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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Arch Toxicol. 2019 Sep 6;93(10):2811–2822. doi: 10.1007/s00204-019-02559-7

Differential metabolism of inorganic arsenic in mice from genetically diverse Collaborative Cross strains

Miroslav Stýblo 1, Christelle Douillet 1, Jacqueline Bangma 2, Lauren A Eaves 2, Fernando Pardo−Manuel de Villena 3, Rebecca Fry 2
PMCID: PMC7244219  NIHMSID: NIHMS1588295  PMID: 31493028

Abstract

Mice have been frequently used to study the adverse effects of inorganic arsenic (iAs) exposure in laboratory settings. Like humans, mice metabolize iAs to monomethyl-As (MAs) and dimethyl-As (DMAs) metabolites. However, mice metabolize iAs more efficiently than humans, which may explain why some of the effects of iAs reported in humans have been difficult to reproduce in mice. In the present study, we searched for mouse strains in which iAs metabolism resembles that in humans. We examined iAs metabolism in male mice from 12 genetically diverse Collaborative Cross (CC) strains that were exposed to arsenite in drinking water (0.1 or 50 ppm) for 2 weeks. Concentrations of iAs and its metabolites were measured in urine and livers. Significant differences in total As concentration and in proportions of total As represented by iAs, MAs, and DMAs were observed between the strains. These differences were more pronounced in livers, particularly in mice exposed to 50 ppm iAs. In livers, large variations among the strains were found in percentage of iAs (15–48%), MAs (11–29%), and DMAs (29–66%). In contrast, DMAs represented 96–99% of total As in urine in all strains regardless of exposure. Notably, the percentages of As species in urine did not correlate with total As concentration in liver, suggesting that the urinary profiles were not representative of the internal exposure. In livers of mice exposed to 50 ppm, but not to 0.1 ppm iAs, As3mt expression correlated with percent of iAs and DMAs. No correlations were found between As3mt expression and the proportions of As species in urine regardless of exposure level. Although we did not find yet a CC strain in which proportions of As species in urine would match those reported in humans (typically 10–30% iAs, 10–20% MAs, 60–70% DMAs), CC strains characterized by low %DMAs in livers after exposure to 50 ppm iAs (suggesting inefficient iAs methylation) could be better models for studies aiming to reproduce effects of iAs described in humans.

Keywords: Arsenic, Metabolism, Genetically diverse mice, The Collaborative Cross

Introduction

Inorganic arsenic (iAs) is a common drinking water and food contaminant (ATSDR 2007; Stanton et al. 2015). Chronic exposures to iAs have been linked to a variety of cancers and other common diseases (Naujokas et al. 2013). Overwhelming evidence from both population and laboratory studies suggests that the capacity to metabolize iAs is one of the key factors that determine susceptibility to diseases associated with iAs exposure (Chung et al. 2009, 2010; de la Rosa et al. 2017; Del Razo et al. 1997; Douillet et al. 2017; Engstrom et al. 2015; Huang et al. 2018a; Spratlen et al. 2018; Tseng 2007).

In humans, iAs is metabolized in a sequence of methylation reactions to form monomethyl-As (MAs) and dimethyl-As (DMAs) metabolites that are excreted primarily in urine (Chen et al. 2011). The urinary profiles of these metabolites, specifically the percentage of total As in urine represented by iAs (%iAs), MAs (%MAs), and DMAs (%DMAs), or DMAs/MAs ratio have been frequently used in epidemiologic studies to assess the efficiency of iAs metabolism (Tseng 2007). Specifically, high %iAs or %MAs and low %DMAs or low DMAs/MAs ratio have been interpreted as indicators of low or impaired capacity to methylate iAs. Because iAs methylation is essential for rapid clearance of As species from the body (i.e., for iAs detoxification), these urinary profiles may also indicate an increased susceptibility to iAs toxicity (Faita et al. 2013; Hernandez and Marcos 2008; Paul et al. 2015; Vahter 2000). For example, low %DMAs and low DMAs/MAs ratio have been associated with increased risk of bladder cancer (Steinmaus et al. 2006) and cardiovascular diseases (Spratlen et al. 2018) in population studies.

The methylation of iAs is catalyzed by the arsenic (+ 3 oxidation state) methyltransferase enzyme (AS3MT) and is thought to take place mainly in the liver (Thomas et al. 2007). In mice, a whole-body knockout of As3mt has resulted in almost complete loss of the ability to methylate iAs and in adverse metabolic phenotype characterized by insulin resistance (Drobna et al. 2009; Douillet et al. 2017). In human populations, AS3MT polymorphisms have been associated with differences in the individual capacity to methylate iAs and in susceptibility to adverse effects of iAs exposure, specifically precancerous skin lesions and cancers (Beebe-Dimmer et al. 2012; Chung et al. 2009; Das et al. 2016; de la Rosa et al. 2017; Engstrom et al. 2015) or diabetes (Drobna et al. 2013). While five intronic AS3MT SNPs that affect urinary profiles of As metabolites have been shown to vary across different ethnicities (Fujihara et al. 2009), two SNPs, AS3MT 12390 (rs3740393) and 14458 (rs11191439), have been consistently related to the capacity to methylate iAs regardless of the geographical regions or populations examined (Agusa et al. 2011).

AS3MT polymorphism appears to be the most important genetic factor affecting iAs metabolism in humans (Antonelli et al. 2014). However, other genes have also been linked to differences in urinary profiles of As metabolites in some studies, including genes encoding N-6-adeninespecific DNA methyltransferase-1, glutathione transferases (GST-P, -M, -T, and -O) (Agusa et al. 2010, 2012; Caceres et al. 2010; Chen et al. 2012, 2017; Chung et al. 2009, 2011; Fu et al. 2014; Harari et al. 2013; Rahbar et al. 2014; Recio-Vega et al. 2016; Rodrigues et al. 2012; Yang et al. 2015), solute carrier organic anion transporter family member 1B1 (Gribble et al. 2013), and enzymes involved in one carbon metabolism (Chung et al. 2010; Lindberg et al. 2007; Schlawicke et al. 2009; Steinmaus et al. 2007), which provides S-adenosylmethionine for AS3MT-catalyzed methylation of iAs. Thus, by affecting iAs metabolism, polymorphisms in these genes may also influence the adverse outcomes associated with chronic exposure to iAs.

Laboratory studies have played an important role in exploring both the pathways for iAs metabolism, as well as adverse effects associated with iAs exposure. Mice have been the preferred animal species used in most of these studies. Like humans, mice metabolize iAs to MAs and DMAs. However, mice convert iAs to DMAs more efficiently than humans as demonstrated by a shorter half-life of iAs and different profiles of As species in urine (Vahter 1999, 2000). DMAs typically represent > 90% of As in urine of mice exposed to iAs, with very low %iAs and %MAs. In contrast, the profiles of As metabolites in human urine are characterized by a lower proportion of DMAs (60–70%) and higher proportions of MAs (10–20%) (Vahter 1999, 2000). These differences may explain why some of the adverse effects reported in population studies have been difficult to reproduce in mice, or why higher doses of iAs were needed in mouse studies to observe similar effects.

A major limitation of previous laboratory studies examining iAs metabolism is a limited genetic diversity among the mouse strains used in these studies. A study published by Hughes et al. (1999) found differences in disposition iAs among adult female mice from C57BL/6, C3H, and B6C3F1 strains after a single oral dose of [73As]arsenate (0.5 or 5 mg As/kg). Still, most of the published studies in which As metabolites were measured in urine or tissues, including studies from our laboratory, used only inbred C57BL/6 mice. The role of genetic background as a key factor affecting iAs metabolism or iAs toxicity has never been systematically examined in mice or in any other animal species. The main goal of the present study was to compare iAs metabolism under conditions of chronic exposure in mice using genetically diverse strains. We selected 12 strains from the Collaborative Cross (CC), a mouse population derived from eight founder strains. The CC is uniquely suited to study the role of genetic diversity in etiology of biological traits (Aylor et al. 2011). We hypothesized (1) that the capacities to methylate iAs would differ among the strains and (2) that the pattern of iAs metabolism (urinary profiles of As species) in some of these strains may resemble those reported in human studies.

Methods

Mice and treatments

Adult males (2–4 months old) CC mice were obtained from the UNC Systems Genetics Core Facility (SGCF). The selection of CC strains and the number of replicates per CC strain were dictated by the SGCF pilot project format (12 strains with 4–6 mice per strain) and by the availability of male mice of CC strains maintained by the SCGF at the start of the study (December 1, 2017). Mice were housed (1–5 per cage) under controlled conditions with 12-h light/dark cycle at 22 ± 1 °C and 50 ± 10% relative humidity. After a 3-day acclimation, mice in each strain were randomly divided into two treatment groups (N = 4–5). One group was exposed to 0.1 ppm and the other to 50 ppm iAs (sodium arsenite, NaAsO2, ≥ 99% pure; Sigma-Aldrich, St. Louis, MO, USA) in drinking (deionized) water ad libitum. Mice in all groups were fed a semi-purified AIN-93G diet (Envigo Teklad, Madison, WI, USA). In our previous studies, the concentrations of iAs in this type of diet ranged from 11 to 50 ppb (Douillet et al. 2017; Huang et al. 2018a, b). Water consumption was monitored weekly. All procedures involving mice were approved by the University of North Carolina Institutional Animal Care and Use Committee.

Sample collection and As analysis

The mice were exposed to iAs in drinking water for 2 weeks. The water with iAs was replaced with fresh water containing iAs after 1st week to limit oxidation of arsenite to arsenate. After 2 weeks, a spot sample of urine was collected from each mouse and frozen at − 80 °C. Mice in all groups were then sacrificed, and livers were dissected, snap-frozen, and stored at − 80 °C. Speciation analysis of As was carried out in urine and liver homogenates prepared in deionized water (10% w/v) using hydride-generation atomic absorption spectrometry coupled with a cryotrap (HG-CT-AAS), as previously described (Hernández-Zavala et al. 2008; Currier et al. 2011). The HG-CT-AAS analysis determined concentrations of total iAs (iAsIII + iAsV), total MAs (MAsIII + MAsV), and total DMAs (DMAsIII + DMAsV). The instrumental limits of detection (LOD) for iAs, MAs, and DMAs using this method are 14, 8, and 20 pg As, respectively, which translates to 0.028, 0.016, and 0.040 ppb for a sample size of 0.5 ml (Hernández-Zavala et al. 2008). An imputed value of square root of LOD for given As species for used for measurements below LOD. Total As content in urine and liver was calculated as the sum of all As species.

As3mt expression analysis

RNA was extracted from samples of liver using Qiagen AllPrepDNA/RNA/miRNA universal kit and QIACube (Qiagen, Hilden, Germany; SN 50797). cDNA was generated from RNA and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in a one-step process using QuantiTect SYBR Green RT-PCR Kit (Qiagenand QuantiTect Primer Assays for As3mt (Mm_As3mt_1_SG) and GAPDH (Mm_Gapdh_3_SG) (both from Qiagen). CT values for As3mt were normalized using Gapdh CT to obtain dCT.

Statistical analysis

The CC strains were randomly assigned a number from 1 to 12; these numbers were used during statistical analyses and in tables and graphs. The concentrations of iAs, MAs, DMAs, and total As in urine and liver were expressed as ppb, i.e., nanogram of As per milliliter and per gram, respectively. The proportions of iAs and its methylated metabolites in urine and liver (%iAs, %MAs, and %DMAs) were calculated to assess iAs methylation capacity. One-way ANOVA was used to assess differences in the concentrations and proportions of As species and As3mt expression among the 12 CC strains. Differences in As3mt expression between exposures to 0.1 and 50 ppm iAs were evaluated using one-tailed t test. Associations between water consumption and the concentrations of total As in urine and liver, as well as associations between As3mt expression and total As or proportions of As species in urine and liver were assessed using Spearman rank correlation. Partial Spearman correlation was used to characterize associations between total As and the proportions of As species in urine and livers while adjusting for the water consumption. The correlation analysis was performed using SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA). The differences between mouse strains and correlations with p values < 0.05 were considered statistically significant.

Data availability

All phenotypic data have been deposited and is available at the Mouse Phenome Database (https://phenome.jax.org/).

Results

CC strains and As3mt haplotypes

Table 1 lists the 12 CC strains that were included in this study, along with the corresponding founder As3mt haplotypes and assigned numbers (No. 1–12) that were used as strain identifiers throughout the study. Detailed information about As3mt polymorphisms present in each founder haplotype has been reported previously (Keane et al. 2011). Variants in exons and variants with predicted effect on gene function are described in Supplemental Table 1.

Table 1.

CC strains included in the study and the corresponding As3mt haplotypes

Assigned numbera CC strain As3mt haplotype
1 CC007/Unc 129S1/SvImJ
2 CC021/Unc NOD/ShiLtJ
3 CC049/TauUnc CAST/EiJ
4 CC011/Unc C57BL/6J
5 CC018/Unc NZO/HlLtJ
6 CC036/Unc C57BL/6J
7 CC053/Unc WSB/EiJ
8 CC027/GeniUnc WSB/EiJ
9 CC033/GeniUnc NOD/ShiLtJ
10 CC008/GeniUnc C57BL/6J
11 CC045/GeniUnc A/J
12 CC038/GeniUnc C57BL/6J
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a

The numbers used throughout the study to identify the CC strains

Water consumption and total As in urine and in liver

The average amount of water consumed by a mouse over the period of 2 weeks differed among the CC strains and depended on the exposure level (Table 2). Water consumption in mice exposed to 0.1 ppm iAs ranged from 28.1 to 81.9 ml, while mice exposed to 50 ppm iAs drank 7.7–34 ml. In all strains, water consumption was reduced in the high exposure cohort, but the level of reduction varied among strains (Table 2). Total As concentrations in urine differed among the CC strains exposed to 0.1 ppm (168–369 ppb; p < 0.0001) (Fig. 1a), as well as strains exposed to 50 ppm (50,071–11,271 ppb; p < 0.0002) (Fig. 1c). Significant differences in total As concentrations were also found in livers at both exposure levels: 0.47–5.02 ppb (p < 0.0001) at 0.1 ppm (Fig. 1b), and 633–3099 ppb (p < 0.002) at 50 ppm (Fig. 1d). The concentration of total As in urine of CC mice exposed to 0.1 ppm iAs positively correlated with water consumption (p = 0.024) (Fig. 1a). No statistically significant correlations were found between water consumption and total As in livers of mice exposed to 0.1 ppm (Fig. 1b), or total As in urine or livers of mice exposed to 50 ppm iAs (Fig. 1c, d).

Table 2.

Number of mice and water consumption in 12 CC strains exposed to 0.1 or 50 ppm iAs in drinking water

Strain number Exposure (ppm) N Water consumeda (ml/mouse)
1 0.1 4 63.1
50 4 (3)b 21.5
2 0.1 5 28.1
50 4 10.7
3 1 4 36.4
50 4 22
4 0.1 5 40.2
50 5 9
5 0.1 4 81.9
50 5 14.4
6 0.1 4 63.15
50 4 24.7
7 0.1 5 28.5
50 4 20.3
8 0.1 4 34.8
50 5 15.7
9 0.1 4 48.9
50 4 18.4
10 0.1 4 63
50 4 34
11 0.1 5 34.6
50 5 13.4
12 0.1 4 42.3
50 4 7.7
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a

Average total amount of water consumed over 2 weeks of exposure (calculated from water volume consumed per cage)

b

One mouse died during the first week of the exposure, leaving three mice in the group

Fig. 1.

Fig. 1

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Spearman correlations between average water consumption and average total As concentration in urine (a, c) and liver (b, d) of mice from 12 CC strains exposed 0.1 (a, b) and 50 ppm (c, d) of iAs in drinking water (N = 3–5 per strain). The assigned strain number is shown with each value

Concentrations and proportions of As species in urine

Figure 2 shows the concentrations of As species in urine of CC mice exposed to 0.1 and 50 ppm iAs. In urine of mice exposed to 0.1 ppm, the concentration of iAs ranged from 3.2 to 7.8 ppb, MAs from 0.5 to 0.9 ppb, and DMAs from 164 to 364 ppb (Fig. 2a–c). Even greater variations were found in the urine of mice exposed to 50 ppm: iAs, 25–255 ppb; MAs, 554–6003; DMAs, 49,429–105,017 ppb (Fig. 2d–f). DMAs was the major urinary As species accounting for 95.7–98.8% and 94.5–99.1% of total As after exposure to 0.1 ppm and 50 ppm, respectively (Fig. 3; Supplemental Figure 1). The differences in %DMAs were statistically significant among the CC strains exposed to 50 ppm (p = 0.009), but not 0.1 ppm iAs (p = 0.094). iAs accounted for 1.1–4.2% of total As in urine of mice exposed to 0.1 ppm, but only for 0.03–0.32% in urine of mice exposed to 50 ppm iAs. An opposite trend was observed in %MAs, which was higher in urine of mice exposed to 50 ppm (0.9–7.1%) as compared to 0.1 ppm (0.2–0.5%). The difference among the CC strain in %MAs was statistically significant after exposure to 50 ppm (p = 0.00009) but not 0.1 ppm iAs (p = 0.094). No statistically significant differences were found in %iAs, regardless exposure levels.

Fig. 2.

Fig. 2

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Concentrations of iAs (a, d), MAs (b, e), and DMAs (c, f) in spot urine samples collected from CC mice exposed to 0.1 ppm (a–c) and 50 ppm (d–f) iAs in drinking water (mean + SE; N = 3–5 per strain)

Fig. 3.

Fig. 3

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Distribution of As species in spot urine samples collected from CC mice exposed to 0.1 ppm (a) and 50 ppm (b) iAs in drinking water (mean + SE; N = 3–5 per strain)

Concentrations and proportions of As species in livers

The concentrations of As species in livers of CC mice exposed to 0.1 and 50 ppm iAs are shown in Fig. 4. The concentrations of iAs varied widely among the strains, ranging from 0.06 to 0.75 ppb after exposure to 0.1 ppm, and from 114 to 697 ppb after exposure to 50 ppm iAs (Fig. 4a, d). Similar variations were observed in concentrations of MAs, 0.06–0.9 ppb and 103–643 ppb (Fig. 4b, e), and DMAs, 0.3–3.4 ppb and 348–1980 ppb (Fig. 4c, f). The distribution of As species in the livers differed from those found in urine. DMAs was still the major metabolite in most strains, but it accounted only for 49.1–72.7% and 29.2–66.3% of total As after exposure to 0.1 and 50 ppm, respectively (Fig. 5; Supplemental Figure 2). The differences in %DMAs among the CC strains were statistically significant after exposure to 50 ppm (p = 0.0004), but not 0.1 ppm iAs (p = 0.221). Unlike in urine, iAs and MAs represented significant fractions of total As in the livers after exposure either to 0.1 ppm or to 50 ppm iAs: iAs accounted for 10.6–41.6% and 15.1–48.3%, respectively, and MAs for 4.2–30.4% and 15.9–28.5%, respectively. The differences among the strains in %iAs and %MAs at both exposure levels were statistically significant (p = 0.000007–0.013).

Fig. 4.

Fig. 4

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Concentrations of iAs (a, d), MAs (b, e), and DMAs (c, f) in livers of CC mice exposed to 0.1 ppm (a–c) and 50 ppm (d–f) iAs in drinking water (mean + SE; N = 3–5 per strain)

Fig. 5.

Fig. 5

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Distribution of As species in livers of CC mice exposed to 0.1 ppm (a) and 50 ppm (b) iAs in drinking water (mean + SE; N = 3–5 per strain)

Correlations between total As and proportions of As species in urine and in liver

Partial Spearman correlation test was used to characterize associations between As species separately in the urine and in the liver, and between the urine and the liver while controlling for the differences between strains in water consumption.

In urine of mice exposed to 0.1 ppm iAs, total As concentration correlated negatively with %iAs and %MAs, and positively with %DMAs (Table 3); these correlations were statistically significant. In contrast, statistically significant positive correlation between total As and %MAs and negative correlation between total As and %DMAs were found in urines of mice exposed to 50 ppm iAs; the correlation between total As and %iAs remained negative, but was only marginally significant. No statistically significant correlations were found between total As and the proportions of As species in the livers of mice exposed to either 0.1 or 50 ppm iAs; a negative correlation between total As and %DMAs after exposure to 50 ppm was only marginally significant.

Table 3.

Partial Spearman correlation between total As and proportions of As species in urine and between total As and proportions of As species in livers of CC mice exposed to 0.1 ppm and 50 ppm iAs in drinking water

Correlation between: 0.1 ppm
 50 ppm
Rho p Rho p
Total As in urine %iAs in urine − 0.446 0.001 − 0.258 0.073
Total As in urine %MAs in urine − 0.413 0.003 0.346 0.015
Total As in urine %DMAs in urine 0.447 0.001 − 0.410 0.004
Total As in liver %iAs in liver 0.119 0.404 0.190 0.191
Total As in liver %MAs in liver − 0.174 0.221 0.214 0.140
Total As in liver %DMAs in liver 0.076 0.594 − 0.266 0.065
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The concentration of total As and proportions of As species for each CC strain were normalized for the average water consumption for the strain at given exposure level. p values for statistically significant correlations are shown in bold font; p values for marginally significant correlations are underlined

No statistically significant correlations between hepatic and urinary %iAs, %MAs and %DMAs were found in CC mice exposed to 0.1 ppm iAs (Table 4). In mice exposed to 50 ppm iAs, %iAs in the urine correlated positively with %iAs and negatively with %DMAs in the liver. Negative correlation was found between %MAs in urine and %MAs in the liver. No statistically significant correlations were found for other combinations.

Table 4.

Partial Spearman correlation of the proportions of As species between urine and livers of CC mice exposed to 0.1 ppm and 50 ppm iAs in drinking water

Correlation between: 0.1 ppm
 50 ppm
Rho p Rho p
%iAs in urine %iAs in liver 0.047 0.744 0.450 0.001
%MAs in urine %MAs in liver 0.129 0.369 − 0.418 0.003
%DMAs in urine %DMAs in liver 0.069 0.633 − 0.219 0.131
%iAs in urine %DMAs in liver − 0.077 0.591 − 0.309 0.031
%DMAs in urine %iAs in liver − 0.025 0.864 − 0.035 0.813
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The concentration of total As and proportions of As species for each CC strain were normalized for the average water consumption for the strain at given exposure level. p values for statistically significant correlations are shown in bold font; p values for marginally significant correlations are underlined

Total As in the liver correlated negatively with total As in the urine of mice exposed to 0.1 ppm and positively with total As in urine of mice exposed to 50 ppm iAs, although the latter correlation was only marginally significant (Table 5). No correlations were found between total As in the liver and %iAs, %MAs, or %DMAs in urine regardless of exposure. Similarly, no statistically significant correlations were found between total As in urine and %iAs or %DMAs in the liver of mice exposed to 0.1 ppm iAs; a positive correlation with %MAs was only marginally significant. In contrast, total As in urine correlated negatively with %MAs and positively with %DMAs in the liver of mice exposed to 50 ppm iAs, and both correlations were statistically significant.

Table 5.

Partial Spearman correlation between total As in livers and urine, between total As in livers and proportions of As species in urine, and between total As in urine and proportions of As species in livers of CC mice exposed to 0.1 ppm and 50 ppm iAs in drinking water

Correlation between: 0.1 ppm
 50 ppm
Rho p Rho p
Total As in liver Total As in urine − 0.356 0.010 0.244 0.092
Total As in liver %iAs in urine − 0.019 0.895 0.051 0.729
Total As in liver %MAs in urine − 0.072 0.614 − 0.148 0.311
Total As in liver %DMAs in urine 0.020 0.888 0.167 0.251
Total As in urine %iAs in liver − 0.135 0.346 − 0.135 0.354
Total As in urine %MAs in liver 0.273 0.053 − 0.349 0.014
Total As in urine %DMAs in liver − 0.087 0.544 0.368 0.009
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The concentration of total As and proportions of As species for each CC strain were normalized for the average water consumption for the strain at given exposure level. p values for statistically significant correlations are shown in bold font; p values for marginally significant correlations are underlined

As3mt mRNA expression in the liver

As3mt mRNA levels were measured in livers collected at sacrifice (Fig. 6). The expression of As3mt varied among the 12 CC strains and this variation was statistically significant for both exposure levels (p < 0.000001). In addition, As3mt expression in the livers of seven CC strains (No. 1, 3, 4, 5, 9, 10, 11, and 12) was significantly higher after exposure to 0.1 ppm than after exposure to 50 ppm iAs; in one strain (No. 10), the expression was higher after 50 ppm vs 0.1 ppm exposure. No statistically significant differences due to exposure were found in As3mt expression in the livers of the other four strains.

Fig. 6.

Fig. 6

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As3mt mRNA expression in livers of mice from 12 CC strains after exposure to 0.1 and 50 ppm iAs in drinking water. dCT (deltaCT) value represents the difference between Ct for As3mt and Ct for GAPDH expression (mean + SD, N = 3–5); *p < 0.05, **p < 0.01 for comparison of 0.1 vs 50 ppm exposure

Correlation between As3mt expression and As species in urine and livers

As3mt expression correlated negatively with %iAs and positively with %DMAs in livers across the 12 CC strains exposed to 50 ppm iAs, and these correlations were statistically significant (Fig. 7). No statistically significant correlations were found between As3mt expression and %MAs or total As concentration in the liver (Supplemental Table 2). No correlations were found between As3mt expression and total As or proportions of As species in urine regardless of exposure (Supplemental Table 2).

Fig. 7.

Fig. 7

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Spearman correlation between As3mt expression (1/dCT) and %iAs (a) and %DMAs (b) in livers of CC mice exposed to 50 ppm iAs in drinking water

Discussion

In population studies, susceptibility to adverse effects of iAs exposure has been linked to differences in iAs metabolism, which in turn are associated with genetic polymorphisms, and AS3MT polymorphisms in particular (Antonelli et al. 2014; Pierce et al. 2012). Studying the adverse effects of iAs exposure in laboratory settings has been hampered by limited information on differences in iAs metabolism among mouse strains with different genetic backgrounds. In addition, there is an ongoing discussion about what exposure levels should be used in laboratory studies to properly model human exposures. While some published studies reported adverse effects of iAs at very low concentrations (0.01–0.1 ppm), results of other studies suggest that high concentrations (50–100 ppm) may be needed to reproduced in mice the effects of iAs exposure that have been observed in human populations. For example, we found that the total As concentrations in the livers of mice that developed diabetes after exposure to 50 ppm iAs (Paul et al. 2007) were in the range of total As concentrations found in liver biopsies from Bangladeshi residents exposed to 0.2–2 ppm iAs in drinking water (Mazumder 2005). This finding is consistent with the fact that mice metabolize iAs more efficiently than humans (Vahter 1999, 2000), and may thus be more resistant to its toxicity.

In the present study, we examined metabolism of iAs in mice exposed to a low (0.1 ppm) and a high (50 ppm) concentration of iAs. The main goal of this study was (1) to determine whether efficiencies and patterns of iAs metabolism differ between genetically diverse mouse strains exposed to these two levels of iAs and (2) to identify a strain in which iAs metabolism (i.e., the profiles of As species in urine) more closely resembles that described in humans. We selected to work with mice from the CC population which has been established to support this type of studies (Aylor et al. 2011). The following are the major findings:

Differences in iAs metabolism among the 12 CC strains depend on the exposure level

We found differences between the strains in the concentration of total As in both urine and livers after exposure to either 0.1 or 50 ppm iAs in drinking water for 2 weeks. Like in our previous studies (Paul et al. 2007, 2011), mice exposed to 50 ppm iAs drank less water than mice exposed to 0.1 ppm. Notably, the water consumption differed among the CC strains (Table 2). However, the fact that the amount of water consumed over the exposure period correlated significantly only with total As in urine and only in mice exposed to 0.1 ppm (Fig. 1) suggests that water consumption was not the only factor that determined As body burden and efficiency of As clearance in the urine, especially not at the high exposure level. The proportions of As species in urine, particularly %DMAs which is thought to characterize the efficiency of iAs methylation in humans, also differed among the CC strains, but these differences were statistically significant only in mice exposed to 50 ppm iAs. In these mice, but not in mice exposed to 0.1 ppm, differences among the CC strains in %DMAs were also found in the liver.

Further evidence that the exposure level determined the pattern of iAs metabolism among the 12 CC strains was provided by the correlation analysis of the associations between the proportions of As species in the urine and the liver or between the urine and the liver. The character of these correlations differed in mice exposed to 0.1 ppm as compared to mice exposed to 50 ppm iAs. For example, the correlation between total As and %DMAs in urine was positive after exposure to 0.1 ppm, but was negative after exposure to 50 ppm iAs (Table 3), suggesting that saturation of the capacity to convert iAs to DMAs may have occurred at 50 ppm exposure. Similar negative correlations between and %DMAs and total As in urine, but at much lower levels of iAs exposure, were observed in population studies (Del Razo et al. 1997; Hopenhayn-Rich et al. 1996a, b; Lindberg et al. 2008). Consistent with the saturation of iAs methylation pathway in the mice exposed to 50 ppm iAs is also the finding that %iAs in urine correlated positively with %iAs and negatively with %DMAs in the liver (Table 4). In addition, total As in urine correlated positively with %DMAs in the liver of mice exposed to 50 ppm iAs (Table 4), suggesting that As excretion at this exposure level depends on the capacity of the liver to convert iAs to DMAs. No such correlation was found in mice exposed to 0.1 ppm iAs.

As3mt polymorphisms and expression in the liver alone do not explain the differences in iAs metabolism among the CC strains

AS3MT polymorphisms are thought to be the single most important genetic factor that affects both the metabolism of iAs in humans (based on urinary profiles of iAs metabolites) and susceptibility to iAs exposure (Antonelli et al. 2014). However, polymorphisms in other genes have also been linked to differences in iAs metabolism. In this study, the 12 CC strains represent seven As3mt haplotypes that are characterized by different SNPs and/ or different 3′UTR structures in the As3mt gene. These differences likely contributed to the variation in As3mt expression and/or in metabolism of iAs among these strains. However, we observed differences in iAs metabolism even between strains with the same As3mt haplotype. For example, the proportions of iAs and its methylated metabolites differed in urine and livers of four CC strains sharing the C57BL/6J haplotype: No. 4 (CC011), No. 6 (CC036), No. 10 (CC008), and No. 12 (CC038). Percent MAs was significantly different in urine (p = 0.0022) and liver (p = 0.0152) after exposure to 50 ppm iAs; statistically significant differences were also found in DMAs/ MAs ratios (p = 0.0006 and p = 0.0086, respectively). Percent iAs was also significantly different in urine of mice exposed to 0.1 ppm (p = 0.028). Thus, polymorphisms in genes other than As3mt must have contributed to the different metabolic phenotypes among these four CC strains.

As3mt knockout in mice has been shown to result in accumulation of iAs in tissues and in very low levels of the methylated As species in tissues and urine (Douillet et al. 2017; Drobna et al. 2009), suggesting that As3mt is the key enzyme in the pathway for methylation of iAs. In the present study, As3mt expression in the liver, the organ that is thought to play a key role in iAs methylation, correlated with %iAs and %DMAs in the liver of mice exposed to 50 ppm, but not 0.1 ppm iAs. Notably, As3mt expression in the liver did not correlate with total As or with the proportions of As species in the urine regardless of exposure level. These data suggest that, while As3mt expression determines the liver capacity to methylate iAs, other tissues and mechanisms contribute significantly to iAs methylation and/or the clearance of iAs and its methylated metabolites in urine.

Limitations

In this study, we examined iAs metabolism only in 12 CC strains. It is possible that iAs metabolism in some of the other CC strains would better resemble that in humans. The 12 selected CC strains covered 7 of the 8 As3mt haplotypes, with only 1 or 2 strains per haplotype, except for C57BL/6J, which was represented by 4 strains. This could limit our ability to characterize the role of As3mt haplotype in the efficiency and patterns of iAs metabolism. The concentrations and proportions of As species were determined only in the liver and urine; while greater strain-dependent differences may occur in other tissues. In addition, the HG-CT-AAS techniques used in this study was not set up for quantification of trimethyl-As species, which have been found in small quantities in tissues of C57BL/6J mice exposed to iAs in our previously published study (Currier et al. 2016). Finally, only male mice were examined in the study; however, the efficiencies of iAs metabolism have been shown to differ between men and women, based on urinary profiles of As metabolites (Harari et al. 2013; Lindberg et al. 2007, 2008). Similarly, we have reported differences in the concentrations and speciation of As in the liver of male and female C57BL/6 mice (Douillet et al. 2017; Huang et al. 2018a, b). Thus, future studies that aim to examine the role of genetics in iAs metabolism in mice should account for sex as an important determinant of iAs metabolism.

Conclusions

To the best of our knowledge, this is the first study that used mice from the CC population to examine the role of genetics in iAs methylation. Results of this study are consistent with data from population studies linking genetic polymorphisms to differences in the urinary profiles of As species. These data also suggest that while the polymorphisms in As3mt play an important role in the capacity of the liver to methylate iAs, particularly at high exposure levels, other factors, e.g., iAs methylation or transport of As metabolites in other tissues, are likely to significantly contribute to the differences in As clearance in urine. The lack of correlations between total As concentration in the liver and %iAs, %MAs, or %DMAs in the urine of the CC mice, both at low and high exposure levels, seems to contradict the common assumption that proportions of As species in urine reflect the capacity of the body to methylate, and thus to detoxify iAs. Finally, while the profiles of As species in urine of the 12 CC strains do not resemble typical profiles found in humans, some of these strains (e.g., No. 2 and CC021) exhibit signs of low capacity to metabolize high levels of iAs, specifically high %iAs and low %DMAs in the liver. These strains may be more susceptible to the adverse effects of iAs exposure than strains typically used in laboratory studies.

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Acknowledgements

This study was supported by a UNC Systems Genetics Core Facility CC Pilot Program grant and by the following grants from NIEHS: R01ES022697 to M.S., R01ES028721 to M.S. and R.F., R01ES029925 to R.F., F.P.M.D.V., and M.S., and by the UNC Nutrition Obesity Research Center grant DK056350 from NIDDK.

Footnotes

Conflict of interest The authors declare that they have no conflicts of interest.

Compliance with ethical standards

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00204-019-02559-7) contains supplementary material, which is available to authorized users.

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All phenotypic data have been deposited and is available at the Mouse Phenome Database (https://phenome.jax.org/).

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