ABSTRACT:

Background: Inorganic arsenic (iAs) is an environmental carcinogen to which millions of people are chronically exposed mainly via drinking water. After ingestion, it goes through a series of reduction and oxidative methylation steps in human body. Trivalent forms of arsenic such as monomethylarsonic acid (MMA) (III) and dimethylarsinic acide (DMA) (III) are considered as the most toxic forms among the metabolized arsenic species. Thus, metabolism of ingested arsenic plays an important role in determining arsenic toxicity, and previous studies reported that proportion of MMA in the total arsenic (%MMA) in urine is associated with a variety of diseases including cancer, diabetes, respiratory syndromes, skin lesions, and peripheral circulatory problems.

Substantial individual variation in the arsenic metabolism and toxicity has been recognized, although the causes of the variation remain to be elucidated. Along with other factors gender plays an important role in arsenic metabolism. Females have shown higher methylation efficiency than males in arsenic-exposed populations in various countries. Gender disparity has been also reported for many arsenic-exposure related diseases such as skin lesion, skin cancer, lung, bladder and kidney cancer and diabetes. The molecular and cellular mechanisms by which arsenic acts as a carcinogen have not been clearly elucidated, but lines of evidence suggest that induction of oxidative stress through the generation of reactive oxygen species (ROS) is one of the important mechanisms. No preceding studies have examined the gender difference in arsenic-induced oxidative stress.

Genetic polymorphisms in arsenic-metabolizing enzymes and DNA repair enzymes are among other factors involved in the metabolism and toxicity of arsenic. Importantly, associations of genetic polymorphism with several diseases depend on sex reported in previous studies. However, the effect of gender and genetic polymorphism on arsenic-induced oxidative stress as well as gender effect on association of genetic polymorphism with arsenic metabolism in arsenic-exposed population has not been evaluated before.

Objectives: This dissertation evaluated the role of gender in arsenic-induced oxidative stress among Bangladeshi arsenic-exposed population. The impact of polymorphisms in arsenic metabolism-related genes as well as those in DNA repair-related genes on arsenic metabolism and oxidative stress, respectively were also evaluated.

Methods: The study protocol was approved by the Ethical Review Committee of the Graduate School of Medicine, University of Tokyo, Japan. Two hundred twenty three Bangladeshi participants aged 18 to 70 years from arsenic-exposed (water As >10ppb) area were participated in this cross-sectional study. Ninety-six males and 126 females giving informed consent were the participants in this study. Water of the tube wells that they were using, urine and peripheral blood samples were collected and analyzed.

Urinary and water arsenic levels were determined by inductively-coupled plasma mass spectrometry (ICP-MS) equipped with a direct reaction cell (Agilent7500ce, Agilent). Arsenic speciation was performed by a HPLC-ICP-MS system. Urinary 8-Hydroxy-2'-deoxyguanosine (8-OHdG) and urinary 15-F(2t)-IsoP levels were determined with commercial ELISA kits.

Genotyping of studied SNPs were carried out by DigiTag2 multiplex PCR assay. Twenty-two SNPs of the following genes were studied: AS3MT, DNMT, MTHFR, GPX1, GSTO1, GSTO2, OGG1, APE1, XRCC1, XRCC3, ERCC5 and LIG4.

Pearson`s correlation, Student t test, Tukey-kramer post hoc, and multiple regression analysis used to evaluate the data. Data were analyzed using JMP statistical software (version-8).

Results: Urinary arsenic (u-As) had positive association with urinary oxidative stress markers- 8-OHdG (r= 0.60; p<0.001) and 15-F(2t)-IsoP (r= 0.46; p<0.001). Arsenic-exposed female had significantly higher 15-F2t-IsoP and %DMA (p<0.05) than males. Gender difference in u-As and urinary 8-OHdG was not found in this study group.

Four AS3MT polymorphisms were significantly associated with As metabolism in this study population (p ≦ 0.01). Exonic SNP of As3MT-rs11191439 significantly associated with arsenic metabolism. While three intronic AS3MT SNPs (rs3740393, rs3740390, and rs11191453) were significantly associated with arsenic metabolism in female group. None of intronic AS3MT polymorphisms affected arsenic methylation in arsenic-exposed male group. DNMT3b (rs2424913) SNP was associated with arsenic metabolism in females.

There were no significant differences in the urinary arsenic metabolites or oxidative stress markers within male or female subjects with different genotypes of MTHFR, GSTO1, GSTO2, OGG1, APE1, XRCC1, XRCC3, ERCC5 and LIG4. No DNA repair genes or arsenic metabolism related genes SNPs were associated with 8-OHdG concentrations in arsenic-exposed population. Three intronic AS3MT SNPs (rs3740393, rs3740390, and rs11191453) were significantly associated with 15-F2t-IsoP in male group.

Discussion: This dissertation reported that arsenic induced oxidative stress in arsenic-exposed population. It also confirmed previous studies that with increase of u-As, 8-OHdG levels increase and males have higher %MMA (p<0.01), indicated less methylation efficiency of arsenic than females. Although females have better methylation ability, females have higher lipid peroxidation than males that indicated As-induced oxidative stress has not depended on arsenic methylation, so other mechanism may be involved. This finding provides certain evidence for women exposed to higher arsenic being at higher risk than men in term of oxidative stress, which may be a leading mechanism for gender disparity in arsenic-related health effects.

Gender stratified analyses showed exonic SNPs of AS3MT-rs11191439 (T/C) hetero type had significantly lower %MMA and higher secondary methylation index (D/M) than TT homo type, especially among arsenic-exposed males. This result indicated that male subjects with hetero type (T/C) carrier had better methylation capability than homotype carrier. While, heterotype of intronic SNPs of AS3MT (rs3740393, rs3740390, and rs11191453) females carrier has lower %MMA indicated better methylation than homotype female carrier. Heterotype of intronic SNPs of AS3MT (rs3740393, rs3740390, and rs11191453) has association higher 15-F(2t)-IsoP in male group. Gender depended association of arsenic-metabolism related genes with arsenic metabolism and oxidative stress markers remain unknown.

This study confirmed previous studies that MTHFR, GSTO1 and GSTO2 SNPs has no association with As metabolism or oxidative stress. In contrast to previous studies this study reported that OGG1, APE1 and XRCC1 SNPs has no association with DNA repair. This gender depended association gene polymorphism with arsenic metabolism and oxidative stress may be another possible mechanism for gender disparity in arsenic-related health effects.

Conclusion: In conclusion, chronic arsenic exposure from drinking water may be related to induction of oxidative stress, as indicated by the increase in urinary 8-OHdG and 15-F(2t)-IsoP levels. The present study also provides evidence that females have higher arsenic metabolism as well as higher As-induced oxidative stress 15-F(2t)-IsoP levels, which may explain the difference in susceptibility to As-induced toxicity.

The present study identified four SNPs in AS3MT and one SNP in DNMT that may affect arsenic methylation in a Bangladeshi population. Hetero-type AS3MT (rs11191439) displayed a greater methylation index in an arsenic-exposed population, particularly in males. Intronic SNPs of AS3MT were associated with oxidative stress markers, particularly in males. Such a gender-specific association of As-metabolism gene polymorphism with altered u-As metabolites has never before been reported. Genotype associations with u-As metabolites as well as 15-F(2t)-IsoP concentrations may depend on exposure level and gender. Further analyses of the association of these genes with arsenic metabolism may help to define the significance of these polymorphisms as a genetic marker of susceptibility to oxidative stress in arsenic-exposed populations.

本学位論文は、2つの酸化ストレスマーカー8-hydroxy-2'-deoxyquanosine (8-OHdG)と15-F2t-isoprostane (13-F2t-IsoP)を用いて、ヒ素曝露された男性および女性の酸化ストレスを比較することにより、バングラデシュのヒ素曝露集団中のヒ素誘発酸化ストレスにおける性別の役割を評価した。また、ヒ素代謝関連の遺伝子、DNA損傷に関わるDNA修復関連遺伝子の遺伝多型も評価し、下記の結果を得ている。

1. 尿中ヒ素およびその代謝物

対象集団において、水中ヒ素濃度と尿中ヒ素濃度の間に正相関(r=0.55;p<0.001)が認められた。女性は男性より低い%MMA(monomethylarsonic acid / total arsenic) (p=0.04)および高い%DMA (dimethylarsinic acid / total arsenic) (p=0.006)と高い第2メチル化インデックスDMA/MMA(p=0.03)を示した。このことは、第2メチル化段階における女性の高いヒ素メチル化能を示唆した。尿中ヒ素代謝物比が尿中総ヒ素、性別、体格指標(BMI)、年齢に依存するかを多変量回帰分析で解析した。年齢、年齢2、BMI、BMI-性別相互作用、いずれの変数もヒ素代謝物比%iAs (inorganic arsenic)、%MMA、%DMAとも有意な関連がなかった。尿中総ヒ素は%MMAとのみ負の関連があり、%iAsまた%DMAとは関連を示さなかった。性別は有意に%DMA(女性>男性)と関連したが、%iAsと%MMAのモデルでは有意な関連はみられなかった。

2. 酸化ストレスマーカー

酸化ストレスのバイオマーカーである8-OHdGおよび15-F2t-IsoPは、対象集団では尿中ヒ素濃度と正に相関した(r=0.60;p<0.001およびr=0.46;p<0.001)。多変量重回帰分析では、尿中総ヒ素は8-OHdGに有意な効果を示したが、性別は8-OHdGに効果を示さなかった。性別と尿中総ヒ素は15-F2t-IsoPに効果があった。多変量重回帰分析において、尿中総ヒ素は男女とも酸化ストレスのバイオマーカーに対して有意な効果があった。しかしながら、%iAsは男性においてのみ15-F2t-IsoPに影響した。他の代謝物比(%iAsと%MMA)は女性において酸化ストレス・マーカーに効果がなかった。

3. 遺伝子分析

5つの単一塩基多型SNP(AS3MT-rs3740393、AS3MT-rs11191453、AS3MT-rs3740390、AS3MT-rs11191439、DNMT1a-rs10854076)が、尿中ヒ素代謝産物の間の統計的に有意な関連性を示した(Tukey-Kramer post-hoc test)。

性別に階層化された分析では、AS3MT-rs3740393、AS3MT-rs3740390、AS3MT-rs11191453の各 SNPが女性において%MMAにと関連を示した。さらに女性において、AS3MT-rs3740390は第2メチル化インデックス(DMA/MMA)と、DNMT1a-rs10854076は第1メチル化インデックス(MMA/iAs)と有意な関連があった。AS3MT-rs11191439を例外として、他のSNPは男性において代謝物比と関連がなかった。

性別に階層化された分析において、男性においてのみAS3MT-rs11191439異型接合性がより高い%MMAおよび第2メチル化インデックス(DMA/MMA)に関係していることが示唆された。対象集団において、As3MT-rs10748835、As3MT-rs1046778、DNMT1a-rs2228611、DNMT1a-rs7253062、DNMT3b-rs2424913、MTHFR-rs1801133、GSTO1-rs4925、GSTO2-rs2297235、GSTO2-rs156697と代謝パターンには関連が認められなかった。

性別によるDNA修復遺伝子多型および8-OHdGの相互作用もまた評価した。DNA修復遺伝子多型(OGG1、XRCC1-rs25487、XRCC1-rs1799782、ERCC5、APE1、LIG4)は、男性か女性のいずれにおいても8-OHdGと関連を示さなかった。更に、酸化的8-OHdG濃度と、ヒ素メチル化あるいは解毒関連遺伝子型の間の相関もみられなかった。DNMT3b-rs2424913 SNPのみが男性において8-OHdGと関連していた。

男性における15-F2t-IsoPについては、AS3MT-rs3740393、AS3MT-rs3740390、AS3MT-rs11191453 SNPは関連を示した。AS3MT-rs3740393およびAS3MT-rs3740390 SNPの異型遺伝子型を持つ男性は、同型遺伝子型より高い15-F2t-IsoPを示しが、AS3MT-rs11191453 SNPの異型遺伝子型を持った男性は、同型遺伝子型より低い15-F2t-IsoPを示した。女性において15-F2t-IsoP濃度とヒ素メチル化あるいは解毒関連遺伝子型には相関がみられなかった。

以上、本研究は、女性がより高いヒ素メチル化能を持ち、同様にヒ素誘発酸化ストレス15-F2t-IsoPが高いことを示した。これはヒ素誘発毒性の感受性の違いを説明し得る。本研究は、バングラデシュ集団においてヒ素メチル化に影響するかもしれないAS3MTの4つのSNPとDNMTの1つのSNPを同定した。異型接合体のAS3MT(rs11191439)は、特に男性において、ヒ素曝露対象集団においてメチル化インデックスが高かった。AS3MTのイントロンSNPは特に男性において酸化ストレスのマーカーと関連があった。このような尿中ヒ素代謝物を変えるヒ素代謝遺伝子多型の性別特異的な関連はいまだかつて報告がない。