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Trang 1Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam
Tetsuro Agusaa,b, Hisato Iwatab,⁎ , Junko Fujiharaa, Takashi Kunitoc, Haruo Takeshitaa, Tu Binh Minhd,
a
Department of Legal Medicine, Shimane University Faculty of Medicine, Enya 89-1, Izumo 693-8501, Japan
b
Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
c
Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan
d
Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Vietnam National University, T3 Building, 334 Nguyen Trai Street, Thanh Xuan District, Hanoi, Vietnam
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 14 September 2009
Revised 29 October 2009
Accepted 6 November 2009
Available online 13 November 2009
Keywords:
Arsenic
Glutathione S-transferase ω1 (GSTO1)
GST ω2 (GSTO2)
GST π1 (GSTP1)
GST μ1 (GSTM1)
GST θ1 (GSTT1)
Genetic polymorphism
Vietnam
To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GSTω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1 (GSTT1) wild/null There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population GSTO1 Glu155del hetero type showed higher urinary concentration of AsVthan the wild homo type Higher percentage of DMAVin urine of GSTM1 wild type was observed compared with that of the null type Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from AsVto
AsIII Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population
© 2009 Elsevier Inc All rights reserved
Introduction
Inorganic arsenic (IA) is known to be a carcinogenic chemical in
human Arsenic contamination in groundwater is one of the most serious
where several areas do not have a public water supply system as yet
Since 2001, our research group has investigated arsenic pollution in the
groundwater and residents from Vietnam and Cambodia (Agusa et al.,
1930μg/l) in the groundwater exceeding 10 μg/l of WHO water standard
exposed to high levels of arsenic through the water consumption
In human, IA ingested through the drinking water and food is
metabolized to dimethyl arsenic Two pathways are hypothesized to
account for the metabolism of IA: a classical scheme consists of a
series of reductions and oxidations coupled with methylations
concept, the reductive methylation by interaction with binding proteins (Hayakawa et al., 2005; Naranmandura et al., 2006) In the biotransformation process, two enzymes, arsenic (+ 3 oxidation
(GSTO), are required in a variety of animals including human
detoxify xenobiotics by catalyzing the conjugation with reduced glutathione GST superfamily includes seven classes,α, μ, ω, π, θ, σ,
GST superfamily (Board et al., 2000) Among GSTO isoforms, GSTO1 is involved in the reduction activities of arsenate (AsV), monomethy-larsonic acid (MMAV), and dimethylarsinic acid (DMAV) (Zakharyan
reduction of MMAVand DMAV, but its activity of DMAVreductase was remarkably lower than that of GSTO1 (Schmuck et al., 2005)
It is anticipated that there is a large variation in susceptibility to toxic effect by IA among individuals and ethnics, depending on the difference
in IA metabolism (Vahter, 2000) Polymorphism(s) in the genes that are responsible for the metabolism of arsenic compounds may contribute to
⁎ Corresponding author Fax: +81 89 927 8172.
E-mail address: iwatah@agr.ehime-u.ac.jp (H Iwata).
0041-008X/$ – see front matter © 2009 Elsevier Inc All rights reserved.
Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / y t a a p
Trang 2the variability in biotransformation of IA (Loffredo et al., 2003; Vahter,
genotypes and haplotypes in AS3MT, which catalyzes the methylation of
arsenite (AsIII) and monomethylarsonous acid (MMAIII) (Lin et al., 2002;
urinary arsenical profile in a Vietnamese population (Agusa et al., 2008,
polymorphisms in GSTO1 and O2 to arsenic metabolism by in vitro
assays (Tanaka-Kagawa et al., 2003; Whitbread et al., 2003; Schmuck et
with biotransformation of arsenic (Chiou et al., 1997; Kile et al., 2005;
Marcos et al., 2006; Zhong et al., 2006; Lin et al., 2007; McCarty et al.,
distribution of gene polymorphisms in GSTs and their relation to the
arsenic metabolism in Vietnamese In the present study, we investigated
whether genetic polymorphisms in the members of GST superfamily,
GSTO1, GSTO2, GSTM1, GSTP1, and GSTT1, can affect arsenic metabolism
in residents from the Red River Delta, Vietnam The co-influence of
genetic polymorphisms in GSTs and other factors (sex, age, body mass
index (BMI), arsenic level in drinking water, and AS3MT genotypes) on
the accumulation and metabolism of arsenic was also examined
Materials and methods
previous work (Agusa et al., 2009b) Well water (n = 28), human hair
(n = 99), urine (n = 100), and blood (n = 100) samples were
randomly collected in March 2006 from Hoa Hau (HH) and Liem
Thuan (LT) in Ha Nam Province, which is located in the Red River Delta, Vietnam The informed consent was obtained from all the participants, and also this study was approved by the Ethical Committee of Ehime University, Japan Data on concentrations of total arsenic in water and human hair, and arsenic compounds in urine (Agusa et al., 2009b), and cumulative arsenic exposure level are
estimated from the As level in groundwater (mg/L), year of tube-well usage (year), annual ingestion rate of groundwater (182.5 days/year), and daily water consumption (3 L/day) All samples were kept at−25
°C in a freezer of the Environmental Specimen Bank (es-BANK) in Ehime University (Tanabe, 2006) until the following analyses
Analyses of arsenic The analytical method of arsenic was described
in more detail elsewhere (Agusa et al., 2009b) Total arsenic (TA) in water and human hair samples was analyzed with an inductively coupled plasma-mass spectrometer (ICP-MS; HP-4500, Hewlett-Packard, Avondale, PA, USA) using internal calibration method
AsIII, and AsVin urine sample were measured with a high performance liquid chromatograph (HPLC; LC10A Series, Shimadzu, Kyoto, Japan)— ICP-MS To separate each arsenical, a polymer-based anion exchange column (Shodex Asahipak ES-502N 7C) was used with 15 mM citric acid (pH 2.0 with nitric acid) (Agusa et al., 2009b)
In the present study, sum of all arsenic compounds and inorganic arsenic (AsIII+ AsV) detected in urine sample are denoted as SA and
IA, respectively Percentages of AB, AsIII, AsV, MMAV, DMAVand IA to
SA in human urine were denoted as %AB, %AsIII, %AsV, %MMAV, %DMAV and %IA, respectively Urinary creatinine was determined at SRL, Inc (Tokyo, Japan) Concentrations of arsenic compounds in urine were expressed asμg As/g on a creatinine basis Because it is considered
Table 1
Information on water and human samples from Hoa Hau and Liem Thuan in Vietnam.
Groundwater
Used period (years) a
TA (μg/l) b 368 (163–502, and 2120 (an outlier)) 1.4 (0.7–6.8) b0.001 c Filtered water
Drinking water e
Subjects
Residential time (years) a
Cumulative arsenic exposure (mg) b 306 (17.6–12800) 4.8 (1.7–13.4) b0.001 c Hair TA (μg/g) b
0.351 (0.028–2.94) 0.232 (0.068–0.690) b0.001 c Urinary SA (μg/g creatinine) b 92.6 (45.2–365) 97.9 (38.6–397) N0.05 c
Urinary DMA V
Urinary MMA V
Urinary As V
(%) a
Abbreviations: TA, total arsenic; BMI, body mass index (weight (kg)/height (m) 2
); SA, sum of arsenic compounds; AB, arsenobetaine; DMA V
, dimethylarsinic acid; MMA V
, monomethylarsonic acid; As III
, arsenite; As V
, arsenate.
a Arithmetic mean and range.
b Geometric mean and range.
c
t-test.
d
χ 2
test.
e filter, filtered water instead of raw groundwater is assumed to be consumed.
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T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362
Trang 3Table 2
Information on primer sequences, annealing temperatures, restriction enzymes, and fragment sizes of the amplified products and frequencies of allele and genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam Gene
symbol
Amino acid
position
rs number a Functional
context
Nucleotide change
Amino acid change
(°C) Restriction enzyme
frequency (%)
Genotype frequency (%)
5′-TGATAGCTAGGAGAAATAATTAC-3′
Ala/Asp: 68, 186, 254 Asp/Asp: 254
Ala: 0.900 Asp: 0.100
Ala/Ala: 0.810 Ala/Asp: 0.180 Asp/Asp: 0.010
5′-GAATTTACCAAGCTAGAGGAGGT-3′
5′-GACCAAGCCAGCATTTTAGG-3′
5′-GCAGGACAGCTTTCTGCTTT-3′
Glu/del: 200, 315, 472 del/del: 315, 472
Glu: 0.955 del: 0.045
Glu/Glu: 0.910 Glu/del: 0.090
5′-CAAAGCGCTTGGCTGTTGATGTC-3′
Glu/Lys: 154, 266, 420 Lys/Lys: 420
Glu: 1.000 Lys: 0
Glu/Glu: 1.000 Glu/Lys: 0 Lys/Lys: 0
5′-CAAAGCGCTTGGCTGTTGATGTC-3′
Thr/Asn: 114, 221, 335 Asn/Asn: 114, 221
Thr: 1.000 Asn: 0
Thr/Thr: 1.000 Thr/Asn: 0 Asn/Asn: 0
5′-CATGCAACCTGAACCTTGGT-3′
Ala/Asp: 116, 192, 308 Asp/Asp: 308
Ala: 1.000 Asp: 0
Ala/Ala: 1.000 Ala/Asp: 0 Asp/Asp: 0
5′-GAGGGACCCCTTTTTGTACC-3′
Asn/Asp: 63, 122, 185 Asp/Asp: 63, 122
Asn: 0.780 Asp: 0.220
Asn/Asn: 0.610 Asn/Asp: 0.340 Asp/Asp: 0.050
5′-TGAGGGCACAAGAAGCCCCT-3′
Ile/Val: 84, 92, 176 Val/Val: 84, 92
Ile: 0.845 Val: 0.155
Ile/Ile: 0.690 Ile/Val: 0.310 Val/Val: 0
5′-GTTGGGCTCAAATATACGGTGG-3′
multiplex PCR
Wild: 210 Null: 0
Wild: 0.580 Null: 0.420
5′-TCACCGGATCATGGCCAGCA-3′
multiplex PCR
Wild: 473 Null: 0
Wild: 0.700 Null: 0.300
a
Rs numbers were cited from NCBI SNP Database (http://www.ncbi.nlm.nih.gov/projects/SNP/).
Trang 4that AsV, IA, and MMAVare metabolized to AsIII, MMA, and DMA,
respectively, in the human body, concentration ratios of AsIII/AsV(III/
used as metabolic index for each arsenical
Genotyping of polymorphisms in GSTO1, GSTO2, GSTP1, GSTM1, and
using a QIAamp DNA mini kit (Qiagen, Chatworth, CA) Reference
sequence of each GST was based on the DNA Data Bank of Japan (DDBJ);
accession numbers of GSTO1, GSTO2, GSTP1, GSTM1, and GSTT1 are
AY817669, AY191318, AY324387, BC024005, and AB057594,
respec-tively DNA was subjected to PCR amplification in 10 μl reaction mixture
containing GoTaq®Green Master Mix (Promega, Madison, WI, USA) and
individual primer pairs corresponding to each mutation of GSTO1
Thr217Asn (threonine to asparagine substitution at amino acid base
217), GSTO1 Ala236Val, GSTP1 Ile105Val, GSTM1 wild/null, and GSTT1
wild/null For the detection of GSTO1 Glu155del and Glu208Lys, the
(15 mM Tris–HCl, 50 mM KCl, pH 8.0), 1.5 mM MgCl2, 0.5μM of each
Applied Biosystems, CA, USA) (Fujihara et al., 2007) A PCR mixture
consisting of PCR buffer, 1.5 mM MgCl2, 0.4μM of each primer, 250 μM
dNTP, and 1 U Takara EX Taq DNA polymerase (Takara, Kyoto, Japan)
was used for genotyping of GSTO1 Ala140Asp and GSTO2 Asn142Asp
Glu208Lys, Thr217Asn, and Ala236Val, GSTO2 Asn142Asp, and GSTP1
Ile105Val were detected by PCR restriction fragment length
polymor-phism (PCR-RFLP) using restriction enzymes Genetic polymorpolymor-phisms in
GSTT1 and M1 (wild or null) were identified by allele specific multiplex
PCR includingβ-globin as a positive control (Sreeja et al., 2005) GSTO1
Glu155del was detected by applying the method of confronting
two-pair primers analysis (CTPP) (Fujihara et al., 2007) The PCR products, which were treated with restriction enzyme or were not treated, were separated in 8% polyacrylamide gel by electrophoresis (300 V, 15 min) and were detected by silver staining The genotyping was carried out in duplicate The representativeness of nucleotide sequences for each
Biosystems Foster, CA, USA) Information on primers, annealing temper-ature, restricted enzyme, and fragment size is presented inTable 2
(ver-sion 5.0, SAS® Institute, Cary, NC, USA), SPSS (ver(ver-sion 12, SPSS, Chicago, IL, USA), and EXCEL Toukei (Version 6.05, Esumi Co., Ltd., Tokyo, Japan) were used for statistical analyses One half of the value
of the respective limits of detection were substituted for those values below the limit of detection and used in statistical analysis Normality
Smirnov's one sample test To adapt parametric analyses, data which showed non-normal distribution was log-transformed Stud-ent's t-test and Tukey–Kramer test were conducted to find differences
in arsenic levels and compositions in the hair and urine among allele
checking sample size distribution in each group category To assess the factors affecting arsenic levels and composition in the urine and hair, and metabolic capacity of arsenic, a stepwise multiple regression analysis was executed Genetic polymorphisms in GST superfamily and cumulative As exposure level as well as SNPs in AS3MT, age, sex,
relationships with arsenic levels and compositions in our previous
analysis as independent variables To apply the regression model, nominal variables (sex and genotypes of GST superfamily and AS3MT)
Fig 1 Frequencies of genotypes of GST superfamily in the Vietnamese and the HapMap populations ( http://www.hapmap.org/index.html.ja ) NA means no available data VN: Vietnamese in this study, CHB (H): Han Chinese in Beijing, China, CHD (D): Chinese in Metropolitan Denver, Colorado, JPT (J): Japanese in Tokyo, Japan, GIH (G): Gujarati Indians in Houston, Texas, MEX (M): Mexican ancestry in Los Angeles, California, CEU (C): Utah residents with Northern and Western European ancestry from the CEPH collection, TSI (T):
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Trang 5Table 3
Concentrations (geometric mean and range) of arsenic compounds in urine and total arsenic in hair for each genotype of GST superfamily in residents from Hoa Hau and Liem Thuan in Vietnam.
Gene and
genotype
MMA V
As III
As V
GSTO1 Ala140Asp
Ala/Asp +
Asp/Asp
GSTO1 Glu155del
GSTO2 Asn142Asp
Asn/Asp +
Asp/Asp
GSTP1 Ile105Val
GSTM1
GSTT1
Abbreviations: AB, arsenobetaine; DMA V , dimethylarsinic acid; MMA V , monomethylarsonic acid; As III , arsenite; As V , arsenate; IA, inorganic arsenic (As III + As V ); SA, sum of arsenic compounds; TA, total arsenic; NA, not available.
⁎ pb0.05.
⁎⁎⁎ pb0.001.
Trang 6were transformed to dummy variables The multicollinearity of independent variables was assessed by calculating the variance
inflation factor (VIF) Linkage disequilibrium and haplotype of SNPs
in GSTO1 were assessed by Haploview (version 4.0, Day Lab at the
indicate statistical significance
Results and discussion Distribution of genetic polymorphisms in GST superfamily Allele and genotype frequencies for each gene are shown in
Thr217Asn, and Ala236Val in this population and thus the data on these mutations were not included for further analysis No mutation homozygotes were found for GSTO1 Glu155del and GSTP1 Ile105Val
significant linkage disequilibrium and haplotype
Genotype frequencies for GST superfamily in this Vietnamese and
GSTO1 Ala140Asp, GSTO1 Glu208Lys, GSTO2 Asn142Asp, and GSTP1 Ile105Val genotypes in the Vietnamese population were similar to those in Asian populations such as CHB (H) (Han Chinese in Beijing, China groups), CHD (D) (Chinese in Metropolitan Denver, Colorado), and JPT (J) (Japanese in Tokyo, Japan) However, even among the Asian populations, frequencies of I/I and I/V genotypes for GSTP1 Ile105Val in the Vietnamese and Chinese (CHB (H) and CHD (D)) were largely different from those in the Japanese (JPT (J)) In addition, although mutant homo types of GSTO1 Glu208Lys and GSTP1 Ile105Val were reported in other populations, no such substitution was detected in the Vietnamese There was no mutation in GSTO1 Ala236Val in the Vietnamese Similarly, low mutation frequencies were reported in other populations except for the Mexican Genotype frequencies of GSTO1 Ala140Asp, Glu155del, and Glu208Lys, and GSTO2 Asn142Asp in the Japanese and Mongolian that have been reported in our previous studies (Fujihara et al., 2007; Takeshita
For GSTO1 Glu155del and Thr217Asn, GSTM1 wild/null, and GSTT1 wild/null, the genotype frequencies of the present study were compared with those in previous studies Ninety-one percent of the Vietnamese analyzed in the present study was the wild type of GSTO1
studies (Whitbread et al., 2003; Fujihara et al., 2007; Paiva et al.,
No mutation allele was detected for GSTO1 Thr217Asn in the present study population (Table 2) Up to date, there is no available information on GSTO1 Thr217Asn mutation in any population, although this type has been registered in NCBI SNP Database as
with the wild type using in vitro assay, although the relevance of this variant in arsenic metabolism is still unclear (Schmuck et al., 2005) Null type frequencies of GSTM1 and T1 in Vietnamese were 42% and 30%, respectively (Table 2) In the review article byMo et al
13.1–54.5% in Caucasians, 46.7% in American-Africans, and 26.9% in Africans for GSTM1 null type and 41.9–52% in Asians, 11.1–28.6% in Caucasians, 26.7% in American-Africans, and 36.6% in Africans for GSTT1 null type Compared with the results in the Asian populations, frequency of GSTT1 deletion in the Vietnamese was lower, while proportion of GSTM1 null type was within the range On the contrary, GSTT1 null type frequency in Taiwanese was 26% (Chiou et al., 1997), which was similar to that in the Vietnamese.Lin et al (2007)found
GSTM1 Wild
GSTT1 Wild
V ,
V ,
III ,
V ,
III +A
V );III/V,
III /As
V ;
V /IA;
V /MMA
V ;
⁎p
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T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362
Trang 7Table 5
Stepwise multiple regression of arsenic concentrations and compositions in urine and hair against sex, age, BMI, TA in drinking water, and polymorphisms in GST superfamily and AS3MT in residents from Hoa Hau and Liem Thuan in Vietnam.
%As III
%As V
GSTP1 Ile105Val (0 = Ile/Ile, 1 = Ile/Val) 0.273 0.002
Abbreviations: AB, arsenobetaine; DMA V
, dimethylarsinic acid; MMA V
, monomethylarsonic acid; As III
, arsenite; As V
, arsenate; IA, inorganic arsenic (As III
+ As V
); III/V, As III
/As V
; M/I, MMA V
/IA; D/M, DMA V
/MMA V
; SA, sum of arsenic compounds; TA, total arsenic; BMI, body mass index (weight (kg)/height (m) 2
).
Trang 8that prevalence of GSTM1 and T1 deletion type were 71% and 35%,
respectively, in Hmong in China, and 42% and 47%, respectively, in Han
in China Therefore, there may be large variations in the frequencies of
GSTM1 and T1 null type even in the Asian populations
Potential effects of genetic polymorphisms in GST superfamily on arsenic
concentration and metabolism in Vietnamese
Since the hair can be a good indicator of chronic arsenic exposure
status, while arsenic level and speciation in the urine can represent
recent exposure and metabolism of arsenic in humans, respectively,
we measured arsenic in the hair and urine to examine their
relationships to genetic polymorphisms in GST isoforms Although
arsenic concentration in drinking water as well as cumulative arsenic
relationships between arsenic metabolism and TA in drinking water
or cumulative arsenic exposure (pN0.05) Thus, the data of all donors
were pooled for analysis of the relationship between arsenic and
genotypes of GSTs Concentrations and compositions of urinary
arsenicals and metabolic index for each genotype of GST superfamily
are shown inTables 3 and 4 Because the sample sizes of the mutation
homo types of GSTO1 Ala140Asp (n = 1) and GSTO2 Asn142Asp
(n = 5) were small for the statistical analysis, these mutations were
included in hetero + homo type group for the further discussion
Concentrations of AsV(p = 0.018) and IA (p = 0.050) in the urine of
than those with the wild type (Table 3) Furthermore, urinary %IAs was
also low in hetero type of GSTO1 Glu155del (p = 0.039,Table 4) On the
other hand, higher M/I value of GSTO1 Glu155del heterozygote was
thiol-transferase and GSH conjugation in mutation of GSTO1 Glu155del than
the wild type in an in vitro study Hence, it might be suggested that if
methylation of IA by AS3MT occurs in the form of arsenic-glutathione as
GSTO1 Glu155del mutation protein enhances the methylation of IAs
However, since the difference in M/I value between them was not so
large in the present study and also the association between GSTO1
Glu155del andfirst methylation index was not obvious in the multiple
regression analysis as shown later (Table 5), further studies are needed
to confirm the association.Schmuck et al (2005)reported that GSTO1
Glu155del protein expressed in Escherichia coli exhibited higher MMAV
and DMAVreductase activities than the wild type Because MMAIIIand
DMAIIIwere not analyzed in the urine sample of the present study,
relationship between genetic polymorphisms in GSTO1 and methylated
trivalent arsenicals remains unclear for the subjects In the case of
human studies, there was no relationship between GSTO1 Glu155del
and urinary arsenical in Mexican (Meza et al., 2005) and Chilean (Paiva
substitu-tion in Mexicans (n = 75) who were exposed to arsenic through the
consumption of drinking water showed unusual urinary arsenic profile;
one who has GSTO1 Glu155del and Glu208Lys substitutions had high %
Ala140Asp substitution in addition to these two substitutions displayed
high %AsIIIand low %DMAVin the urine compared to the rest of the study
population In the present study, no such haplotype was found
As for GSTM1, concentration (p = 0.018,Table 3) and composition
(p = 0.028,Table 4) of AB in the null type were significantly greater
than those in the wild type, although the reason was not clear
1989), the relationship for %AB might be partially due to a negative
(p = 0.009) was low in GSTM1 null compared with that in the wild type (Table 4), implying that GSTM1 null may affect %DMAVin the Vietnamese However, the result was not consistent with those in
urinary %IA for the null genotype of GSTM1 in Taiwanese In the workers occupationally exposed to arsenic in Chile (Marcos et al.,
between GSTM1 genotype and methylation ratios in Bangladesh people with skin lesions No relation of GSTM1 genotype to arsenic level in toenail was reported in subjects from arsenic-endemic region
in Bangladesh (Kile et al., 2005), whereas arsenic concentrations in urine and hair were high in GSTM1 null carriers exposed to indoor combustion of high arsenic coal in China (Lin et al., 2007)
We found that GSTP1 Ile105Val homozygote had higher concen-trations of AB, MMAV, AsIII, IA and SA, and %AsIIIand %IA in the urine than the heterozygote, whereas the opposite trend was observed for %
Ile105Val hetero type was significantly higher than that in the wild type (p = 0.002,Table 4) Urinary III/V in the heterozygote of GSTP1 Ile105Val was about half of that in the wild homozygote (p = 0.027,
erythrocyte of GSTP1 Ile105Val wild type was higher than that of the mutation type in the healthy Chinese, but AsVreduction activity
variant homo type showed a slightly (p = 0.085) higher %DMAVin the urine than the wild type in Chileans (Marcos et al., 2006), the GSTP1 Ile105Val variant homo type was not observed in the population of the present study
There were no significant associations of GSTO1 Ala140Asp, GSTO2 Asn142Asp, and GSTT1 wild/null with concentrations and composi-tions of arsenic in the urine and hair (pN0.05,Tables 3 and 4) The result for the polymorphism of GSTO1 Ala140Asp was consistent with the results reported in several in vitro studies; activities of MMAV
activities of thioltransferase and GST conjugation between GSTO1
According to the study ofMukherjee et al (2006), protein expression level of GSTO1 Ala140Asp mutation was similar to that of the wild type, although the activity was not measured In human studies, there was no association of GSTO1 Ala140Asp with arsenic metabolism in the Mexican (Meza et al., 2005), Hungarian, Romanian, and Slovakian
Like GSTO1, GSTO2 has six exons that are separated within 7.5 kb nucleotide length on chromosome 10q24.3 The homology of amino
compared to GSTO1 Furthermore, GSTO2 Asn142Asp polymorphism does not affect those reductase activities This result may support our results on GSTO2 Asn142Asp which showed no association with arsenic metabolism (Tables 3 and 4)
Argentina (Steinmaus et al., 2007), and Chinese (Lin et al., 2007) subjects, GSTT1 wild/null polymorphism had no relevance to urinary arsenic in Vietnamese of the present study (Tables 3 and 4) However,
drinking water, %DMAVin urine of subjects with GSTT1 null type was higher than that in the wild type Also, the interaction between GSTT1 wild type and secondary methylation ratio might increase the risk of
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T Agusa et al / Toxicology and Applied Pharmacology 242 (2010) 352–362
Trang 9of As in the nail of GSTT1 null type carriers in Bangladesh.
difference (Table 1, pb0.001) and was positively correlated with TA
cumulative arsenic exposure level (pb0.001) Therefore, the
associ-ation of hair TA level with polymorphisms in GST superfamily was
assessed by ANCOVA, having correction with concentration of TA in
drinking water and cumulative arsenic exposure as covariates, but no
significant results were found (pN0.05,Table 3) Similar to our results,
human hair and toenail and polymorphisms of GSTM1 and T1 in the
contrast,Lin et al (2007) found high concentrations of arsenic in
both hair and urine of GSTM1 null carriers who were exposed to
arsenic from indoor coal combustion in Southwest Guizhou, China,
and arsenic levels in hair and urine
Potential effects of genetic polymorphisms in GST superfamily and AS3MT,
sex, age, BMI, arsenic level in drinking water, and cumulative arsenic
exposure level on arsenic concentration and metabolism in Vietnamese
In the previous study (Agusa et al., 2009b), we investigated the
influence of various factors on arsenical concentration and
composi-tion in the urine and hair in this Vietnamese populacomposi-tion, including age,
sex, BMI, occupation, residential years, alcohol and smoking habits,
and TA level in drinking water, and 13 SNPs in AS3MT such as a4602g
(a to g substitution at nucleotide base 4602), t4740c, t5913c, a6144t,
g7395a, t8979a, g12390c, t12590c, c14215t, t14458c, t35587c,
g35991a, and g37853a, and revealed significant effects of sex, age,
BMI, concentration of TA in drinking water, and several SNPs in
AS3MT Therefore, those factors might co-affect the results on
relationships between arsenic and GST genotypes examined in this
regression analyses was conducted to assess whether the
distribu-tions of urinary arsenic metabolites were dependent on sex, age, BMI,
selenium, and gene polymorphisms of GSTO1, AS3MT, and
methyle-netetrahydrofolate reductase (MTHFR) Here, we attempted to detect
the effects of sex, age, BMI, concentration of arsenic in water,
cumulative arsenic exposure status, and polymorphisms in GST
superfamily and AS3MT on arsenic level and profile in the Vietnamese
using a stepwise multiple regression analysis Prior to the analysis, we
numbers of each sex, age and BMI in each genotype of GST
superfamily (pN0.05) The multicollinearity of independent variables
for multiple regression analysis was examined by calculating the VIF
The result showed that there was no significant multicollinearity (all
VIFs were less than 10)
The result of stepwise multiple regression analysis is shown in
of univariate analysis in the present study (Tables 3 and 4) and our
previous results (Agusa et al., 2009b): associations between GSTO1
Ile105Val and concentrations of AB, MMAV, AsIII, IA and SA, %DMAV,
%AsIII, %IA, III/V and M/I in urine; between GSTM1 wild/null and %DMAV
in urine; between age and M/I; between sex and concentrations of
MMAV, AsIII and IA, %MMAV, %AsIII, %IA and D/M in urine, and
concentration of TA in hair; between BMI and concentrations of
DMAV, AsV, IA and SA in urine, and TA in hair; between concentrations
of TA in drinking water and hair; between AS3MT t4740c and
between AS3MT t14458c and M/I in urine; between AS3MT g35991a
GSTM1 wild/null with D/M in urine; of age with DMAVlevel, SA level and %DMAVin urine, and with TA level in hair; of sex with %AB and M/I in urine; of AS3MT t4740c with AsIIIlevel and M/I in urine; of AS3MT t5913c with AsVlevel and D/M in urine; of AS3MT a6144t with
%MMAVin urine; of AS3MT g7395a with AsVlevel and %AsVin urine;
of AS3MT c14215t with III/V in urine; of AS3MT t35587c with AB and
Factors influencing metabolic capacity of arsenic were also character-ized as follows: lower III/V in c/c homo type of AS3MT c14215t and Ile/Val hetero type of GSTP1 Ile105Val; lower M/I in a/a homo type of AS3MT g35991a, Ile/Ile homo type of GSTP1 Ile105Val, t/t homo type
of AS3MT t5913c, t/t homo type of AS3MT t14558c, male, younger people, and t/t homo type of AS3MT t4740c; and lower D/M in g/g homo type of AS3MT g12390c, male, null type of GSTM1, and c/c + t/
c types of AS3MT t5913c
Although we statistically detected co-effects of genetic polymorph-isms in GST superfamily as well as sex, age, BMI, TA in drinking water and AS3MT genotypes on the arsenic concentration and metabolism, adjusted determination coefficient (Radj2 ) in multiple regression equa-tion was not so high (up to 0.344 for M/I) (Table 5) Thus, it seems possible that there are other significant factors to account for the variation in arsenic level and metabolism Other SNP sites in GSTO1
determined in the present study, might also be involved in the variation.Mukherjee et al (2006)found large variations of protein expression levels of GSTO1 Cys32Tyr and GSTO2 Val41Ile, Cys130Tyr, and L158Ile as well as those investigated in the present study (GSTO1 Ala140Asp, Glu155del, Glu105Lys, and Ala236Val, and GSTO2 Asn142Asp), indicating the possibility of significant variations in the expression levels and catalytic functions among polymorphisms in GSTO1 and GSTO2 Also, recombinant Arg173Trp and Thr306Ile
activity and immunoreactive protein compared to the wild type
quite low (up to 5%) in African-Americans, Caucasian-Americans, Han
relationship among the population at small sample scale Many SNPs
in the intron regions of GSTO1, GSTO2 and AS3MT have been reported
Database, and the linkage between the intronic polymorphisms and metabolism of arsenic is of interest Alternatively, other genetic polymorphisms such as MTHFR Ala222Val and Glu429Ala, which may associate with arsenic metabolism in human (Lindberg et al., 2007;
considered Further studies at larger scale are required to detect more rigid relationships between genetic polymorphisms and arsenic
selenium and vitamins C and E are known to modify toxicity of arsenic
metabolism for residents in developing countries
In summary, we suggest here that genotypes of GSTO1 Glu155del, GSTP1 Ile105Val, and GSTM1 wild/null affect arsenic metabolism in a Vietnamese population Interestingly, GSTP1 Ile105Val polymorphism,
on which there is little information in association with arsenic
with urinary arsenic In addition to the genetic polymorphisms of GST superfamily, sex, age, BMI, TA in the drinking water, and various SNPs
in AS3MT were also related to arsenic level and profile in the
indicating the associations between genetic polymorphisms of GSTs and arsenic metabolism in a Vietnamese population
Trang 10We wish to thank Dr A Subramanian, CMES, Ehime University,
Japan for critical reading of the manuscript The authors express
thanks to the staff of the CETASD, Hanoi University of Science and Dr
H Sakai, CMES (current affiliation; Laboratory of Structure-Function
Biochemistry, Department of Chemistry, Faculty of Science, Kyushu
University, Japan) for their help in sample collection We also
acknowledge Ms H Touma and Ms N Tsunehiro, staff of the
es-BANK, CMES for their support in sample management and Ms Y Fujii,
Department of Legal Medicine, Shimane University Faculty of
Shimane University Faculty of Medicine, Japan) for her technical
assistant This study was supported by Japan Society for the
Promotion of Science (JSPS) for the cooperative research program
under the Core University Program between JSPS and Vietnamese
Academy of Science and Technology (VAST) Financial support was
also provided by grants from Research Revolution 2002 (RR2002; to
H.I.) Project for Sustainable Coexistence of Human, Nature and the
20221003; to S.T.) and (A) (No 19209025; to H.T.) from JSPS, and
21st Century and Global COE Programs from the Ministry of
Education, Culture, Sports, Science, and Technology (MEXT), Japan
and JSPS The award of the JSPS Post Doctoral Fellowship for
Researchers in Japan to T Agusa (No 207871) is also acknowledged
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