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. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2022 Sep 24;454:116255. doi: 10.1016/j.taap.2022.116255

Zinc supplementation prevents mitotic accumulation in human keratinocyte cell lines upon environmentally relevant arsenic exposure

Mayukh Banerjee a,*, Kavitha Yaddanapudi b,c,d, J Christopher States a
PMCID: PMC9683715  NIHMSID: NIHMS1851322  PMID: 36162444

Abstract

Disrupted cell cycle progression underlies the molecular pathogenesis of multiple diseases. Chronic exposure to inorganic arsenic (iAs) is a global health issue leading to multi-organ cancerous and non-cancerous diseases. Exposure to supratherapeutic concentrations of iAs causes cellular accumulation in G2 or M phase of the cell cycle in multiple cell lines by inducing cyclin B1 expression. It is not clear if iAs exposure at doses corresponding to serum levels of chronically exposed populations (~100 nM) has any effect on cell cycle distribution. In the present study we investigated if environmentally relevant iAs exposure induced cell cycle disruption and mechanisms thereof employing two human keratinocyte cell lines (HaCaT and Ker-CT), flow cytometry, immunoblots and quantitative real-time PCR (qRT-PCR). iAs exposure (100 nM; 24 hours) led to mitotic accumulation of cells in both cell lines, along with the stabilization of ANAPC11 ubiquitination targets cyclin B1 and securin, without affecting their steady state mRNA levels. This result suggested that induction of cyclin B1 and securin is modulated at the level of protein degradation. Moreover, zinc supplementation successfully prevented iAs-induced mitotic accumulation and stabilization of cyclin B1 and securin without affecting their mRNA levels. Together, these data suggest that environmentally relevant iAs exposure leads to mitotic accumulation possibly by displacing zinc from the RING finger subunit of anaphase promoting complex/cyclosome (ANAPC11), the cell cycle regulating E3 ubiquitin ligase. This early cell cycle disruptive effect of environmentally relevant iAs concentration could underpin the molecular pathogenesis of multiple diseases associated with chronic iAs exposure.

1. Introduction

Exposure to xenobiotics poses high potential for disease development (Zhu et al., 2017). Dubbed as the “largest mass poisoning in history” (Sen and Biswas, 2013; Mori et al., 2018), chronic exposure to inorganic arsenic (iAs) is a public health issue affecting >230 million individuals across 108 countries (Podgorski and Berg, 2020; Shaji et al., 2021). Chronic iAs exposure leads to non-cancerous, pre-cancerous and cancerous diseases spanning multiple organs and tissues (Kapaj et al., 2006; Banerjee, 2011; IARC., 2012; Hong et al., 2014). Skin cancer is the most common malignant outcome of chronic iAs exposure (Hunt et al., 2014). Exposed populations in Asia show high incidence of iAs-induced cancers (Smith et al., 1992; Khan et al., 2003), while those in western countries commonly develop lifestyle associated disorders such as diabetes, obesity, and cardiovascular diseases (Navas-Acien et al., 2008; Frediani et al., 2018; Sobel et al., 2020).

Dietary zinc inadequacy is associated with higher risk of developing iAs-induced diseases including skin lesions (Deb et al., 2013). Zinc inadequacy has also been linked to higher risk of developing diseases that can also be caused by chronic iAs exposure (Ho, 2004; Liu et al., 2013; Cao et al., 2019; Farooq et al., 2020; Knez and Glibetic, 2021; Nakatani et al., 2021). Zinc intake for ~10% of the US population is lower than half the recommended amount (Wakimoto and Block, 2001). Furthermore, iAs exposed populations both in Asia and USA suffer from low serum zinc levels (Bhowmick et al., 2015; Rahman et al., 2016; Dashner-Titus et al., 2018), making them highly susceptible to the hazardous health effects of iAs toxicity. Together, these epidemiological observations suggest that interplay between iAs and zinc plays a key role in molecular pathogenesis of iAs-induced multi-organ toxicity as well as inter-individual disease susceptibility.

The molecular mechanism by which the interplay between iAs and zinc modulates the disease development is poorly understood and represents an important open question in the field. One study on iAs-exposed population in USA reported weak borderline epidemiological association between low dietary zinc intake with high urinary primary methylation ratio for arsenic (Steinmaus et al., 2005). Unfortunately, this observation has not been corroborated by further studies in any other population so far. Interestingly, several groups including ours, have demonstrated that iAs physically interacts with several classes of zinc finger proteins that contain C3H1 or C4 motifs, displacing zinc and disrupting their function (Ding et al., 2009; Zhou et al., 2011; Sun et al., 2014; Zhou et al., 2014; Zhou et al., 2015; Huestis et al., 2016; Ding et al., 2017; Wong et al., 2019; Banerjee et al., 2020; Vergara-Geronimo et al., 2021). This mechanism becomes especially important since iAs does not interact with nucleic acids (Salnikow and Zhitkovich, 2008) but is known to affect myriads of pathways culminating in transcriptome and proteome-wide differential expression (Lantz et al., 2007; Bailey et al., 2014; Mir et al., 2017; Ferragut Cardoso et al., 2022).

Zinc finger proteins represent 3% of the entire human proteome (Abbehausen, 2019). These proteins regulate some of the most fundamental biological processes in almost all cell and tissue types (Cassandri et al., 2017). Critical cellular processes regulated by zinc finger proteins include cell cycle progression, DNA repair, transcription, splicing, miRNA degradation and protein degradation (Cassandri et al., 2017; Buhimschi and Crews, 2019; Shi et al., 2020). Published literature unequivocally demonstrates that iAs can bind to and displace zinc from several zinc finger proteins involved in many of these processes (Vergara-Geronimo et al., 2021). Thus, iAs-mediated zinc displacement from zinc finger proteins that regulate one or more of these essential processes could represent a central unifying mechanism modulating its multi-organ toxicity.

Cell cycle is a fundamental biological process responsible for growth, replication, and division of eukaryotic cells. Proper cell cycle progression maintains genome integrity, tissue homeostasis and regeneration (Krafts, 2010; Zhivotovsky and Orrenius, 2010). Smooth cell cycle progression is orchestrated by modulating the stabilization and degradation of cyclin proteins by cognate E3 ubiquitin ligases (Dang et al., 2021). Two RING finger containing E3-ubiquitin ligases are responsible for ubiquitinating cyclins for degradation in cell cycle phase specific manner; SKP-Cullin-Fbox controls G1-S and S-G2 transitions, while anaphase promoting complex/cyclosome (APC/C) regulates G2-M and M-G1 transitions (Dang et al., 2021). Disruption of phase-specific expression and stabilization of cyclins or E3 ubiquitin ligase function leads to cell cycle dysregulation and diseases (Boehm and Nabel, 2003; Zhivotovsky and Orrenius, 2010; Visconti et al., 2016; Otto and Sicinski, 2017; Deng et al., 2020; Joseph et al., 2020; Dang et al., 2021). RING finger domains in RBX1 and ANAPC11 are the ubiquition transferring subunits of SKP-Cullin-Fbox and APC/C, respectively. RBX1 is a target for iAs-mediated zinc displacement (Jiang et al., 2018), which would compromise its function leading to cell cycle dysregulation.

Disruption of cell cycle progression is an early effect of iAs exposure that is central to the etiology of iAs-induced diseases including cancers, across multiple organs (States et al., 2002; McCollum et al., 2005; McNeely et al., 2006; Sidhu et al., 2006; Taylor et al., 2006; McNeely et al., 2008a; McNeely et al., 2008b; Sakai et al., 2014; States, 2015; Al-Eryani et al., 2017; Sage et al., 2017; Xiong et al., 2018; Ganapathy et al., 2019; Medda et al., 2021). Exposure to iAs at environmental to supratherapeutic concentrations, almost universally leads to cellular accumulation in G2 or M phase of the cell cycle (Park et al., 2001; Yih et al., 2005; Yih et al., 2006; Li et al., 2009; Zhang et al., 2012; Xiong et al., 2018; Ganapathy et al., 2019), characterized by induction of cyclin B1 (Cai et al., 2003; Ganapathy et al., 2019).

It is not clear how iAs exposure leads to cyclin B1 induction resulting in cellular accumulation in G2 or M phase of the cell cycle. Moreover, almost all the studies so far have employed high concentrations (μM-mM) of iAs to investigate its effect on cell cycle (McCollum et al., 2005; McNeely et al., 2006; McNeely et al., 2008a; McNeely et al., 2008b; Sakai et al., 2014). To put this into perspective, basal blood iAs level of unexposed populations is ~20 nM (Takayama et al., 2021), while that of chronically exposed populations is ~100 nM (Gonsebatt et al., 1992; Pi et al., 2000; Wu et al., 2001). This distinction is important because the molecular and disease outcomes from iAs exposure are seldom linear but mostly J-shaped (Snow et al., 2005; Bodwell et al., 2006; Ahn et al., 2020) and conclusions drawn from high exposure may not be successfully extrapolated to a lower dose. Thus, there is a considerable knowledge gap in understanding if and how environmentally and toxicologically relevant iAs exposure affects cell cycle distribution.

In the current study, we investigated the effect of environmentally relevant iAs exposure (100 nM) on cell cycle distribution in two human keratinocyte cell lines, HaCaT and Ker-CT. We also investigated whether iAs exposure could induce cyclin B1 and securin expression in these cells and if this induction is modulated at the mRNA or protein level. Finally, we explored if modulating the zinc:iAs ratio favoring zinc could prevent iAs-induced cell cycle dysregulation.

2. Materials and methods

2.1. Chemicals and reagents

Sodium arsenite (NaAsO2; CAS No. 7784–0698) and zinc acetate [Zn(CH3COO)2; CAS No. 557-34-6] were obtained from ThermoFisher Scientific Inc (Waltham, MA). Filter sterilized stock solutions of sodium arsenite and zinc acetate (both 1000X) were prepared in ultrapure DNase/RNase-free distilled water (ThermoFisher Scientific Inc.) immediately before use. Minimum Essential Media (MEM) alpha modification media, trypsin, ethylenediaminetetraacetic acid (EDTA), Trypsin-EDTA (0.05% trypsin-0.02% EDTA) and penicillin/streptomycin were purchased from ThermoFisher Scientific Inc. Fetal bovine serum (FBS; characterized) was procured from Hyclone (Logan, UT). KGM™ Gold Keratinocyte Growth media and KGM™ SingleQuots™ Supplement Pack were obtained from Lonza (Basel, Switzerland).

2.2. Cell Culture

HaCaT cells were the kind gift from Dr. Tai Hao Quan (University of Michigan), while Ker-CT cells were purchased from ATCC (CRL-4048, Manassas, VA). Identity of HaCaT cells was ascertained by short tandem repeat (STR) mapping analysis, outsourced to a commercial vendor (Labcorp, Burlington, NC) as described previously (Ferragut Cardoso et al., 2022). For each experiment, cells (HaCaT or Ker-CT) were cultured in independent triplicates as per previously published protocols (Ferragut Cardoso et al., 2022; Nail et al., 2022). Briefly, HaCaT cells were cultured in MEM alpha modification media supplemented with 10% fetal bovine serum, 100 units/mL penicillin/100 μg/mL streptomycin and 2 mM glutamine. Ker-CT cells were cultured in KGM™ Gold Keratinocyte Growth Media supplemented with the KGM™ SingleQuots™ Supplement Pack. Both the cell lines were maintained in a 37°C humidified tissue culture incubator with 5% CO2. None of the media by themselves had any discernible concentration of zinc. However, supplementation of HaCaT media with 10% FBS leads to a final concentration of ~4 μM of zinc, while Ker-CT cells were grown in serum free media. Importantly, most of the zinc in the HaCaT media is sequestered by binding to albumin and other ligands (Bozym et al., 2010) leaving very low level of free zinc in the media (pM-low nM) to interact with the zinc finger proteins (Bozym et al., 2010).

For iAs dose response experiments, 24 h post-seeding, cells (HaCaT or Ker-CT) were exposed to increasing concentrations of iAs (0–1 μM) for 24 h followed by washing with 1X PBS (thrice) and cell harvest by trypsinization. For each sample, 106 cells were fixed for flow cytometry analysis, 106 cells were used for RNA isolation and the rest were used for preparation of whole cell lysates. For zinc supplementation studies, 24 h post-seeding, cells (HaCaT or Ker-CT) were simultaneously exposed to iAs (0 or 0.1 μM) and zinc (0 or 1 μM) for 24 h, followed by cell harvest as described above.

2.3. Flow cytometry cell cycle analysis

Flow cytometry analysis was employed to determine cell cycle distribution as described previously with minor modifications (Al-Eryani et al., 2017) . Briefly, 106 cells/sample were fixed in 100% ethanol overnight, followed by centrifugation at 1500 rpm at room temperature for 10 minutes and resuspension in 475 μL of 1X PBS. The suspension was treated with RNase A (final concentration of 1 mg/mL) to degrade all RNA and subsequently stained with propidium iodide (final concentration of 50 μg/mL) for 30 minutes in the dark at room temperature. Fluorescence signal was acquired by flow cytometry employing a BD FACScanto II flow cytometer and 20,000 cells were analyzed per sample. The raw data were analyzed employing FlowJo® v.10.2 to determine cell cycle distribution.

2.4. Immunoblot analysis

Immunoblot analysis was employed to investigate the steady state levels of cell cycle related proteins as well as the expression of a mitosis specific marker. Cell lysis, sample preparation, total protein quantification (by BCA assay), immunoblotting, blocking, stripping and image acquisition were performed as described in detail elsewhere (Ferragut Cardoso et al., 2022). Details regarding the antibodies used, their dilutions and optimized incubation conditions are presented in Supplementary Table 1. Raw data for densitometric analysis were generated from the images employing the Image J software (Schneider et al., 2012).

2.5. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis

Total RNA isolation from HaCaT and Ker-CT cells, quality control assessment and cDNA generation were performed as described previously (Banerjee et al., 2020; Ferragut Cardoso et al., 2022). qRT-PCR was performed as described before with GAPDH as housekeeping gene (Banerjee et al., 2020). Cyclin B1 (assay Hs.PT.56a.39564933) and securin (assay Hs.PT.58.4436796.g) were amplified using PrimeTime® qPCR primers (Integrated DNA Technologies, Coralville, IA) and PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Inc.) as per manufacturer’s protocol. For each assay, fold change was calculated compared to unexposed control employing the ΔΔCT method (Banerjee et al., 2020).

2.6. Statistical analysis

Data for concentration-response experiments were analyzed by one-way ANOVA with Dunnett’s multiple comparisons post-hoc test. Data for effect of zinc supplementation on iAs exposure were analyzed by two-way ANOVA with Tukey’s multiple comparisons post-hoc test. p<0:05 was considered significant for every test. All statistical analyses were performed employing GraphPad Prism (version 9.0.2; GraphPad).

3. Results

3.1. iAs exposure leads to mitotic accumulation in human keratinocyte cell lines

iAs exposure for 24 h, starting at 0.1 μM resulted in significant accumulation of cells at the G2 or M phase of the cell cycle compared to unexposed control in each cell line (Fig. 1A–B; p<0.05 by one-way ANOVA). However, exposure to 20 nM iAs for 24 h had no effect on cell cycle distribution compared to unexposed control in either cell line (Supplementary Fig. 1). Since standard flow cytometry cannot distinguish between cells in G2 or M phase of the cell cycle, we employed immunoblot analysis for mitosis specific marker phosphorylated histone 3 (H3-pSer10) for this purpose (Tsuta et al., 2011). iAs exposure at all concentrations (0.1–1 μM) in each cell line showed strong signals for H3-pSer10, while no signal was observed in the unexposed controls (Fig 1C–D). These data unequivocally demonstrate that iAs exposure at environmentally relevant concentration of 0.1 μM leads to mitotic accumulation in human keratinocytes.

Fig. 1.

Fig. 1.

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iAs exposure induces mitotic accumulation in human keratinocytes. (A) Cell cycle analysis following iAs dose response (0–1 μM; 24 h) in HaCaT cells. B. Cell cycle analysis following iAs dose response (0–1 μM; 24 h) in Ker-CT cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for Dunnett’s test following one-way ANOVA for both panels. (C) Immunoblot for mitotic marker H3-pSer10 (loading control: total H3) following iAs dose response (0–1 μM; 24 h) in HaCaT cells. (D) Immunoblot for mitotic marker H3-pSer10 (loading control: total H3) following iAs dose response (0–1 μM; 24 h) in Ker-CT cells. Densitometry was not performed for panels C-D as no signal was observed for H3-pSer10 in the unexposed cells.

3.2. iAs-induced mitotic accumulation is associated with stabilization of cyclin B1 and securin

Degradation of cyclin B1 and securin ubiquitinylated by APC/C is a mandatory before cells can progress from metaphase to anaphase (Chang et al., 2003). Given that iAs exposure leads to mitotic accumulation of human keratinocytes, we next investigated the steady state levels of cyclin B1 and securin. Our data demonstrate that iAs exposure at 0.1 μM and above (0.1–1 μM) induced cyclin B1 and securin expression in each cell line (Fig. 2).

Fig. 2.

Fig. 2.

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iAs exposure stabilizes cyclin B1 and securin protein expression. (A) Immunoblot for cyclin B1 and securin expression (loading control: GAPDH) following iAs dose response (0–1 μM; 24 h) in HaCaT cells. All the three proteins were developed on the same blot. (B) Densitometry of cyclin B1 expression for panel A. (C) Densitometry of securin expression for panel A. (D) Immunoblot for cyclin B1 and securin expression (loading control: GAPDH) following iAs dose response (0–1 μM; 24 h) in Ker-CT cells. All the three proteins were developed on the same blot. (E) Densitometry of cyclin B1 expression for panel D. (F) Densitometry of securin expression for panel D. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for Dunnett’s test following one-way ANOVA (panels B-C and E-F).

3.3. iAs exposure does not affect cyclin B1 or securin mRNA levels

To investigate if iAs-induced induction of cyclin B1 and securin is regulated at the level of transcription, we employed RT-qPCR. The data are presented in Fig. 3. We did not observe any difference in the steady state cyclin B1 or securin mRNA levels upon iAs exposure compared to unexposed control in either cell line (Fig. 3). These data strongly suggest that iAs-induced cyclin B1 and securin induction is not regulated transcriptionally but is likely due to suppressed APC/C-mediated degradation.

Fig. 3.

Fig. 3.

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iAs exposure does not affect steady state of cyclin B1 and securin mRNA levels. (A) qRT-PCR analysis of steady state cyclin B1 mRNA following iAs dose response (0–1 μM; 24 h) in HaCaT cells. (B) qRT-PCR analysis of steady state securin mRNA following iAs dose response (0–1 μM; 24 h) in HaCaT cells. (C) qRT-PCR analysis of steady state cyclin B1 mRNA following iAs dose response (0–1 μM; 24 h) in Ker-CT cells. (D) qRT-PCR analysis of steady state securin mRNA following iAs dose response (0–1 μM; 24 h) in Ker-CT cells.

3.4. Zinc supplementation prevents iAs-induced mitotic accumulation in human keratinocyte cell lines

Mitotic degradation of cyclin B1 and securin requires ubiquitination by the RING finger containing E3-ubiquitin ligase APC/C (Brown et al., 2014; Wang et al., 2019). iAs is known to displace zinc from RING finger proteins abrogating their function (Vergara-Geronimo et al., 2021). Furthermore, we and others have demonstrated that iAs-induced functional disruption of zinc finger proteins can be prevented by zinc supplementation (Sun et al., 2014; Banerjee et al., 2020). Since we observed stabilization of cyclin B1 and securin upon iAs exposure, we investigated if zinc supplementation could prevent this stabilization and mitotic accumulation. Zinc supplementation prevented iAs-induced mitotic accumulation in both cell lines (Fig. 4) as is evident from flow cytometric analysis (Fig. 4A and 4C) and concomitant loss of mitosis specific H3-pSer10 signal in presence of both supplemental zinc and iAs (Fig. 4B and 4D). The zinc supplementation also suppressed stabilization of APC/C targets cyclin B1 and securin back to basal levels in each cell line (Fig. 5A–C and 5F–H). Additionally, there was no difference in cyclin B1 and securin mRNA levels with either iAs exposure or zinc supplementation compared to unexposed control (Fig. 5D–E and 5I–J).

Fig. 4.

Fig. 4.

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Zn2+ supplementation prevents iAs-induced mitotic accumulation. (A) Cell cycle analysis in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (B) Immunoblot analysis for H3-pSer10 (loading control: total H3) in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (C) Cell cycle analysis in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (D) Immunoblot analysis for H3-pSer10 (loading control: total H3) in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). **p<0.01, ***p<0.001, ****p<0.0001 for Tukey’s pos-hoc test following 2-way ANOVA (panels A and C). Densitometry was not performed for panels H3-pSer10 in panels B and D as no signal was observed for H3-pSer10 in the unexposed cells.

Fig. 5.

Fig. 5.

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Zn2+ supplementation prevents iAs-induced stabilization of cyclin B1 and securin. (A) Immunoblot analysis for cyclin B1 (loading control: GAPDH) and securin (loading control: GAPDH) in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (B) Densitometric analysis of cyclin B1 expression in panel A. (C) Densitometric analysis of securin expression in panel A. (D) qRT-PCR analysis of steady state cyclin B1 mRNA in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (E) qRT-PCR analysis of steady state securin mRNA in HaCaT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (F) Immunoblot analysis for cyclin B1 and securin (loading control: GAPDH) in Ker-CT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). All the three proteins were developed on the same blot. (G) Densitometric analysis of cyclin B1 expression in panel F. (H) Densitometric analysis of securin expression in panel F. (I) qRT-PCR analysis of steady state cyclin B1 mRNA in Ker-CT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). (J) qRT-PCR analysis of steady state securin mRNA in Ker-CT cells exposed to iAs (0 or 0.1 μM; 24 h) and supplemented with Zn2+ (0 or 1 μM; 24 h). **p<0.01, ***p<0.001, ****p<0.0001 for Tukey’s pos-hoc test following 2-way ANOVA (panels B-E and panels G-J).

4. Discussion

Dysregulated cell cycle lies at the heart of pathogenesis for innumerable diseases spanning every tissue and organ (Boehm and Nabel, 2003; Zhivotovsky and Orrenius, 2010; Williams and Stoeber, 2012; Toettcher, 2013; Visconti et al., 2016; Otto and Sicinski, 2017; Sherr and Bartek, 2017; Joseph et al., 2020). In addition to endogenous factors such as DNA damage and ER stress, cell cycle disruption is also a common outcome of xenobiotic exposure (Waalkes et al., 2000; Glahn et al., 2008). Multiple studies have demonstrated that iAs exposure leads to disrupted cell cycle progression, albeit at therapeutic and supratherapeutic concentrations (Halicka et al., 2002; McCollum et al., 2005; McNeely et al., 2006; McNeely et al., 2008a; McNeely et al., 2008b; Yu et al., 2008; Muenyi et al., 2014; Muenyi et al., 2015; Kim et al., 2021), which do not necessarily reflect the mechanisms operative at much lower environmentally relevant concentrations.

This is the first study to our knowledge to demonstrate that exposure to iAs reflecting serum levels of chronically exposed populations (100 nM) but not that reflecting basal blood iAs levels in unexposed populations (20 nM) leads to mitotic accumulation of cells as early as 24 hours across two different human keratinocyte cell lines. Given that HaCaT and Ker-CT cell lines take ~20 and 24 h respectively for each cell cycle (Ramirez et al., 2003; Gábor, 2012), our data suggests that iAs exposure causes mitotic accumulation as early as the first cell cycle, post exposure. Our group had previously demonstrated that chronic exposure to 100 nM iAs for 7 weeks leads to accumulation of HaCaT cells in G2 or M phase of the cell cycle (Al-Eryani et al., 2017). Results from the current study thus corroborate these earlier findings while also showing that: (i) similar cell cycle disruptive effects of iAs exposure across two different human keratinocyte cell lines make it unlikely that this is a cell line specific effect; (ii) iAs exposure induces cell cycle disruption at levels that reflect the serum iAs concentrations in chronically exposed populations (Gonsebatt et al., 1992; Pi et al., 2000; Wu et al., 2001); (iii) cell cycle disruption induced by iAs exposure is an extremely early event evident at 24 hours of exposure; (iv) iAs exposure leads to cellular accumulation exclusively in mitosis (Fig. 1C–D); (v) iAs-induced mitotic accumulation is associated with stabilization cyclin B1 and securin specifically at the protein level, but not mRNA (Fig. 2–3). Our data is consistent with a previous study that demonstrated that iAs exposure (500 nM) suppressed ubiquitination-mediated cyclin B1 protein degradation (Ganapathy et al., 2019). However, another previous study on SVEC4–10 mouse endothelial cells showed that supratherapeutic concentrations of iAs (12–16 μM; 24 hours) suppressed securin expression with concomitant accumulation of cells in the G2 or M phase (Chao et al., 2006). This observation is paradoxical as the authors did not explain why they observed the cells accumulating in G2 or M phase when securin depletion effectively propels the cells from metaphase to anaphase (Malumbres, 2015), which is also borne out by their data on faulty chromatid segregation. Moreover, the difference between this study and ours also highlights the non-linear effect of iAs exposure, as well as cell line and organismal differences in iAs handling and metabolism (Drobna et al., 2010).

Given our results, it is important to consider how iAs exposure could lead to increased expression of both cyclin B1 and securin. Under normal circumstances, both cyclin B1 and securin are degraded at metaphase (Thornton and Toczyski, 2003; Malumbres, 2015). Cyclin B1 degradation inactivates CDK1, while securin degradation releases separase, the protease that cleaves cohesion, triggering separation of sister chromatids (Sullivan and Morgan, 2007). Degradation of cyclin B1 and securin are mandatory for metaphase to anaphase transition and eventually, mitotic exit (Malumbres, 2015). While cyclin B1 and securin control different processes that culminate in mitotic exit, both of them are degraded in metaphase by the E3 ubiquitin ligase complex, APC/C (Nakayama and Nakayama, 2006). ANAPC11 is the catalytic subunit of APC/C that transfers the ubiquitin to the substrates, targeting them for degradation (Tang et al., 2001; Watson et al., 2019). ANAPC11 contains a RING finger type zinc finger domain that is essential for its ubiquitination function (Tang et al., 2001). iAs has been demonstrated to bind to RING finger domains, displacing zinc and disrupting their function (Vergara-Geronimo et al., 2021). Thus, it is possible that iAs exposure is inhibiting ANAPC11 function by physically interacting with its RING finger motif, leading to stabilization of its mitotic substrates, cyclin B1 and securin, trapping the cells in metaphase.

Interestingly, RBX1, the catalytic subunit of SKP-Cullin-FBOX, the E3 ubiquitin ligase modulating G1-S and S-G2 transitions is highly homologous to ANAPC11 and contains a RING finger domain (Nakayama and Nakayama, 2006; Wei and Sun, 2010; Gilberto and Peter, 2017; Fouad et al., 2019). RBX1 RING finger has been shown to be targeted by iAs, displacing the zinc (Jiang et al., 2018), albeit at high concentration (5 μM). However, the effect of iAs exposure on cell cycle regulating function of RBX1 through direct binding and zinc displacement has never been explored. Thus, it was possible that iAs exposure could lead to cellular accumulation in any phase of the cell cycle, depending on whether it preferentially affects RBX1 (accumulation in G1 or S) or ANAPC11 (Accumulation in G2 or M). This possibility explains why we chose to employ unsynchronized cells for this study, as opposed to synchronizing cells. Employing unsynchronous cell populations provided us with the most unbiased approach to determine the cell cycle phase that is most susceptible to effects of iAs exposure. It is possible that depending on the phase at which cells would be synchronized, we might observe different effects of iAs exposure on cell cycle distribution, which might not truly reflect its effect.

iAs exposure at environmental to supratherapeutic concentrations, almost universally leads to accumulation of cells in G2 or M phase of the cell cycle, not in G1 or S (States, 2015). This observation strongly indicates that under cellular conditions with low iAs exposure, iAs might preferentially target ANAPC11 over RBX1. Sequence alignment of ANAPC11 and RBX1 shows that the third zinc finger motif of RBX1 consists of C2HD (C75-H77-C94-D97) rather than canonical C3H1 residues as in ANAPC11 (C51-H53-C73-C76) (Tang et al., 2001). iAs can displace zinc from C3H1/C4 zinc finger motifs, but not C2HD (90). Thus, C2HD domain in RBX1 is unlikely to bind iAs, making it less susceptible to iAs-induced zinc displacement. In the future, biophysical and cellular studies comparing the relative affinities of iAs and zinc towards the RING finger domains of ANAPC11 and RBX1 would be important to experimentally dissect the molecular mechanisms involved in iAs-induced mitotic accumulation. In addition, it would be critical to evaluate the impact of iAs and zinc binding and mutual displacement on the substrate ubiquitination properties of ANAPC11 and RBX1 at environmentally relevant concentrations.

The hypothesis that iAs-induced mitotic accumulation is mediated by zinc displacement from ANAPC11 is reinforced by our results unequivocally demonstrating that zinc supplementation prevented it in both cell lines (Fig. 4–5). Zinc supplementation suppressed iAs-induced stabilization of APC/C targets cyclin B1 and securin back to basal levels (Fig. 5A–C and 5F–H) without affecting their steady state mRNA levels (Fig. 5D–D and 5I–J). This strongly suggests that levels of cyclin B1 and securin are being regulated by ubiquitination-mediated protein degradation, possibly via restoration of ANAPC11 ubiquitin transfer function. Our data are consistent with previous publications from our group and others demonstrating that excess zinc can displace zinc finger bound iAs, restoring function (Sun et al., 2014; Banerjee et al., 2020).

Interestingly, as compared to the previous study (Sun et al., 2014), we employed much lower concentration of zinc (1 μM vs. 5 μM). Our choice of zinc concentration (1 μM) is both toxicologically safe as well as physiologically relevant. The basal serum zinc levels of healthy human populations range between 10–17.5 μM (Wieringa et al., 2015; Alves et al., 2016). Multiple clinical trials for zinc supplementation have employed 20–30 mg zinc/day in human subjects without any deleterious effects (Ranjbar et al., 2013; Rathnayake et al., 2016; Afzali et al., 2021). HaCaT cells can tolerate zinc exposures to ~100 μM without any toxic effect. (Emri et al., 2015). Thus, the dosage of zinc used in this study is much lower than basal serum zinc levels. Additionally, we demonstrated that this concentration of zinc (1 μM) can successfully prevent iAs-mediated dysregulated TRA2B splicing by ZRANB2 (Bastick et al., 2022), further justifying the choice of zinc dosage. Thus, although we used much lower zinc concentration for supplementation studies, we still observed complete prevention of effects of iAs exposure (Fig 4–5). In the cellular system, the zinc finger proteins are in a state of dynamic equilibrium with available iAs and zinc. The proportion of any zinc finger protein bound to zinc or iAs will depend on the relative concentrations of iAs and zinc in the system as well as on the relative affinity of the zinc finger domain for iAs and zinc. Ultimately, it is possible that modulating the zinc:iAs ratio favoring zinc (10:1) could displace iAs bound to zinc finger proteins, restoring their functions and perhaps alleviating, or at least decelarating the effects of iAs exposure. Our data are generated from in vitro systems, which might behave differently than experimental organisms. However, given that rodents do not develop any characteristic skin lesions from arsenic exposure even at very high doses (Waalkes et al., 2007; States et al., 2011), there is no other model system known to us to investigate the mechanisms of arsenic-induced cell cycle disruption in skin cells, in vivo.

Results from this study firmly demonstrate that environmentally relevant iAs exposure leads to mitotic accumulation in two different human keratinocyte cell lines as early as 24 hours, possibly by inhibition of APC/C mediated degradation of cyclin B1 and securin (Fig. 6). This iAs-mediated mitotic accumulation could be one of the central players behind the diverse range of disease outcomes that arise upon chronic iAs exposure. Our results further strengthen the hypothesis that zinc supplementation could be an accessible and inexpensive way to combat effects of chronic iAs exposure (Fig. 6).

Fig. 6.

Fig. 6.

Open in a new tab

Proposed mechanism of action by which iAs exposure induces mitotic accumulation. iAs exposure stabilizes cyclin B1 and securin preventing cells from progressing to anaphase from metaphase. iAs suppressed ubiquitination mediated protein degradation of cyclin B1 and securin possibly by displacing zinc from ANAPC11 RING finger (steps affected by iAs are shown in orange arrows). The orange “?” represents that the exact nature of physical interaction between ANAPC11 and iAs is currently unknown. Zinc supplementation restores basal degradation of cyclin B1 and securin thus alleviating iAs-induced mitotic accumulation (effects of iAs exposure that are alleviated by zinc supplementation are shown in purple arrows). Zinc possibly achieves this by preventing iAs binding to ANAPC11 ring finger, although the exact nature of this interaction is currently unknown and is hence represented by the purple “?”.

Supplementary Material

Supplementary Table 1
Supplementary Figure 1

Supplementary Fig. 1. iAs exposure at 0.2 μM for 24 h does not affect cell cycle distribution. (A) Cell cycle analysis in HaCaT cells exposed to iAs (0 or 0.2 μM; 24 h). (B) Cell cycle analysis in Ker-CT cells exposed to iAs (0 or 0.2 μM; 24 h).

Acknowledgements

The authors thank Mr. Omar Sadi Sarkar and Mr. Lewis C. Chew for their technical help in performing flow cytometry.

Funding

This work was supported by a Career Development award to MB through the National Institutes of Health (NIH)/National Institute of Environmental Health Sciences grant P30ES030283 (JCS), NIH/NIEHS grant R01ES027778 (JCS) and NIH/NIGMS P20GM135004 Project Grant to KY.

Footnotes

Declaration of Competing Interest

The authors declare no competing financial interest.

CRediT Author Statement

Mayukh Banerjee: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision, Project Administration, Funding Acquisition.

Kavitha Yaddanapudi: Methodology, Validation, Formal Analysis, Investigation, Resources, Data Curation, Writing – Review & Editing.

J. Christopher States: Conceptualization, Resources, Writing – Review & Editing, Funding Acquisition.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1
NIHMS1851322-supplement-Supplementary_Table_1.docx (14.1KB, docx)
Supplementary Figure 1

Supplementary Fig. 1. iAs exposure at 0.2 μM for 24 h does not affect cell cycle distribution. (A) Cell cycle analysis in HaCaT cells exposed to iAs (0 or 0.2 μM; 24 h). (B) Cell cycle analysis in Ker-CT cells exposed to iAs (0 or 0.2 μM; 24 h).

NIHMS1851322-supplement-Supplementary_Figure_1.tif (1.2MB, tif)

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