Environmental Research 164 (2018) 18–23

Contents lists available at ScienceDirect

Environmental Research
journal homepage: www.elsevier.com/locate/envres

Arsenic methylation capacity in relation to nutrient intake and genetic T

polymorphisms in one-carbon metabolism
Brenda Gamboa-Loiraa, César Hernández-Alcaraza, A. Jay Gandolfib, Mariano E. Cebriánc,
⁎
Ana Burguete-Garcíaa, Angélica García-Martíneza,1, Lizbeth López-Carrilloa,
a
Instituto Nacional de Salud Pública, Av. Universidad 655, Col. Santa María Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, México
b
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, USA
c
Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Del. Gustavo
A. Madero, C.P. 07360, Ciudad de México, México

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Nutrients and genetic polymorphisms participating in one-carbon metabolism may explain inter-
Inorganic arsenic individual differences in inorganic arsenic (iAs) methylation capacity, which in turn may account for variations
Arsenic metabolism in susceptibility to iAs-induced diseases.
Micronutrients Objectives: 1) To evaluate the association between polymorphisms in five one-carbon metabolism genes (FOLH1
Polymorphisms
c.223 T > C, MTHFD1 c.1958 G > A, MTHFR c.665 C > T, MTR c.2756 A > G, and MTRR c.66 A > G) and iAs
Northern Mexico
methylation capacity; 2) To assess if previously reported associations between nutrient intake and iAs methy-
lation capacity are modified by those polymorphisms.
Methods: Women (n = 1027) exposed to iAs in Northern Mexico were interviewed. Blood and urine samples
were collected. Nutrient dietary intake was estimated using a validated food frequency questionnaire. iAs me-
thylation capacity was calculated from urinary iAs species (iAs, monomethylarsonic acid [MMA] and di-
methylarsinic acid [DMA]) measured by high performance liquid chromatography (HPLC-ICP-MS). One poly-
morphism in each of the five genes evaluated was genotyped by allelic discrimination. Multivariable linear
regression models were used to evaluate if genetic polymorphisms modified the associations between iAs me-
thylation capacity parameters and nutrient intake.
Results: The median (min-max) concentration of total arsenic (TAs) was 20.2 (1.3–2776.0) µg/g creatinine in the
study population. Significant interactions for iAs metabolism were only found with FOLH1 c.223 T > C poly-
morphism and vitamin B12 intake, so that CT and CC genotype carriers had significantly lower %iAs, and higher
DMA/iAs with an increased vitamin B12 intake, as compared to carriers of wild-type TT.
Conclusion: Differences in dietary nutrient intake and genetic variants in one-carbon metabolism may jointly
influence iAs methylation capacity. Confirmation of these interactions in other populations is warranted.

1. Introduction (monomethylarsinous acid [MMA+3] and monomethylarsonic acid

Several studies have suggested one-carbon metabolism related nu-
trient intake (e.g. folate, choline, methionine, betaine, etc.) is associated
with inorganic arsenic (iAs) elimination efficiency through the gen-
eration of S-Adenosylmethionine (SAM) which serves as a methyl donor
for iAs metabolism.
Ingested iAs is eliminated through urine as dimethylated metabo-
lites (dimethylarsonuous acid [DMA+3] and dimethylarsinic acid
[DMA+5]) in greater proportion than the monomethylated
[MMA+5]) products (60–70% vs. 10–20%), and the fraction that re-
mains as iAs (10–30%) (Shen et al., 2016). The intermediates MMA+3
and DMA+3 are highly toxic and may be partially responsible for ar-
senic toxicity (Moe et al., 2016; Petrick et al., 2000; Vahter, 2002).
Higher %MMA has been associated with increased risk of cancer and
cardiovascular disease (Kuo et al., 2017). Our research group pre-
viously found that women with higher capacity to methylate iAs to
MMA and/or a lower capacity to further methylate MMA to DMA were
at higher risk for breast cancer (López-Carrillo et al., 2014). Individual

⁎
Correspondence to: Instituto Nacional de Salud Pública. Av. Universidad 655, Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, México.
E-mail addresses: brenda.gamboa@espm.insp.mx (B. Gamboa-Loira), cesar.hernandez@insp.mx (C. Hernández-Alcaraz), gandolfi@pharmacy.arizona.edu (A.J. Gandolfi),
mcebrian@cinvestav.mx (M.E. Cebrián), aburgete@insp.mx (A. Burguete-García), angelica.garcia67@gmail.com (A. García-Martínez), lizbeth@insp.mx (L. López-Carrillo).
1
Un Kilo de Ayuda A.C. Av. Paseo de la Reforma 1110, Col. Lomas de Chapultepec, Del. Miguel Hidalgo, C.P. 11000, Ciudad de México, México.

https://doi.org/10.1016/j.envres.2018.01.050
Received 19 May 2017; Received in revised form 22 January 2018; Accepted 31 January 2018
Available online 17 February 2018
0013-9351/ © 2018 Elsevier Inc. All rights reserved.
B. Gamboa-Loira et al.
methylation capacity depends not only on age, sex, body mass index
(BMI), smoking, and exposure level but also on nutrient intake and
polymorphisms in one-carbon metabolism (Engström et al., 2009; Hall
et al., 2012; Tseng, 2009).
Observational studies have reported a negative association between
urinary %MMA and dietary intake and/or plasma levels of nutrients
involved in one-carbon metabolism. In several studies, an increase in %
DMA was observed in relation to greater consumption of these nutrients
(Basu et al., 2011; Gamble et al., 2005; Heck et al., 2007; López-Carrillo
et al., 2016; Spratlen et al., 2017; Steinmaus et al., 2005a). Results from
a randomized clinical trial showed that folic acid supplementation in
folate-deficient adults was significantly associated with higher %DMA
and lower %MMA in urine (Gamble et al., 2006), as well as with lower
concentrations of iAs and MMA in blood (Gamble et al., 2007).
Additionally, one-carbon metabolism polymorphisms have been
associated with iAs methylation capacity. Consistently, studies have
shown that carriers of TT genotype in methylene tetrahydrofolate re-
ductase (MTHFR) c.665 C > T polymorphism have impaired iAs me-
thylation capacity, i.e. increased %iAs and %MMA, and decreased %
DMA (Deng et al., 2007; Engström et al., 2007; Lindberg et al., 2007;
Steinmaus et al., 2007). In contrast, scarce and inconsistent information
is available regarding methionine synthase (MTR) c.2756 A > G and
methionine synthase reductase (MTRR) c.66 A > G polymorphisms
(Engström et al., 2009, 2007; Porter et al., 2010). The joint effect of
nutrient intake and genetic variants on iAs metabolism has not been
studied previously. In addition to the aforementioned polymorphisms,
folate hydrolase 1 (FOLH1 c.223 T > C) and methylenetetrahydrofolate
dehydrogenase 1 (MTHFD1 c.1958 G > A) have a key role in folate
absorption and metabolism (Krajinovic, 2008; O’Keefe et al., 1998),
although no information is available regarding their relationships with
iAs metabolism.
Therefore, the aims of this study are 1) to evaluate the association
between polymorphisms in one-carbon metabolism and iAs methylation
capacity; 2) to assess if previously reported associations between nu-
trient intake (methionine, choline, vitamin B12, folate, riboflavin, vi-
tamin B6, and betaine) and iAs methylation capacity (López-Carrillo
et al., 2016) are modified by those polymorphisms, among Mexican
women residing in Northern Mexico, where iAs concentrations in water
have been reported to be in the range of 7–740 μg/l (Camacho et al.,
2011).
2. Materials and methods
2.1. Study population
A cross-sectional study was undertaken among 1027 healthy
Mexican women (only one pregnant) aged at least 20 years and residing
for one or more years in any of the following northern states:
Chihuahua, Coahuila, Durango, Nuevo Leon, and Sonora, during the
period 2007–2011 (López-Carrillo et al., 2014). Women were identified
with the Master Sampling System for National Health Surveys in
Mexico, which provides a list of homes located in both urban and rural
areas. Detailed information about sample procedures is published
elsewhere (López-Carrillo et al., 2016). The response rate was 99.6%
(1027/1031).
Pending informed consent, participants were interviewed face to
face in one occasion, at their homes by trained interviewers about so-
ciodemographic characteristics, diet, alcohol, and tobacco consump-
tion. Anthropometric measurements for calculating the BMI were also
obtained. Participants donated one blood sample and a first-morning
void urine sample.
The project was approved by the Research, Biosecurity and
Bioethics Committees at the National Institute of Public Health
(Mexico).
19
Environmental Research 164 (2018) 18–23
2.2. Urinary arsenic determination
Samples were collected in a sterile disposable polypropylene urine
collection cup, stored in a fridge and maintained at least for two years
at −70 °C until analysis. Concentrations (µg/L) of urinary species ar-
senite (As+3), arsenate (As+5), MMA+5, DMA+5 and arsenobetaine
(AsB) were determined by high performance liquid chromatography
inductively coupled plasma mass spectrometry (HPLC-ICP-MS), ac-
cording to methodology previously described (Gilbert-Diamond et al.,
2011). Measurements below the limit of detection (LOD) (AsB: 24.25%;
As+3: 19.28%; As+5: 56.28%; MMA+5: 1.95%; DMA+5: 0.49%) were
given the corresponding LOD: AsB: 0.08; As+3: 0.15; As+5: 0.41;
MMA+5: 0.12 and DMA+5: 0.12, divided by the square root of two
(LOD/√2), as suggested by Barr et al. (2006). The urinary concentration
of creatinine (mg/dl) was measured using an enzymatic method kit
(Randox, Antrim County, UK). Coefficients of variation were: MMA+5
= 8%, DMA+5 = 9%, As+3 = 8%, AsB = 18% and creatinine =
2.76%. iAs concentration was the sum of As+3 and As+5. Arsenic ex-
posure was assessed as the sum of iAs, MMA+5, and DMA+5 (total ar-
senic [TAs]), excluding AsB.
In order to evaluate iAs methylation capacity, we calculated the
following parameters: 1) percentages of iAs (%iAs), MMA+5 (%MMA)
and DMA+5 (%DMA) with respect to TAs and 2) methylation ratios:
first = MMA+5/iAs (MMA/iAs); second = DMA+5/MMA+5 (DMA/
MMA); and total = DMA+5/iAs (DMA/iAs).
2.3. Nutrient intake evaluation
Daily consumption over the last year of 119 foods and 14 dishes was
estimated using a validated semi-quantitative food frequency ques-
tionnaire (Galván-Portillo et al., 2011). Based on the frequency of food
consumption reported by participants and tables of nutrient composi-
tion No. 20 of the United States Department of Agriculture (USDA), the
daily intake of total energy was estimated, as well as that of riboflavin,
vitamin B6, folate, vitamin B12, choline, methionine and betaine
(López-Carrillo et al., 2016).
2.4. Genotyping of FOLH1 c.223T > C, MTHFD1 c.1958 G > A, MTHFR
c.665C > T, MTR c.2756A > G and MTRR c.66A > G
The following single nucleotide polymorphisms involved in one-
carbon metabolism were included in this study: FOLH1 c.223T > C
(rs202676); MTHFD1 c.1958 G > A (rs2236225); MTHFR c.665C > T
(rs1801133); MTR c.2756A > G (rs1805087) and MTRR c.66A > G
(rs1801394). They were genotyped by allelic discrimination, using
TaqMan® assays and an ABI 7900 Sequence Detection System (Applied
Biosystems, Foster City, CA), under the following conditions: 95 °C per
10 min, 40 cycles to 95 °C during 15 s and 60 °C per 1 min. Conditional
upon DNA availability, samples were analyzed in duplicate (kappa
coefficients were: FOLH1 c.223T > C = 1.00; MTHFD1 c.1958 G > A
= 1.00; MTHFR c.665C > T = 1.00; MTR c.2756A > G = 0.31; MTRR
c.66A > G = 0.17). Negative controls were included on each plate.
Sample amplification failures were as follows: 18 for FOLH1
c.223T > C; 23 for MTHFD1 c.1958 G > A; 7 for MTHFR c.665C > T; 6
for MTR c.2756A > G and, 13 for MTRR c.66A > G, which rendered
final sample sizes for subsequent analysis of: 1009 (FOLH1), 1004
(MTHFD1), 1020 (MTHFR), 1021 (MTR) and 1014 (MTRR).
2.5. Statistical analysis
Selected characteristics of the study population were described
using measures of central tendency and dispersion.
The observed distributions of the study genotypes were assessed by
the Hardy-Weinberg Equilibrium test. Linkage Disequilibrium between
MTHFR c.665 C > T and MTR c.2756 A > G genetic variants was eval-
uated by the D’ statistic.
B. Gamboa-Loira et al. Environmental Research 164 (2018) 18–23

We calculated the adjusted iAs methylation capacity means using Table 1

multiple regression models, and compared them by genotypes of in- Selected characteristics of the study population (n = 1027).
terest with Wald's test. Each model included one percentage or iAs
Variable Median (P10, P90)
methylation ratio transformed to log scale, one polymorphism of in-
terest, and the following covariates: age (years), total energy intake Age (years) 54 (37, 71)
(kcal/day) and TAs (µg/g creatinine). Covariates were selected if they BMI (kg/m2) 29.95 (23.44, 38.54)
TAs (µg/l) 12.09 (3.41, 56.93)
correlated significantly with any of the percentages or iAs methylation
% ≥ 35 µg/l 17.3
ratios and/or correlated significantly with energy-adjusted nutrients of TAs (µg/g creatinine) 20.22 (6.48, 100.04)
interest, and were not significantly correlated with each other. BMI was AsB (µg/l)a 0.53 (0.06, 19.38)
not included because it was highly correlated with total energy intake. Creatinine (mg/dl) 64.00 (18.49, 161.50)
Likewise, the association between urinary creatinine and iAs methyla- Total energy intake (kcal/day) 1948.58 (1279.16, 3039.00)
Riboflavin (mg/day) 1.28 (0.75, 1.97)
tion capacity was evaluated adjusting for age (years), total energy in-
DRI (% < DRI) 0.9b (18.99)
take (kcal/day) and TAs (µg/l). Vitamin B6 (mg/day) 1.48 (0.95, 2.41)
To evaluate if genetic variants modify the nutrient-iAs methylation DRI (% < DRI) 1.2b (23.37)
capacity association, linear regressions were performed according to Folate (µg/day) 313.64 (164.63, 542.54)
DRI (% < DRI) 460b (81.6)
the genetic inheritance models: co-dominant and dominant (the wild-
Vitamin B12 (µg/day) 1.82 (0.71, 5.10)
type genotype was the reference category), and recessive (the wild and DRI (% < DRI) 3b (73.52)
heterozygous genotypes comprised the reference category). Linear re- Choline (mg/day) 245.59 (148.31, 386.51)
gression models included one single nutrient adjusted for energy re- DRI (% < DRI) 425c (94.94)
siduals (continuous), one genetic variant, the respective nutrient x ge- Betaine (mg/day) 17.39 (7.16, 35.37)
DRI (% < DRI) –
netic variant interaction term and the mentioned covariates. To reduce
Methionine (mg/kg/day) 16.75 (9.93, 27.07)
the likelihood of type I error due to multiple comparisons, False DRI (% < DRI) 15c,d (37.78)
Discovery Rate corrected p-values (Benjamini and Hochberg, 1995)
a
were considered significant at < 0.05. All statistical analyses were AsB is 4.4% of TAs.
b
performed in Stata 13 software (Stata Corp, College Station, TX, USA). Average for Mexican women aged 19–70 years (Bourges et al., 2009).
c
Average for US women aged 19–70 years (IOM, 2006).
d
DRI for methionine + cysteine.
3. Results

B12 = 0.995, 95% CI = 0.978, 1.012). No other genetic variants

The median age of the population was approximately 54 years at the
time of the study, and the median BMI was at the lower limit of obesity
(30 kg/m2). Median TAs in urine was 12.09 µg/l and about 17% of
women had concentrations ≥ 35 µg/l. AsB was 4.4% of TAs. Median
urinary creatinine was 64.00 mg/dl. Women in our study had daily
dietary intakes lower than the dietary reference intake (DRI) for ribo-
flavin (18.99% less than 0.9 mg/day), vitamin B6 (23.37% less than
1.2 mg/day), folate (81.60% less than 460 µg/day), vitamin B12
(73.52% less than 3.0 µg/day), choline (94.94% less than 425 mg/day),
and methionine (37.78% less than 15 mg/kg/day) (Bourges et al., 2009;
Institute of Medicine, 2006) (Table 1).
The distribution of genotypes of interest was found in Hardy-
Weinberg equilibrium, except for FOLH1 which was marginally sig-
nificant (p = 0.046). MTHFR c.665 C > T and MTR c.2756 A > G ge-
netic variants were not in linkage disequilibrium (D' = 0.05, p = 0.23).
FOLH1 c.223 T > C CC carriers had significantly lower %iAs than TT
and CT carriers, and higher DMA/iAs than CT carriers. MTHFD1
c.1958 G > A GG carriers had lower %iAs than GA and AA carriers, and
higher DMA/iAs than GA carriers. Also, MTR c.2756 A > G GG carriers
had lower %iAs than AA and AG carriers (Table 2). Urinary creatinine
was positively associated with %MMA (β = 0.0010, p < 0.001) and %
DMA (β = 0.0002, p = 0.055), and negatively with %iAs (β =
−0.0008, p = 0.013) (data not included in the table).
Geometric mean ratios between FOLH1 c.223 T > C and vitamin
B12 intake on urinary arsenic methylation capacity parameters are
presented in Table 3. Under the co-dominant (p for interaction =
0.027) and dominant (p for interaction < 0.001) genetic inheritance
models, one µg increase in vitamin B12 intake significantly reduced %
iAs among variant allele carriers (Co-dominant: GMRCT x vitamin B12 =
0.964, 95% CI = 0.945, 0.984; GMRCC x vitamin B12 = 0.971, 95% CI =
0.937, 1.006. Dominant: GMR(CT + CC) x vitamin B12 = 0.963, 95% CI =
0.947, 0.980) compared to wild-type homozygous (GMRTT x vitamin B12
= 1.005, 95% CI = 0.991, 1.019). Also, one µg increase in vitamin B12
intake increased DMA/iAs among variant allele carriers (Co-dominant:
GMRCT x vitamin B12 = 1.048, 95% CI = 1.021, 1.076; GMRCC x vitamin B12
= 1.036, 95% CI = 0.990, 1.085; p for interaction = 0.040. Dominant:
GMR(CT + CC) x vitamin B12 = 1.049, 95% CI = 1.025, 1.073; p for in-
teraction < 0.001) compared to wild-type homozygous (GMRTT x vitamin
modified the association between the intake of nutrients involved in the
one-carbon metabolism pathway and iAs metabolism (Supplementary
Table 1).
4. Discussion
Our results suggest that iAs metabolism is jointly affected by both
genetic variants and dietary intake of nutrients involved in one-carbon
metabolism. In a previous report by this research group, we found in-
creased vitamin B12 intake was associated with a greater iAs methy-
lation capacity in this population (López-Carrillo et al., 2016). In this
study, we show that this relationship is modified by FOLH1 c.223 T > C
polymorphism: the association of vitamin B12 intake was greater
among variant allele carriers compared to wild-type allele carriers
under the dominant inheritance model.
The FOLH1 gene codifies folate hydrolase 1 enzyme, responsible for
folate intestinal absorption. The c.223 T > C polymorphism results in a
Tyr75His amino acid change that impairs its intestinal absorption
(O’Keefe et al., 1998). Previously we reported that dietary folate intake
decreased %iAs, and increased DMA/iAs (López-Carrillo et al., 2016). A
high percentage of our population had low intake of folate, which may
have limited the power to detect an interaction with FOLH1
c.223 T > C polymorphism on iAs methylation capacity in this report.
However, we found that vitamin B12, a co-factor in the homocysteine
methylation reaction for SAM synthesis, interacted with FOLH1
c.223 T > C on iAs methylation capacity. The FOLH1 variant genotype
may increase the efficiency of vitamin B12 in the one-carbon cycle;
further studies are needed to elucidate the underlying mechanism.
Variations in folate and vitamin B12 intake as well as genotype dis-
tributions may explain why plasma vitamin B12 has been inconsistently
related with iAs methylation capacity (Gamble et al., 2005; Hall et al.,
2009a, 2009b; Li et al., 2008).
The study variants are found in five genes whose enzymes converge
in the synthesis of SAM (Brody et al., 2002; Hall et al., 2012; Ludwig
and Matthews, 1997; Olteanu et al., 2002). Information on the re-
lationship between these genes and iAs methylation capacity is scarce.
A small previous study did not find a significant relationship between %

20
B. Gamboa-Loira et al. Environmental Research 164 (2018) 18–23

Table 2
Adjusted means of urinary arsenic methylation capacity parameters by genotypes of interest.

Genotypes n Parameters

%iAs %MMA %DMA MMA/iAs DMA/MMA DMA/iAs

Mean (95% CI) Mean (95% CI) Mean (95% CI) Mean (95% CI) Mean (95% CI) Mean (95% CI)

FOLH1 c.223T > Ca

TT 528 11.7 (9.6, 14.2)* 10.0 (8.6, 11.6) 73.9 (68.6, 79.5) 0.8 (0.7, 1.0) 7.4 (6.1, 8.9) 6.3 (4.9, 8.1)
CT 421 12.5 (10.3, 15.2)* 10.4 (9.0, 12.1) 72.1 (67.0, 77.6) 0.8 (0.7, 1.0) 6.9 (5.7, 8.3) 5.8 (4.5, 7.4)*
CC 60 9.8 (7.8, 12.3)* 9.4 (7.9, 11.2) 76.8 (70.6, 83.6) 0.9 (0.7, 1.2) 8.2 (6.6, 10.1) 7.8 (5.8, 10.5)*
MTHFD1 c.1958 G > A
GG 198 10.6 (8.7, 12.9)* 9.9 (8.5, 11.5) 75.7 (70.2, 81.7) 0.9 (0.7, 1.1) 7.6 (6.3, 9.3) 7.1 (5.5, 9.2)*
GA 490 12.2 (10.0, 14.8)* 10.3 (8.9, 12.0) 72.3 (67.2, 77.9) 0.8 (0.7, 1.0) 7.0 (5.8, 8.5) 5.9 (4.6, 7.6)*
AA 316 11.9 (9.8, 14.6)* 10.2 (8.8, 11.9) 73.2 (67.9, 79.0) 0.8 (0.7, 1.0) 7.1 (5.9, 8.7) 6.1 (4.7, 8.0)
MTHFR c.665 C > T
CC 244 12.2 (10.0, 14.9) 10.0 (8.6, 11.7) 73.5 (68.2, 79.2) 0.8 (0.7, 1.0) 7.3 (6.0, 8.9) 6.0 (4.7, 7.8)
CT 532 11.6 (9.5, 14.0) 10.2 (8.8, 11.9) 73.1 (68.0, 78.6) 0.9 (0.7, 1.1) 7.1 (5.9, 8.6) 6.3 (4.9, 8.1)
TT 244 11.3 (9.2, 13.8) 9.7 (8.4, 11.4) 74.9 (69.5, 80.8) 0.9 (0.7, 1.1) 7.7 (6.3, 9.3) 6.6 (5.1, 8.6)
MTR c.2756 A > G
AA 676 12.1 (10.0, 14.6)* 10.3 (8.9, 11.9) 72.9 (67.8, 78.3) 0.8 (0.7, 1.0) 7.1 (5.9, 8.5) 6.0 (4.7, 7.7)
AG 300 12.2 (10.0, 14.8)* 10.3 (8.8, 11.9) 73.8 (68.6, 79.5) 0.8 (0.7, 1.0) 7.2 (5.9, 8.7) 6.1 (4.7, 7.8)
GG 45 9.7 (7.6, 12.4)* 8.7 (7.2, 10.5) 76.8 (70.1, 84.2) 0.9 (0.7, 1.2) 8.8 (6.9, 11.1) 7.9 (5.7, 10.8)
MTRR c.66 A > G
AA 576 11.8 (9.7, 14.4) 10.2 (8.8, 11.8) 73.0 (67.9, 78.6) 0.8 (0.7, 1.0) 7.2 (5.9, 8.7) 6.1 (4.8, 7.9)
AG 380 11.8 (9.7, 14.3) 10.1 (8.7, 11.7) 73.8 (68.6, 79.4) 0.8 (0.7, 1.0) 7.3 (6.1, 8.8) 6.3 (4.9, 8.1)
GG 58 11.1 (8.8, 14.1) 10.6 (8.9, 12.7) 75.4 (69.0, 82.3) 0.9 (0.7, 1.2) 7.1 (5.6, 8.9) 6.8 (5.0, 9.2)

Means were adjusted for age (years), total energy intake (kcal/day) and TAs (µg/g creatinine).
* FOLH1 c.223T > C TT vs. CC, and CT vs. CC; MTHFD1 c.1958 G > A GG vs. GA, and GG vs. AA; MTR c.2756 A > G AA vs. GG, and AG vs. GG comparison of urinary arsenic
methylation capacity means FDR corrected p-value < 0.05.
a
Hardy-Weinberg Equilibrium test p= 0.046.

iAs, %MMA nor %DMA and MTR c.2756 A > G variant (Porter et al.,
2010). Another study reported a marginal significant decrease in %
MMA between AG carriers compared to AA carriers (Engström et al.,
2007). MTHFR c.665 C > T genetic variant has been previously asso-
ciated with a reduction of iAs methylation capacity (Deng et al., 2007;
Engström et al., 2007; Lindberg et al., 2007; Steinmaus et al., 2007),
whereas other studies did not find significant associations (Agusa et al.,
2008; Engström et al., 2009; Porter et al., 2010). Engström et al. (2009)
reported carriers of GA and GG genotypes in MTRR c.66 A > G to have
lower %iAs than AA carriers. We found FOLH1 c.223 T > C, MTR
c.2756 A > G variant homozygous, and MTHFD1 c.1958 G > A wild
type homozygous had better iAs methylation capacity; however, it
cannot be ruled out that the other variants have a role in iAs methy-
lation capacity, due to the small sample size.
Results of this study should be interpreted with caution considering
its methodological limitations. AsB may be converted to DMA (Navas-
Acien et al., 2011). The correlation between AsB and the sum of in-
organic and methylated species in this population was 0.2365
(p < 0.05), which may suggest some formation of DMA from AsB. This
may not have had a major impact in our results due to the low per-
centage of AsB (about 4% of TAs), that reflects a low seafood con-
sumption (that did not vary by genotype). We controlled for potential
confounders in iAs methylation capacity such as age, total energy in-
take, and TAs (Tseng, 2009). Another potential confounder is urine
dilution variation. Creatinine concentration is an indicator of such di-
lution, however, the optimal way to control this factor is a matter of
debate. Some authors suggest dividing urinary concentrations of the
metabolite of interest by creatinine concentration (Barr et al., 2004). As
the calculation of the percentages and ratios under study eliminates the
concentration of creatinine in the numerator and in the denominator,

Table 3
Geometric mean ratiosa between FOLH1 c.223T > C and vitamin B12 intake on urinary arsenic methylation capacity parameters.

Genetic inheritance model for FOLH1 %iAs %MMA %DMA MMA/iAs DMA/MMA DMA/iAs
c.223 T > C GMR (95% CI) GMR (95% CI) GMR (95% CI) GMR (95% CI) GMR (95% CI) GMR (95% CI)

Co-dominant
TT 1.005 (0.991, 0.996 (0.986, 1.000 (0.995, 0.991 (0.977, 1.004 (0.990, 0.995 (0.978,
1.019)* 1.007) 1.005) 1.005) 1.017) 1.012)*
CT 0.964 (0.945, 0.989 (0.974, 1.011 (1.003, 1.026 (1.004, 1.022 (1.002, 1.048 (1.021,
0.984)* 1.005) 1.019) 1.048) 1.042) 1.076)*
CC 0.971 (0.937, 0.986 (0.960, 1.006 (0.993, 1.016 (0.979, 1.020 (0.985, 1.036 (0.990,
1.006)* 1.014) 1.020) 1.055) 1.056) 1.085)*
Dominant
TT 1.005 (0.991, 0.996 (0.986, 1.000 (0.995, 0.991 (0.977, 1.004 (0.990, 0.995 (0.978,
1.019)* 1.007) 1.005) 1.005) 1.017) 1.012)*
CT + CC 0.963 (0.947, 0.987 (0.974, 1.010 (1.004, 1.025 (1.006, 1.023 (1.006, 1.049 (1.025,
0.980)* 1.001) 1.017) 1.044) 1.041) 1.073)*
Recessive
TT + CT 0.992 (0.981, 0.994 (0.985, 1.003 (0.999, 1.002 (0.990, 1.009 (0.998, 1.011 (0.997,
1.003) 1.002) 1.008) 1.014) 1.021) 1.026)
CC 0.971 (0.937, 0.986 (0.960, 1.006 (0.993, 1.016 (0.979, 1.020 (0.985, 1.036 (0.990,
1.006) 1.014) 1.020) 1.055) 1.056) 1.085)

a
Adjusted for age (years), total energy intake (kcal/day), and TAs (µg/g creatinine).
* Interaction between FOLH1 c.223T > C and vitamin B12 intake FDR corrected p-value < 0.05.

21
B. Gamboa-Loira et al.
only TAs concentrations were creatinine adjusted. In addition, because
creatinine has been associated with a reduction of %iAs and an increase
of %DMA (Basu et al., 2011; Peters et al., 2014), as it was observed in
this study, other authors recommend including creatinine as an ad-
justment variable in multivariate models. We obtained the same results
adjusting TAs for creatinine vs. including creatinine as an independent
variable (Supplementary Table 2).
iAs methylation capacity evaluation with a single urine sample is a
good estimate of the chronic exposure, considering that the source of
exposure is the water of daily consumption, together with the evidence
showing the existence of stable methylation patterns (Steinmaus et al.,
2005b) with small variations throughout the day (< 5%) (Concha et al.,
2002). However, the percentage of samples with iAs species un-
detectable was high in this study (As+3: 19.28% and As+5: 56.28%).
For future studies this situation might be prevented following the re-
commendation to oxidize all As+3 to As+5 using H2O2 prior to analysis,
that allowmeasuring combined iAs species and minimize the number of
undetectable samples (Scheer et al., 2012).
The presence of random error in the estimation of reported food
consumption is an inherent limitation to the information obtained
through interviews, thus it should not be ruled out that our results are
underestimated in some degree. Random measurement error could also
lead to higher variation in nutrient intake in some of the subgroups
defined by genotypes than in others, which in turn would lead to obtain
spurious interactions. In contrast, we did not identify reasons why there
would be systematically different dietary reports among comparison
groups that would have produced a differential error. In addition,
random error in genotyping may have reduced the respective associa-
tions of MTR and MTRR with iAs methylation capacity parameters
considering that the genotyping duplicate analysis for them show kappa
coefficients lower than one.
Regarding representativeness of our sample, we observed that the
medians of total energy, protein, carbohydrate, total fat and folate
consumption were similar to the Mexican national medians for women
aged 20–59 years reported in the National Health Survey And Nutrition
2006 (ENSANUT 2006) (Barquera et al., 2009). There is no data
available from ENSANUT or other studies in relation to other nutrients
evaluated in this work.
In conclusion, our results confirm that both diet and genetic dif-
ferences in one-carbon metabolism contribute to iAs methylation ca-
pacity, and may help to identify genetically susceptible individuals in
populations exposed to iAs worldwide. Future interventions should
consider both nutrient intake and gene variants in one-carbon meta-
bolism to mitigate health effects associated to iAs exposure.
Acknowledgments
This study was conducted with funding from CONACyT: Fondo
Sectorial de Investigación en Salud y Seguridad Social (2005-2-14373,
2009-1-111384, 2010-1-140962 and 2016-1-272632). Additional sup-
port was obtained from Dean Carter Binational Center for
Environmental Health Sciences (NIH ES-04940). The analyses for ar-
senic metabolites were performed by the Analytical Section of the
Hazard Identification Core, College of Pharmacy, University of Arizona.
We wish to acknowledge Michael Kopplin for performing the analyses
and Rosa María Hernández for technical support.
Conflicts of interest
The authors declare they have no conflicts of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.envres.2018.01.050.
22
Environmental Research 164 (2018) 18–23
References
Agusa, T., Fujihara, J., Minh, T.B., Trang, P.T.K., Takeshita, H., Iwata, H., Viet, P.H.,
Tanabe, S., 2008. Genetic polymorphisms influencing arsenic metabolism in human:
evidence from Vietnam. Interdiscip. Stud. Environ. Chem. Responses Chem. Pollut.
179–185.
Barquera, S., Hernández-Barrera, L., Campos-Nonato, I., Espinosa, J., Flores, M.,
Barriguete, A., Rivera, J.A., 2009. A Energy and nutrient consumption in adults:
analysis of the Mexican National Health and Nutrition Survey 2006. Salud Publica
Mex. 51 (Suppl 4), S562–S573.
Barr, D.B., Landsittel, D., Nishioka, M., Thomas, K., Curwin, B., Raymer, J., Donnelly,
K.C., McCauley, L., Ryan, P.B., 2006. A survey of laboratory and statistical issues
related to farmworker exposure studies. Environ. Health Perspect. 114, 961–968.
http://dx.doi.org/10.1289/ehp.8528.
Barr, D.B., Wilder, L.C., Caudill, S.P., Gonzalez, A.J., Needham, L.L., Pirkle, J.L., 2004.
Urinary creatinine concentrations in the U.S. population: implications for urinary
biologic monitoring measurements. Environ. Health Perspect. 113, 192–200. http://
dx.doi.org/10.1289/ehp.7337.
Basu, A., Mitra, S., Chung, J., Guha Mazumder, D.N., Ghosh, N., Kalman, D., von
Ehrenstein, O.S., Steinmaus, C., Liaw, J., Smith, A.H., 2011. Creatinine, diet, mi-
cronutrients, and arsenic methylation in West Bengal, India. Environ. Health
Perspect. 119, 1308–1313. http://dx.doi.org/10.1289/ehp.1003393.
Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and
powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300. http://dx.doi.
org/10.2307/2346101.
Bourges, R.H., Casanueva, E., Rosado, J., 2009. Recomendaciones de Ingestión de
Nutrimentos para la población mexicana. Bases Fisiológicas. Médica Panamericana.
Brody, L.C., Conley, M., Cox, C., Kirke, P.N., McKeever, M.P., Mills, J.L., Molloy, A.M.,
O’Leary, V.B., Parle-McDermott, A., Scott, J.M., Swanson, D.A., 2002. A poly-
morphism, R653Q, in the trifunctional enzyme methylenetetrahydrofolate dehy-
drogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthe-
tase is a maternal genetic risk factor for neural tube defects: report of the Birth
Defects Research Group. Am. J. Hum. Genet. 71, 1207–1215. http://dx.doi.org/10.
1086/344213.
Camacho, L.M., Gutiérrez, M., Alarcón-Herrera, M.T., Villalba, M.D.L., Deng, S., 2011.
Occurrence and treatment of arsenic in groundwater and soil in northern Mexico and
southwestern USA. Chemosphere 83, 211–225. http://dx.doi.org/10.1016/j.
chemosphere.2010.12.067.
Concha, G., Vogler, G., Nermell, B., Vahter, M., 2002. Intra-individual variation in the
metabolism of inorganic arsenic. Int. Arch. Occup. Environ. Health 75, 576–580.
http://dx.doi.org/10.1007/s00420-002-0361-1.
Deng, F., Guo, X., Chen, L., Wang, Z., Zhang, K., 2007. Relationship between 5,10-me-
thylenetetrahydrofolate reductase genetic polymorphism and arsenic metabolism.
Beijing Da Xue Xue Bao. 39, 149–152.
Engström, K.S., Broberg, K., Concha, G., Nermell, B., Warholm, M., Vahter, M., 2007.
Genetic polymorphisms influencing arsenic metabolism: evidence from Argentina.
Environ. Health Perspect. 115, 599–605. http://dx.doi.org/10.1289/ehp.9734.
Engström, K.S., Nermell, B., Concha, G., Strömberg, U., Vahter, M., Broberg, K., 2009.
Arsenic metabolism is influenced by polymorphisms in genes involved in one-carbon
metabolism and reduction reactions. Mutat. Res. - Fundam. Mol. Mech. Mutagen.
667, 4–14. http://dx.doi.org/10.1016/j.mrfmmm.2008.07.003.
Galván-Portillo, M., Torres-Sánchez, L., Hernández-Ramírez, R.U., Anaya-Loyola, M.A.,
2011. [Validity and reproducibility of a food frequency questionnaire to estimate
folate intake in a Mexican population]. Salud Publica Mex. 53, 237–246.
Gamble, M.V., Liu, X., Ahsan, H., Pilsner, J.R., Ilievski, V., Slavkovich, V., Parvez, F.,
Chen, Y., Levy, D., Factor-Litvak, P., Graziano, J.H., 2006. Folate and arsenic meta-
bolism: a double-blind, placebo-controlled folic acid-supplementation trial in
Bangladesh. Am. J. Clin. Nutr. 84, 1093–1101.
Gamble, M.V., Liu, X., Ahsan, H., Pilsner, R., Ilievski, V., Slavkovich, V., Parvez, F., Levy,
D., Factor-Litvak, P., Graziano, J.H., 2005. Folate, homocysteine, and arsenic meta-
bolism in arsenic-exposed individuals in Bangladesh. Environ. Health Perspect. 113,
1683–1688.
Gamble, M.V., Liu, X., Slavkovich, V., Pilsner, J.R., Ilievski, V., Factor-Litvak, P., Levy, D.,
Alam, S., Islam, M., Parvez, F., Ahsan, H., Graziano, J.H., 2007. Folic acid supple-
mentation lowers blood arsenic. Am. J. Clin. Nutr. 86, 1202–1209.
Gilbert-Diamond, D., Cottingham, K.L., Gruber, J.F., Punshon, T., Sayarath, V., Gandolfi,
A.J., Baker, E.R., Jackson, B.P., Folt, C.L., Karagas, M.R., 2011. Rice consumption
contributes to arsenic exposure in US women. Proc. Natl. Acad. Sci. USA 108,
20656–20660. http://dx.doi.org/10.1073/pnas.1109127108.
Hall, M.N., Gamble, M.V., 2012. Nutritional manipulation of one-carbon metabolism:
effects on arsenic methylation and toxicity. J. Toxicol. 2012, 595307. http://dx.doi.
org/10.1155/2012/595307.
Hall, M.N., Liu, X., Slavkovich, V., Ilievski, V., Mi, Z., Alam, S., Factor-Litvak, P., Ahsan,
H., Graziano, J.H., Gamble, M.V., 2009a. Influence of cobalamin on arsenic meta-
bolism in Bangladesh. Environ. Health Perspect. 117, 1724–1729. http://dx.doi.org/
10.1289/ehp.0900734.
Hall, M.N., Liu, X., Slavkovich, V., Ilievski, V., Pilsner, J.R., Alam, S., Factor-Litvak, P.,
Graziano, J.H., Gamble, M.V., 2009b. Folate, cobalamin, cysteine, homocysteine, and
arsenic metabolism among children in Bangladesh. Environ. Health Perspect. 117,
825–831. http://dx.doi.org/10.1289/ehp.0800164.
Heck, J.E., Gamble, M.V., Chen, Y., Graziano, J.H., Slavkovich, V., Parvez, F., Baron, J.A.,
Howe, G.R., Ahsan, H., 2007. Consumption of folate-related nutrients and metabo-
lism of arsenic in Bangladesh. Am. J. Clin. Nutr. 85, 1367–1374.
Institute of Medicine, 2006. Dietary Reference Intakes: the Essential Guide to Nutrient
Requirements. Dietary Reference Intakes.
B. Gamboa-Loira et al.
Krajinovic, M., 2008. MTHFD1 gene: role in disease susceptibility and pharmacogenetics.
Pharmacogenomics 9, 829–832. http://dx.doi.org/10.2217/14622416.9.7.829.
Kuo, C.-C., Moon, K.A., Wang, S.-L., Silbergeld, E., Navas-Acien, A., 2017. The association
of arsenic metabolism with cancer, cardiovascular disease, and diabetes: a systematic
review of the epidemiological evidence. Environ. Health Perspect. 125, 87001.
http://dx.doi.org/10.1289/EHP577.
Li, L., Ekström, E.-C., Goessler, W., Lönnerdal, B., Nermell, B., Yunus, M., Rahman, A., El
Arifeen, S., Persson, L.A., Vahter, M., 2008. Nutritional status has marginal influence
on the metabolism of inorganic arsenic in pregnant Bangladeshi women. Environ.
Health Perspect. 116, 315–321. http://dx.doi.org/10.1289/ehp.10639.
Lindberg, A.-L., Kumar, R., Goessler, W., Thirumaran, R., Gurzau, E., Koppova, K.,
Rudnai, P., Leonardi, G., Fletcher, T., Vahter, M., 2007. Metabolism of low-dose in-
organic arsenic in a central European population: influence of sex and genetic
polymorphisms. Environ. Health Perspect. 115, 1081–1086. http://dx.doi.org/10.
1289/ehp.10026.
López-Carrillo, L., Gamboa-Loira, B., Becerra, W., Hernández-Alcaraz, C., Hernández-
Ramírez, R.U., Gandolfi, A.J., Franco-Marina, F., Cebrián, M.E., 2016. Dietary mi-
cronutrient intake and its relationship with arsenic metabolism in Mexican women.
Environ. Res. 151, 445–450. http://dx.doi.org/10.1016/j.envres.2016.08.015.
López-Carrillo, L., Hernández-Ramírez, R.U., Gandolfi, A.J., Ornelas-Aguirre, J.M.,
Torres-Sánchez, L., Cebrian, M.E., 2014. Arsenic methylation capacity is associated
with breast cancer in northern Mexico. Toxicol. Appl. Pharmacol. 280, 53–59. http://
dx.doi.org/10.1016/j.taap.2014.07.013.
Ludwig, M.L., Matthews, R.G., 1997. Structure-based perspectives on B12-dependent
enzymes. Annu. Rev. Biochem. 66, 269–313. http://dx.doi.org/10.1146/annurev.
biochem.66.1.269.
Moe, B., Peng, H., Lu, X., Chen, B., Chen, L.W.L., Gabos, S., Li, X.-F., Le, X.C., 2016.
Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species de-
termined using real-time cell sensing. J. Environ. Sci. 49, 113–124. http://dx.doi.org/
10.1016/j.jes.2016.10.004.
Navas-Acien, A., Francesconi, K.A., Silbergeld, E.K., Guallar, E., 2011. Seafood intake and
urine concentrations of total arsenic, dimethylarsinate and arsenobetaine in the US
population. Environ. Res. 111, 110–118. http://dx.doi.org/10.1007/s11103-011-
9767-z.Plastid.
O’Keefe, D.S., Su, S.L., Bacich, D.J., Horiguchi, Y., Luo, Y., Powell, C.T., Zandvliet, D.,
Russell, P.J., Molloy, P.L., Nowak, N.J., Shows, T.B., Mullins, C., Vonder Haar, R.A.,
Fair, W.R., Heston, W.D., 1998. Mapping, genomic organization and promoter ana-
lysis of the human prostate-specific membrane antigen gene. Biochim. Biophys. Acta
1443, 113–127.
Olteanu, H., Munson, T., Banerjee, R., 2002. Differences in the efficiency of reductive
activation of methionine synthase and exogenous electron acceptors between the
common polymorphic variants of human methionine synthase reductase.
23
Environmental Research 164 (2018) 18–23
Biochemistry 41, 13378–13385.
Peters, B.A., Hall, M.N., Liu, X., Neugut, Y.D., Pilsner, J.R., Levy, D., Ilievski, V.,
Slavkovich, V., Islam, T., Factor-Litvak, P., Graziano, J.H., Gamble, M.V., 2014.
Creatinine, arsenic metabolism, and renal function in an arsenic-exposed population
in Bangladesh. PLoS One 9, 1–21. http://dx.doi.org/10.1371/journal.pone.0113760.
Petrick, J.S., Ayala-Fierro, F., Cullen, W.R., Carter, D.E., Vasken Aposhian, H., 2000.
Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human
hepatocytes. Toxicol. Appl. Pharmacol. 163, 203–207. http://dx.doi.org/10.1006/
taap.1999.8872.
Porter, K.E., Basu, A., Hubbard, A.E., Bates, M.N., Kalman, D., Rey, O., Smith, A., Smith,
M.T., Steinmaus, C., Skibola, C.F., 2010. Association of genetic variation in cy-
stathionine-beta-synthase and arsenic metabolism. Environ. Res. 110, 580–587.
http://dx.doi.org/10.1016/j.envres.2010.05.001.
Scheer, J., Findenig, S., Goessler, W., Francesconi, K.A., Howard, B., Umans, J.G., Pollak,
J., Tellez-Plaza, M., Silbergeld, E.K., Guallar, E., Navas-Acien, A., 2012. Arsenic
species and selected metals in human urine: validation of HPLC/ICPMS and ICPMS
procedures for a long-term population-based epidemiological study. Anal. Methods 4,
406–413. http://dx.doi.org/10.1039/C2AY05638K.
Shen, H., Niu, Q., Xu, M., Rui, D., Xu, S., Feng, G., Ding, Y., Li, S., Jing, M., 2016. Factors
affecting arsenic methylation in arsenic-exposed humans: a systematic review and
meta-analysis. Int. J. Environ. Res. Public Health 13. http://dx.doi.org/10.3390/
ijerph13020205.
Spratlen, M.J., Gamble, M.V., Grau-Perez, M., Kuo, C.C., Best, L.G., Yracheta, J.,
Francesconi, K., Goessler, W., Mossavar-Rahmani, Y., Hall, M., Umans, J.G., Fretts,
A., Navas-Acien, A., 2017. Arsenic metabolism and one-carbon metabolism at low-
moderate arsenic exposure: Evidence from the Strong Heart Study. Food Chem.
Toxicol. 105, 387–397. http://dx.doi.org/10.1016/j.fct.2017.05.004.
Steinmaus, C., Carrigan, K., Kalman, D., Atallah, R., Yuan, Y., Smith, A.H., 2005a. Dietary
intake and arsenic methylation in a U.S. population. Environ. Health Perspect. 113,
1153–1159. http://dx.doi.org/10.1289/ehp.7907.
Steinmaus, C., Moore, L.E., Shipp, M., Kalman, D., Rey, O.A., Biggs, M.L., Hopenhayn, C.,
Bates, M.N., Zheng, S., Wiencke, J.K., Smith, A.H., 2007. Genetic polymorphisms in
MTHFR 677 and 1298, GSTM1 and T1, and metabolism of arsenic. J. Toxicol.
Environ. Health, Part A 70, 159–170. http://dx.doi.org/10.1080/
15287390600755240.
Steinmaus, C., Yuan, Y., Kalman, D., Atallah, R., Smith, A.H., 2005b. Intraindividual
variability in arsenic methylation in a U.S. population. Cancer Epidemiol. Biomark.
Prev. 14, 919–924. http://dx.doi.org/10.1158/1055-9965.EPI-04-0277.
Tseng, C.H., 2009. A review on environmental factors regulating arsenic methylation in
humans. Toxicol. Appl. Pharmacol. http://dx.doi.org/10.1016/j.taap.2008.12.016.
Vahter, M., 2002. Mechanisms of arsenic biotransformation. Toxicology 181–182,
211–217.