co-exposed cells (BEAS-2B-As+BaP) formed significantly more suspension culture spheres than cells exposed to arsenic or BaP alone. Moreover, this result was confirmed by using another assay (ALDEFLUOR assay) based on the fact that CSCs have high aldehyde dehydrogenase (ALDH) activity (Sugihara and Saya, 2013). As shown in Supplementary Figure 1 (Fig. S1), arsenic and BaP co-exposure-transformed cells contain significantly more ALDH positive cells. Together, these results suggest that arsenic and BaP co-exposure synergizes in inducing CSC-like property.

CSCs are considered as tumor initiating and maintaining cells (Nguyen et al., 2012). We next determined whether arsenic and BaP co-exposure-transformed cells exhibit a significantly stronger tumorigenicity. Ours and others previous studies showed that subcutaneous inoculation of 2 to 5 million of chronic arsenic or BaP exposure alone-transformed human bronchial epithelial cells produce xenograft tumors in nude mice (Wang et al., 2011; Zhang et al. 2012; Zhao et al., 2012). In this study, we injected significantly less amount of cells and found that subcutaneous inoculation of 0.25 million of passage-matched control cells or arsenic exposure alone-transformed cells did not produce any xenograft tumors in nude mice (0 tumor from 5-injected mice). Only two small tumors were produced from 2 of 5 mice injected with 0.25 million of BaP exposure alone-transformed (BEAS-2B-BaP) cells (Fig. 1D). In sharp contrast, all 5 mice injected with arsenic and BaP co-exposure-transformed (BEAS-2B-As+BaP) cells produced bigger tumors (Fig. 1D). These results are consistent with that shown in Fig. 1B–C and Fig. S1, providing additional evidence showing that arsenic and BaP co-exposure synergizes in inducing CSC-like property to enhance tumorigenesis.

To further determine whether arsenic and BaP co-exposure acts synergistically in inducing lung tumors, we performed lung tumorigenesis studies using A/J mice. Since general populations are commonly exposed to arsenic and BaP through arsenic-contaminated drinking water and BaP-contained food, arsenic and BaP were administered to A/J mice via drinking water and oral gavage, respectively. The detailed arsenic and BaP exposure schedules are described in Materials and Methods and summarized in Supplementary Figure 2 (Fig. S2). It was found that arsenic exposure through drinking water started from gestation day 18 was well-tolerated by the pregnant mice as evidenced by no significant changes in the survival and body weight of their offspring, which is consistent with previous reports (Waalkes et al., 2003, 2007; Tokar et al., 2011). Arsenic exposure alone throughout the experiment caused no significant changes in mouse survival and body weight (Fig. 1E). Mice exposed to BaP alone or arsenic plus BaP all survived but showed about 10% decreases in body weight compared to the control group mice at the end of the experiment (p>0.05, Fig. 1E). No lung tumors or other types of tumors were found in control and arsenic exposure alone group. One mouse from BaP exposure alone group and one mouse from arsenic plus BaP co-exposure group had stomach tumor. Analysis of lung tissue sections by board-certified pathologists Drs. Kazuyoshi Kumagai and Jack Harkema revealed that all mice in BaP exposure alone group and arsenic and BaP co-exposure group developed lung adenoma (Fig. 1E). Moreover, three out of ten mice in arsenic and BaP co-exposure group also developed lung adenocarcinoma (Fig. 1E). Further quantifications of mouse lung tumors indicated that lung tumor multiplicity and tumor burden in arsenic and BaP co-exposed mice are significantly higher than that in BaP-exposed alone mice (Fig. 1F–H). These results demonstrate that arsenic exposure through drinking water together with BaP exposure via oral ingestion act synergistically in inducing lung tumorigenesis in mice.

Figure 2.

Figure 2.

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Arsenic and BaP co-exposure does not act synergistically in increasing BPDE-DNA adduct formation but synergizes in inducing the epigenetic change. A. Representative Western blot analysis of CYP-1A1 and 1B1 protein levels in passage-matched control cells (BEAS-2B-Control), arsenic exposure alone-transformed cells (BEAS-2B-As), BaP exposure alone-transformed cells (BEAS-2B-BaP) and arsenic plus BaP co-exposure-transformed cells (BEAS-2B-As+BaP). Similar results were observed in the repeated Western blots. B. BPDE-DNA adduct levels (mean ± SD, n=3) in passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells. C. Representative Western blot analysis of H3K9me2 and SUV39H1 protein levels in passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells. Similar results were observed in the repeated Western blots. D-E. Representative images of IHC staining of H3K9me2 (D) or SUV39H1 (E) in mouse lung tumor sections from mouse exposed BaP alone (BaP) or arsenic plus BaP (As+BaP). Scale bar: 100 μm.

3.2. Arsenic and BaP co-exposure does not act synergistically in producing more BPDE-DNA adducts but synergizes in inducing the epigenetic change

Next, we started investigating the mechanism of how arsenic and BaP co-exposure acts synergistically in inducing cell transformation, CSC-like property and tumorigenesis. It is known that BaP metabolic activation by cytochrome P450–1A1 (CYP-1A1) and 1B1 and subsequent formation of benzo(a)pyrene diol epoxide (BPDE)-DNA adduct play a critical role in BaP tumorigenicity (Hecht, 2003; Baird et al. 2005). We thus first determined whether arsenic and BaP co-exposure acts synergistically in regulating CYP-1A1 and 1B1 expression and the BPDE-DNA adduct formation. Western blot analysis showed that arsenic and BaP co-exposure transformed cells (BEAS-2B-As+BaP) express a higher level of CYP-1A1 than arsenic or BaP exposure alone-transformed cells (Fig. 2A). No significant change of CYP-1B1 expression level was detected (Fig. 2A). However, Further analysis using a BPDE-DNA Adduct ELISA Assay Kit revealed that no significant difference of BPDE-DNA adduct levels was detected between BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells (Fig. 2B). These results suggest that other mechanism may play an important role in the observed synergistic tumorigenic effect of arsenic and BaP co-exposure

Epigenetic dysregulation has been shown to play critical roles in producing cancer stemn cells and tumorigenesis (Baylin and Jones, 2016; Langevin et al., 2015). Since both arsenic and BaP have been reported to alter histone H3 posttranslational modifications (Chu et al., 2011; Sadikovic et al., 2008), a key mechanism of epigenetics, we next investigated whether arsenic and BaP co-exposure has a synergism in deregulating histone H3 posttranslational modifications. We screened several histone H3 posttranslational modifications and found while chronic arsenic or BaP exposure alone had no significant effect on the level of histone H3 lysine 9 (H3K9) dimethylation (H3K9me2), arsenic plus BaP co-exposure showed a synergistic effect in drastically increasing H3K9me2 level (Fig. 2C). Further Western blot analysis revealed that chronic arsenic and BaP co-exposure also significantly increases the expression level of the histone-lysine N-methyltransferase SUV39H1 (suppressor of variegation 3-9 homolog 1) (Fig. 2C). Moreover, IHC staining showed that mouse lung tumors from arsenic and BaP co-exposure group have much higher levels of H3K9me2 (Fig. 2D) and SUV39H1 (Fig 2E) than mouse lung tumors from BaP exposure alone group. Together, these results indicate that arsenic and BaP co-exposure synergizes in causing epigenetic dysregulation in both arsenic and BaP co-exposure-transformed cells and co-exposure-induced mouse lung tumors.

3.3. Stably knocking down SUV39H1 expression level in arsenic and BaP co-exposure-transformed cells significantly reduces their transformed-phenotype and CSC-like property

Next, we wanted to determine whether up-regulation of SUV39H1 expression level in arsenic and BaP co-exposure-transformed cells (BEAS-2B-As+BaP) play an important role in their transformed-phenotype and enhanced-CSC-like property. We generated SUV39H1 stable knockdown cells using SUV39H1 specific-targeting shRNAs. As shown in Fig. 3A, efficient knockdown of SUV39H1 expression level was confirmed by Western blot analysis. Moreover, the level of H3K9me2 in SUV39H1 stable knockdown (BEAS-2B-As+BaP-SUV39H1 shRNA) cells was greatly reduced compared to the Control shRNA cells (Fig. 3A), indicating that the high level of H3K9me2 in arsenic and BaP co-exposure-transformed cells is mainly mediated by the up-regulated expression of SUV39H1.

Figure 3.

Figure 3.

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Stably knocking down SUV39H1 expression greatly reduces H3K9me2 level in arsenic and BaP co-exposure-transformed cells and their transformed-phenotype and CSC-like property. A. Representative Western blot analysis of SUV39H1 and H3K9me2 levels in arsenic and BaP co-exposure-transformed cells stably expressing a non-targeting control shRNA or SUV39H1-specific targeting shRNA. Similar results were observed in the repeated Western blots. B-C. Effect of stably knocking down SUV39H1 expression in arsenic and BaP co-exposure-transformed cells on their capability of forming soft agar colonies (B) and suspension culture spheres (C) (mean ± SD, n=3). *p<0.05, compared to BEAS-2B-As+BaP-Control shRNA cells.

Further soft agar colony formation assay and serum-free suspension culture sphere formation assay revealed that arsenic and BaP co-exposure-transformed cells with SUV39H1 stable knockdown formed significantly less soft agar colonies (Fig. 3B) and suspension spheres (Fig. 3C) than the control shRNA cells. These results suggest that up-regulation of SUV39H1 expression level plays a critical role in maintaining the transformed-phenotype and the enhanced-CSC-like property of arsenic and BaP co-exposure-transformed cells.

3.4. Arsenic and BaP co-exposure up-regulates SUV39H1 expression by synergistically activating aryl hydrocarbon receptor (AhR)

We next determined the mechanism by which chronic arsenic and BaP co-exposure up-regulates SUV39H1 expression. Q-PCR analysis showed while either arsenic or BaP exposure alone slightly increase SUV39H1 mRNA level (Fig. 4A); arsenic and BaP co-exposure greatly up-regulates SUV39H1 mRNA level in more than an additive way (Fig. 4A). These results suggest that arsenic and BaP co-exposure may act synergistically in increasing SUV39H1 gene transcription. We then did bioinformatics analysis of SUV39H1 gene promoter and found that SUV39H1 promoter region has binding sites for a number of transcription factors including aryl hydrocarbon receptor (AhR). We decided to first investigate the role of AhR in SUV39H1 up-regulation by arsenic and BaP co-exposure based on the following two important facts: (i) AhR is a transcription factor playing important roles in tumor initiation and progression (Murray et al. 2014; Tsay et al. 2013); (ii) Previous studies showed that arsenic or BaP is capable of activating AhR with distinct mechanisms: while BaP is a ligand for AhR and activates AhR through the direct ligand-receptor interaction; arsenic activates AhR through a ligand-independent mechanism probably by inducing oxidative stress (Wu et al. 2009; Murray et al. 2014; Tsay et al. 2013).

Figure 4.

Figure 4.

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Arsenic and BaP co-exposure up-regulates SUV39H1 expression by synergistically activating aryl hydrocarbon receptor (AhR). A. The mRNA level of SUV39H1 in passage-matched control cells (BEAS-2B-Control), arsenic exposure alone-transformed cells (BEAS-2B-As), BaP exposure alone-transformed cells (BEAS-2B-BaP) and arsenic plus BaP co-exposure-transformed cells (BEAS-2B-As+BaP) analyzed by q-PCR (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells; # p<0.05, compared to arsenic exposure alone-transformed cells; $ p<0.05, compared to BaP exposure alone-transformed cells. B. Effect of arsenic and BaP exposure on AhR nuclear localization. The representative IF staining images are overlaid images of AhR staining in red fluorescence with nuclear DAPI staining in blue fluorescence. White arrows point to cells with strong nuclear AhR staining. Scale bar: 100 μm. The quantification of AhR nuclear localization was calculated by analyzing 300 cells of each of triplicate staining from each of 4 groups of cells (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells; # p<0.05, compared to arsenic exposure alone-transformed cells; $ p<0.05, compared to BaP exposure alone-transformed cells. C. ChIP-q-PCR analysis of AhR enrichment in SUV39H1 promoter region in passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells. The results are expressed as Percent Input (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells; # p<0.05, compared to arsenic exposure alone-transformed cells; $ p<0.05, compared to BaP exposure alone-transformed cells. D. Representative Western blot analysis of SUV39H1 and AhR levels in arsenic and BaP co-exposure-transformed cells transfected with control siRNA oligoes or AhR-specific targeting siRNA oligoes (100 nM). Cells were collected for Western blot analysis 48 h after siRNA transfection. Similar results were observed in the repeated Western blots.

We first carried out Western blot analysis and found that chronic arsenic and BaP co-exposure does not significantly change the expression level of AhR. AhR is a transcription factor localizing in cytoplasm when it is inactivated. AhR nuclear localization is one of parameters that indicate its transcriptional activity activation. We then performed AhR immunofluorescence (IF) staining to determine its cellular localization. As shown in Fig. 4B, AhR positive staining (red color) is mostly observed in cytoplasm in passage-matched control cells; increased AhR nuclear staining is seen in arsenic or BaP exposure alone-transformed cells. However, a significantly more and stronger AhR nuclear staining (pink color, overlaid from AhR staining red color and nuclear DNA DAPI staining blue color) is detected in arsenic and BaP co-exposure-transformed cells (Fig. 4B). These results strongly suggest that arsenic and BaP co-exposure acts synergistically in activating AhR.

To further demonstrate that arsenic and BaP co-exposure up-regulates SUV39H1 expression by increasing AhR activation, we next performed AhR chromatin immunoprecipitation (ChIP)-SUV39H1 promoter q-PCR assay to determine the enrichment of AhR in SUV39H1 gene promoter region. In consistent with SUV39H1 expression level and AhR IF staining results, our AhR-SUV39H1 ChIP-q-PCR analysis revealed increased enrichment of AhR in SUV39H1 gene promoter region in arsenic or BaP exposure alone-transformed cells (Fig. 4C). However, arsenic and BaP co-exposure resulted in a significantly more AhR enrichment in SUV39H1 gene promoter region (Fig. 4C), providing additional evidence showing that arsenic and BaP co-exposure synergizes in activating AhR to increase SUV39H1 expression. Furthermore, siRNA knocking down the expression level of AhR greatly reduced SUV39H1 expression level in arsenic and BaP co-exposure-transformed cell (Fig. 4D). Together these results indicate that arsenic and BaP co-exposure up-regulates SUV39H1 expression by synergistically activating AhR.

3.5. Up-regulation of SUV39H1 by arsenic and BaP co-exposure leads to down regulation of SOCS3 to promote cell transformation and CSC-like property

After demonstrating that arsenic and BaP co-exposure up-regulates the expression level of SUV39H1 and H3K9me2, which play an important role in arsenic and BaP co-exposure-induced cell transformation and CSC-like property, we next wanted to investigate how up-regulation of SUV39H1 and H3K9me2 promotes arsenic and BaP co-exposure-induced cell transformation and CSC-like property. Since H3K9me2 is one of repressive H3 posttranslational modifications, an important epigenetic mechanism that suppresses gene expression (Smolle and Workman, 2013; Greer and Shi, 2012), we hypothesized that high levels of SUV39H1 and H3K9me2 cause down-regulation of important tumor suppressive gene expression to promote arsenic and BaP co-exposure-induced cell transformation and CSC-like property.

It is known that both arsenic and BaP exposure are capable of increasing inflammatory cytokine signaling, which may be caused either by increased inflammatory cytokine production or by reduced expression of the suppressors of cytokine signaling (SOCSs). SOCS3 has been shown to function as a tumor suppressor; and epigenetic down-regulation of SOCS3 is often found in lung cancer (Baltayiannis et al. 2008; He et al. 2003). Currently, little is known about the effect of arsenic or BaP exposure on the level of SOCS3 expression. Our Western blot analysis revealed that chronic arsenic exposure alone has no significant effect on SOCS3 protein expression level and chronic BaP exposure decreases SOCS3 protein level (Fig. 5A). However, arsenic and BaP co-exposure reduced the protein level of SOCS3 more significantly than BaP exposure alone did (Fig. 5A), suggesting that arsenic and BaP co-exposure synergizes in down-regulating SOCS3 expression.

Figure 5.

Figure 5.

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Up-regulation of SUV39H1 and increase of H3K9me2 levels by arsenic and BaP co-exposure cause down-regulation of suppressor of cytokine signaling 3 (SOCS3). A. Representative Western blot analysis of SOCS3 expression level in in passage-matched control cells (BEAS-2B-Control), arsenic exposure alone-transformed cells (BEAS-2B-As), BaP exposure alone-transformed cells (BEAS-2B-BaP) and arsenic plus BaP co-exposure-transformed cells (BEAS-2B-As+BaP). Similar results were observed in the repeated Western blots. B. Representative Western blot analysis of SOCS3 protein level in vector control and SOCS3 stably overexpressing BEAS-2B-As+BaP cells. Similar results were observed in the repeated Western blots. C-D. Effect of stably expressing SOCS3 in arsenic and BaP co-exposure-transformed cells on their capability of forming soft agar colonies (C) and suspension culture spheres (D) (mean ± SD, n=3). *p<0.05, compared to BEAS-2B-As+BaP-pLenti6.3 vector control cells. E. The mRNA level of SOCS3 in passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells analyzed by q-PCR (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells; # p<0.05, compared to arsenic exposure alone-transformed cells; $ p<0.05, compared to BaP exposure alone-transformed cells. F. ChIP-q-PCR analysis of H3K9me2 enrichment in SOCS3 promoter region in passage-matched control cells, arsenic exposure alone-transformed cells, BaP exposure alone-transformed cells and arsenic plus BaP co-exposure-transformed cells. The results are expressed as Percent Input (mean ± SD, n=3). *p<0.05, compared to the passage-matched control cells; # p<0.05, compared to arsenic exposure alone-transformed cells; $ p<0.05, compared to BaP exposure alone-transformed cells. G. Representative Western blot analysis of SOCS3 protein level in arsenic and BaP co-exposure-transformed cells stably expressing a non-targeting control shRNA or SUV39H1-specific targeting shRNA. Similar results were observed in the repeated Western blots.

To determine whether down-regulation of SOCS3 expression level in arsenic and BaP co-exposure-transformed cells (BEAS-2B-As+BaP) plays an important role in their transformed-phenotype and enhanced-CSC-like property, we stably overexpressed SOCS3 in arsenic and BaP co-exposure-transformed cells. SOCS3 overexpression was confirmed by Western blot analysis (Fig. 5B). Soft agar colony formation assay and serum-free suspension culture sphere formation assay revealed that arsenic and BaP co-exposure-transformed cells with SOCS3 stable overexpression formed significantly less soft agar colonies (Fig. 5C) and suspension spheres (Fig. 5D) than the vector control cells (BEAS-2B-As+BaP-pLenti6.3). These results suggest that down-regulation of SOCS3 expression level plays an important role in maintaining the transformed-phenotype and enhanced-CSC-like property of arsenic and BaP co-exposure-transformed cells.

Next, we further determined how arsenic and BaP co-exposure down-regulates SOCS3 expression. Q-PCR analysis showed that arsenic and BaP co-exposure reduces SOCS3 mRNA level more significantly than arsenic or BaP exposure alone (Fig. 5E). To determine whether up-regulation of SUV39H1 and H3K9me2 play a role in SOCS3 down-regulation by arsenic and BaP co-exposure, we first performed H3K9me2 ChIP-SOCS3 promoter a-PCR analysis. It was found that arsenic exposure alone does not have a significant effect on the enrichment of H3K9me2 in SOCS3 promoter region (Fig. 5F). Increased enrichment of H3K9me2 was detected in SOCS3 promoter region of BaP exposure alone-transformed cells (Fig. 5F). However, arsenic and BaP co-exposure increased the enrichment of H3K9me2 in SOCS3 promoter region significantly more than BaP exposure alone (Fig. 5F). Furthermore, stably knocking down the expression of SUV39H1, which greatly decreased the level of H3K9me2 (Fig. 3A), drastically increased SOCS3 expression level in arsenic and BaP co-exposure-transformed cells (Fig. 5G). Together, these results indicate that arsenic and BaP co-exposure down-regulates SOCS3 expression through up-regulated-expression of SUV39H1 and H3K9me2.

3.6. Down-regulation of SOCS3 by arsenic and BaP co-exposure causes a stronger activation of Akt and Erk1/2 to promote cell transformation and CSC-like property

We next wanted to further investigate how SOCS3 down-regulation promotes arsenic and BaP co-exposure-induced cell transformation and CSC-like property. SOCS3 is one of the key negative regulators that inhibit cytokine signaling-triggered activation of Akt and extracellular-signal-regulated kinase 1 and 2 (Erk1/2) (Culig, 2013; Inagaki-Ohara et al. 2013). Moreover, Akt and Erk1/2 activation has been shown to promote cancer cell survival, proliferation and induction of CSCs or CSC-like cells (Martelli et al. 2010; Dreesen and Brivanlou, 2007). We thus determined the phosphorylation (activation) status of Akt and Erk1/2 in control, arsenic or BaP exposure alone and co-exposure-transformed cells. It was found while arsenic or BaP exposure alone increases the phosphor-levels of Akt and Erk1/2 (Fig. 6A), arsenic and BaP co-exposure synergizes in further drastically increasing the phosphor-levels of Akt and Erk1/2 (Fig. 6A). Moreover, the greatly increased phosphor-levels of Akt and Erk1/2 were also detected in arsenic and BaP co-exposure-caused mouse lung tumor compared to that in BaP exposure alone-induced lung tumor (Fig. 6B–C). Together, these results suggest that arsenic and BaP co-exposure causes a significantly much stronger activation of Akt and Erk1/2.

Figure 6.

Figure 6.

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Arsenic and BaP co-exposure synergizes in activating Akt and Erk1/2 and inhibition of Akt and Erk1/2 significantly reduces transformed phenotype and CSC-like property of co-exposure-transformed cells. A. Representative Western blot analysis of phosphor- and total Akt and Erk1/2 levels in passage-matched control cells (BEAS-2B-Control), arsenic exposure alone-transformed cells (BEAS-2B-As), BaP exposure alone-transformed cells (BEAS-2B-BaP) and arsenic plus BaP co-exposure-transformed cells (BEAS-2B-As+BaP). Similar results were observed in the repeated Western blots. B-C. Representative IHC staining images of phosphor-Akt (B) or phosphor-Erk1/2 (C) in mouse lung tumors from BaP alone or arsenic plus BaP (As+BaP) group. Scale bar: 100 μm. D. Representative Western blot analysis of phosphor- and total Akt and Erk1/2 levels in vector control and SOCS3 stably overexpressing BEAS-2B-As+BaP cells. Similar results were observed in the repeated Western blots. E-F. Effect of inhibition of Akt and/or Erk1/2 on the capability of arsenic and BaP co-exposure-transformed cell forming soft agar colonies (E) and suspension culture spheres (F) (mean ± SD, n=3). *p<0.05, compared to vehicle control-treated cells; # p<0.05, compared to the Akt inhibitor (AKTi) alone-treated cells; $ p<0.05, compared to MEK inhibitor (U0126) alone-treated cells.

Next we determined whether enhanced activation of Akt and Erk1/2 by arsenic and BaP co-exposure is due to the down-regulation of SCOS3. Western blot analysis showed stable overexpression of SOCS3 in arsenic and BaP co-exposure-transformed cells drastically reduces the phosphor-levels of Akt and Erk1/2 (Fig. 6D), confirming that SOCS3 down-regulation leads to highly activation of Akt and Erk1/2.

Finally, we determine whether highly activated Akt and Erk1/2 in arsenic and BaP co-exposure-transformed cells (BEAS-2B-As+BaP) plays a critical role in their transformed-phenotype and enhanced-CSC-like property. We treated arsenic and BaP co-exposure-transformed cells with a specific Akt inhibitor (AKTi), a MEK inhibitor (U0126) that causes inhibition of Erk1/2 or both (AKTi + U0126). Their specific inhibitory effects on AKT or Erk1/2 phosphorylation are shown in Supplementary Figure 3 (Fig. S3). Soft agar colony formation assay showed that inhibition of either Akt or Erk1/2 significantly decreases the number of colonies formed by arsenic and BaP co-exposure-transformed cells (Fig. 6E). Simultaneous inhibition both Akt and Erk1/2 displayed a much stronger effect in reducing the number of soft agar colonies (Fig. 6E), indicating that both Akt and Erk1/2 activation play an important role in anchorage-independent growth of arsenic and BaP co-exposure-transformed cells. In contrast, serum-free suspension culture sphere formation assay showed that only inhibition of Akt activity drastically reduces the number of suspension spheres formed by arsenic and BaP co-exposure-transformed cells (Fig. 6F), while inhibition of Erk1/2 activity has no significant effect on sphere formation (Fig. 6F). Moreover, simultaneous inhibition of both Akt and Erk1/2 exhibited a similar effect to that of inhibition of Akt alone on sphere formation (Fig. 6F). Together, these results suggest while both Akt and Erk1/2 activation contribute significantly to anchorage-independent growth of arsenic and BaP co-exposure-transformed cells; it is Akt activation that plays a significant role in promoting their CSC-like property.

4. Discussion

Arsenic and BaP are among the most common environmental carcinogens and humans are frequently exposed to arsenic and BaP together; however, the combined effect and mechanism of arsenic and BaP co-exposure remain largely unknown. In this study, we found that arsenic and BaP co-exposure acts synergistically in inducing cell malignant transformation, cancer stem cell (CSC)-like property and tumorigenesis. Moreover, arsenic and BaP co-exposure via oral ingestion also synergizes in inducing lung tumorigenesis in mice. Mechanistically, arsenic and BaP co-exposure acts synergistically in (i) activating AhR to increase the expression of a histone H3 lysine 9 methyltransferase SUV39H1; (ii) up-regulation of SUV39H1 increases repressive epigenetic modification of H3K9 dimethylation (H3K9me2) suppressing the expression of tumor suppressive SOCS3; and (iii) down-regulation of SOCS3 leads to a significantly stronger activation of Akt and Erk1/2, which enhances cell transformation, CSC-like property and tumorigenesis.

One unique feature of this study is arsenic and BaP co-exposure route in mice. The most common route of general population arsenic exposure is through consuming arsenic-contaminated drinking water; and one common route of non-smoker general population BaP exposure is via consuming BaP-contaminated food. Hattemer-Frey and Travis reported that the food chain is the dominant pathway of human BaP exposure, accounting for about 97% of the total daily intake of BaP (Hattemer-Frey and Travis, 1991). However, the combined effect of arsenic and BaP co-exposure via oral ingestion is unknown. Previous studies showed that arsenic (arsenic trioxide) and BaP co-exposure via intratracheal instillations significantly increases lung tumorigenesis in male Wistar-King albino rats (Ishinishi et al., 1977) and male Syrian golden hamsters (Pershagen et al., 1984). However, arsenic exposure via respiratory route usually occurs and limits mainly in occupational environment. The findings from our study indicate that arsenic exposure through drinking water with BaP co-exposure via oral ingestion could significantly increase lung cancer risk in general population. Moreover, since BaP is one of major carcinogens in cigarette smoke, our results also provide important mechanistic insight for early epidemiology study findings showing that arsenic-exposed (via drinking water) people who were cigarette smokers had a significantly higher lung cancer risk than those who were non-smokers: it could be due to the synergistic lung tumorigenic effect of arsenic and BaP co-exposure.

An important point needs to be further clarified is about mouse arsenic exposure level used in this study in drinking water. Our mice were exposed to 20 ppm of sodium arsenite via drinking water, which is much higher than current US EPA Drinking Water Arsenic Standard (10 ppb) but could still be human exposure relevant. This is due to the following facts: (i) About 1% of 34,000 drinking water samples from Bangladesh were found to have arsenic levels higher than 1 ppm with the highest concentration measured at 4.7 ppm (IARC 2004). Arsenic concentration in drinking water used in this study is about 4.3 to 20 times higher than the reported high arsenic levels that general populations could possibly be exposed to. Using such a range of higher dose in chemical carcinogenesis animal studies is common since animals were exposed to chemicals only for a certain amount of time; however human exposure could be lifetime (Waalkes et al, 2007). (ii) Previous studies showed that the levels of inorganic arsenic (12.1 μg/L) and mono-methylated arsenic (MMAs 20.2 μg/L) in the whole blood of fetal mice from mothers exposed to 42.5 ppm arsenic in drinking water during gestation day 8 to 18 are very close to plasma arsenic levels (inorganic arsenic 8.2 μ g/L; MMAs 20.7 μg/L) of a human population exposed to drinking water containing 0.41 ppm arsenic (Waalkes et al., 2007; Devesa et al., 2006; Pi et al., 2002); (iii) It was reported that the levels of total speciated-arsenic in livers of mice exposed to 25 and 50 ppm sodium arsenite in drinking water were 423 and 1165 μg arsenic/kg, respectively (Paul et al., 2007), which are very close to liver arsenic levels (100 to 1,200 μg arsenic/kg) of a general population exposed to 0.22 to 2 ppm of arsenic in drinking water (Mazumder, 2005). These studies strongly suggest that mice have much stronger capabilities in metabolizing inorganic arsenic and excreting inorganic arsenic metabolites than humans. Therefore, much higher arsenic exposure levels or longer exposure time are needed in mice in order to reproduce chronic arsenic toxicity observed in humans (Paul et al., 2007).

Previous studies showed that arsenic acts as a co-carcinogen increasing the solar-simulation ultraviolet radiation (UVR) exposure-induced skin carcinogenesis probably by inhibiting DNA damage repair (Rossman et al., 2002; Wu et al., 2005). How arsenic exposure increases BaP lung tumorigenic effect remains largely unknown. It is generally accepted that the tumorigenicity of BaP mainly depends on its metabolic activation via CYP-1A1 and 1B1 and subsequent formation of BPDE-DNA adduct (Hecht, 2003; Baird et al. 2005). It is reasonable to speculate that arsenic may enhance BaP tumorigenicity by increasing BPDE-DNA adduct formation via increasing its metabolic activation. However, the reported effects of arsenic on BaP exposure-induced BPDE-DNA adduct levels are inconsistent and controversial. For example, some studies reported that arsenic and BaP co-exposure increased BPDE-DNA adduct levels in cultured cells and mouse tissues (Maier et al., 2002; Evans et al., 2004). Others found no change of BPDE-DNA adduct levels in arsenic and BaP co-exposed mice compared to animals exposed to BaP alone (Fischer et al., 2005). Moreover, some studies showed that arsenic exposure alone is able to increase cellular CYP-1A1 expression (Wu et al., 2009; Kann et al., 2005); others reported that arsenic and BaP co-exposure has no effect on or even reduces cellular CYP-1A1 level (Evans et al., 2004; Vakharia et al., 2001). These controversial findings strongly suggest that other mechanism may play an important role in the synergistic tumorigenic effect of arsenic and BaP co-exposure.

In this study we found that arsenic and BaP co-exposure does not synergize in increasing BPDE-DNA adduct formation; but rather synergistically activate AhR to increase SUV39H1 expression and histone H3 repressive methylation modification (H3K9me2). Ours and other recent studies showed that up-regulation of SUV39H1 expression and SUV39H1-mediated histone H3 repressive methylation modifications (H3K9me2 and H3K9me3) play important roles in enhancing cancer stemness property (Wang et al., 2018; Yang et al., 2017). Our recent study showed that upregulation of histone-lysine methyltransferases including SUV39H1 plays a causal role in hexavalent chromium exposure-induced CSC-like property and cell transformation (Wang et al., 2018). Yang et al. (2017) reported that SUV39H1 (KMT1A) is highly expressed and responsible for the increase of H3K9me3 level in bladder cancer stem cells (BCSCs) and SUV39H1 depletion diminishes the formation of BCSC tumorspheres and xenograft tumors. Further mechanistic studies revealed that H3K9me3 modification on the promoter region of the GATA3 represses the transcription of GATA3 leading to upregulated expression of STAT3, which increases self-renewal of BCSCs (Yang et al., 2017). In this study we found that SUV39H1-mediated H3K9me2 enrichment in the promoter region of SOCS3 suppresses the expression of tumor suppressive SOCS3, resulting in a strong activation of Akt and Erk1/2 to promote cell transformation, CS-like property and tumorigenesis.

Our finding that arsenic and BaP co-exposure acts synergistically in inducing CSC-like property is novel, providing an important mechanistic insight for understanding the increased lung cancer risk from arsenic and BaP co-exposure. CSCs are considered as cancer initiating and maintaining cells, which means that more CSCs produce more and bigger tumors (Nguyen et al., 2012). The results of our cell culture studies showing that arsenic and BaP co-exposure is capable of generating more CSC-like cells match well with the findings from of our mouse studies showing that arsenic and BaP co-exposure produces more and bigger lung tumors. Moreover, our mechanistic study findings also have translational values. For example, our identified signaling pathway leading to enhanced Akt and Erk1/2 activation offers excellent opportunities for the prevention and treatment of lung cancer resulting from arsenic and BaP co-exposure. We are screening natural and synthetic compounds that are capable of inhibiting both Akt and Erk1/2 activity and test their efficacy to reduce arsenic and BaP co-exposure-induced lung tumorigenesis.

In summary, we showed by cell culture studies that chronic arsenic and BaP co-exposure acts synergistically in inducing cell transformation, CSC-like property and tumorigenicity. Moreover, we also found in animal studies using general population-relevant oral exposure route that arsenic and BaP co-exposure synergizes in inducing lung tumorigenesis. Mechanistic studies reveal that arsenic and BaP co-exposure does not significantly increase the level of BPDE-DNA adduct compared to BaP exposure alone. Instead, arsenic and BaP co-exposure synergizes in causing epigenetic dysregulations as evidenced by significantly up-regulating the expression of a histone H3 K9 methyltransferase SUV39H1 and increasing H3 K9 dimethylation (H3K9me2), which in turn down-regulates the expression of tumor suppressive SOCS3 leading to enhanced activation of Akt and Erk1/2 to promote cell transformation, CSC-like property and tumorigenesis. These findings imply that arsenic and BaP co-exposure in generation population via oral ingestion may significantly increase lung cancer risk.

Supplementary Material

1

Highlights.

  • Arsenic and BaP co-exposure acts synergistically in inducing CSC-like property

  • Arsenic and BaP combined oral exposure acts synergistically in increasing lung tumorigenesis

  • Arsenic and BaP co-exposure synergizes in causing epigenetic dysregulation

  • Arsenic and BaP co-exposure down-regulates SOCS3 to enhance Akt and Erk1/2 activation

Acknowledgements

This research was supported by National Institute of Environmental Health Sciences grants 1R01ES028256, 1P30ES026529-01A1 and The University of Kentucky Center for Appalachian Research in Environmental Sciences Career Development Award. This research was also supported by the Shared Resources Facilities on Biostatistics and Bioinformatics and Biospecimen Procurement and Translational Pathology at University of Kentucky Markey Cancer Center (P30CA177558).

Footnotes

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Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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