NATIONAL CHIAYI UNIVERSITY

Department of Microbiology, Immunology & Biopharmaceuticals

Analysis on urinary bladder cancer of human and mice, chronically exposed to

Arsenic in drinking water.

Advisor:Yi-Wen Liu, PhD. Professor

Speaker:Mezbahul HaqueMaster’s Student

Date: 2014. 05.17

Introduction

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Arsenic is a chemical element with symbol As and atomic number 33. Arsenic occurs in many minerals, usually in conjunction with sulfur and

metals, and also as a pure elemental crystal. It was first documented by Albertus Magnus. Arsenic is a naturally occurring element that exists in the environment in

a number of different forms, each with its own unique physical, chemical,

and toxicological characteristics. Arsenic is considered as the

group -1 carcinogen.

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Arsenic

http://en.wikipedia.org/wiki/Arsenic

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Arsenic-induced carcinogenicityInduces MAPK signal transduction, which activates transcription factors such as AP-1 and NF-kB to alter various gene expression profile.Kinase activation, which mediates several downstream signaling pathways such as integrin, Src, Rho, Grb2, EGFR, ERK, and cadherins. These pathways are involved in cell adhesion, cell migration, cell survival, cell cycle & carcinogenesis. Arsenic provokes proliferation of bladder epithelial cells and upregulates proto-onco-gene expression such as c-fos, c-jun, and EGR-1, which may collectively contribute to bladder cancer.

Toxicol Int. 2011 Jul-Dec; 18(2): 87–93.

A major exposure for humans to arsenic occurs through the ingestion of drinking water contaminated with inorganic arsenic.

Inorganic arsenic, most often in trivalent form (arsenite, AsIIIi ) or pentavalent form (arsenate, AsVi), is the most abundant form of arsenic in nature, and is commonly present in soil, water, and food.

Inorganic arsenic is easily absorbed into the blood and taken up by cells in tissues, primarily the liver, where it undergoes a series of reductions and oxidations.

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Inorganic Arsenic

S. Suzuki et al. / Toxicology 299 (2012) 155– 159

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Inorganic Arsenic Carcinogenesis

Cellular damage derived from arsenic biotransformation can occur through generation of reactive oxygen species (ROS), And through epigenetic mechanisms: changes in DNA methylation patterns (by depletion of cellular pools of methyl group), histone modification, and altered expression of microRNAs (miRNAs).

Journal of Toxicology Volume 2011, Article ID 431287, 13 pages

S-adenosylmethionine (SAM) Glutathione (GSH).

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Inorganic Arsenic Methylation

Arsenic methyltransferase (AS3MT) catalyzes the oxidative methylation of arsenite using s-adenosylmethionine (SAM) as the methyl donor, forming methylarsonic acid (MMAV), and s-adenosylhomocysteine (SAH).

MMAV is reduced to methylarsonous acid (MMAIII) before a subsequent oxidative methylation step yielding dimethylarsinic acid (DMAV) and SAH.

Journal of Toxicology, Volume 2012 (2012), Article ID 595307, 11 pages

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Organic Arsenic

Dimethylarsinic acid (DMAV)Monomethylarsonic acid (MMAV)

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water is carcinogenic to the skin, urinary bladder and lungs of humans and possibly other tissues. The inorganic arsenic is then methylated to the organic pentavalent arsenicals, monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV), which are more easily execrated in the urine.Trivalent forms of these methylated arsenicals are highly toxic to a variety of tissues, and are now considered to be toxicologically active and possibly intermediates in the induction of cancer in humans and in animal models.

S. Suzuki et al. / Toxicology 299 (2012) 155– 159S.M. Cohen et al. / Toxicology and Applied Pharmacology 222 (2007) 258–263

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DMA Dimethylarsinic acid (DMAV) (also called Cacodylic acid) is the chemical compound with the formula (CH3)2AsO2H. Derivatives of cacodylic acid, cacodylates, were frequently used as herbicides.

Dimethylarsinic acid (DMAV)

Health effects: DMAV is highly toxic by ingestion, inhalation, or skin contact. Once thought to be a byproduct of inorganic arsenic detoxification, it is now believed to have serious health consequences of its own. It has been shown to be teratogenic in rodents, most

http://en.wikipedia.org/wiki/Cacodylic_acid

often damage embryonic development but also fetal fatality at high doses. It has been shown to be genotoxic in human cells, causing apoptosis and also decreased DNA production and shorter DNA strands. While not itself a strong carcinogen, cacodylic acid does promote tumors in the presence of carcinogens in organs such as the kidneys and liver.

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DMA is the reactive organic intermediate. Possible mechanisms of arsenic-induced cytotoxicity include

oxidative stress secondary to free radical formation, interaction with cellular proteins via reaction with sulfhydryl groups, and/or depletion of cellular glutathione.

Because of the high doses required to induce rat bladder tumors and the unique metabolism and kinetics of DMAV, it is highly unlikely that DMAV poses a carcinogenic risk at anticipated exposures in humans.

DMAV carcinogenesis in the rat should be modeled using a nonlinear dose-response relationship, based on a margin of exposure.

Mechanism of DMA

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Effect of DMAV in Male and Female

Toxicology and Applied Pharmacology 222 (2007) 258–263

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Effects of dietary administration of DMAV

In a 2-year bioassay involving dietary administration of DMAV to male and female rats, the incidences of hyperplasia and urothelial tumors were increased. It is evident that there is a significantly greater response in females compared to males, with a statistically significantly increased incidence of bladder tumors occurring only in the females at 100 ppm, and a significant increase of hyperplasia at 40 ppm of the diet.

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Mode of action for arsenical-induced urinary bladder carcinogenesis.

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Carcinogenic Effect of AsIII

S. Suzuki et al. / Toxicology 261 (2009) 41–46

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Histopathology of urothelial bladder epithelia treated by ASIII

Urothelial bladder epithelia in control rat (A) and treated with AsIII (B). Simple hyperplasia was seen in (B).

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Carcinogenic Effect of AsIII in comparison with control

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Carcinogenic Effect of AsIII in comparison with control

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Carcinogenic Effect of AsV

S. Suzuki et al. / Toxicology 299 (2012) 155– 159

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Carcinogenic Effect of AsV in comparison with control

female rats

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Causing hyperplasia by the treatment with DMAV

Chem. Res. Toxicol. 2002, 15, 1150-1157

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Cell surface change with the treatment of DMA

Dimethylarsinic acid (DMAV) is carcinogenic to the rat urinary bladder when administered at high doses in the diet or drinking water. At a dietary dose of 100 ppm (íg/g), it produces cytotoxicity within 6 h and increased proliferation (hyperplasia) by 7 days of administration. Chem. Res. Toxicol. 2002, 15, 1150-1157

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In Vitro Cytotoxicity of various arsenic

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Arsenic Metabolism & Excretion

Critical Reviews in Toxicology, 36:99–133, 2006

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Disposition and Intracellular Metabolism of Trivalent Versus Pentavalent Inorganic Arsenic

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Metabolites found in the urine

48 hours after an exogenously administered dose of DMAV. Dose 50 mg/kg body weight; 4.0% of metabolites excreted was reported to be Asi. Dose 40 mg As/kg BW-DMAv ; 57% of total dose excreted in the urine. Dose 40 mg As/kg BW-DMAv ; 69% of total dose excreted in the urine. Dose 0.1 mg As/kg BW-DMAv ; 84% of total dose excreted in the urine.

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Metabolism of different arsenic compounds

Urine of humans four days after exogenous ingestion of 500 μg arsenic as either sodium arsenite (Asi), MMAV, or DMAV. a, 45.1% of total dose excreted in urine; b, 78.3% of total dose excreted in urine; c, 75.1% of total dose excreted in urine.

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BBN with DMAV

Cancer Letters 134 (1998) 29±36

Bromodeoxyuridine

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Metabolites in Urine when DMA with BBN

WhenNCI–Black–Reiter (NBR) rats were exposed for 4 weeks to drinking water containing 0.05% (BBN), followed by exposure for 32 weeks to drinking water containing 100 ppm DMAV,

the concentrations of arsenic metabolites measured in urine were: 31.1 mg/L DMAV, 17 mg/L TMAO, 7.4 mg/L of two unidentified arsenic compounds, 0.15 mg/L arsenobetaine (AsBe), 0.09 mg/L Asvi , 0.05 mg/L MMAV,

In a similar experiment that exposed only 50 ppm DMAV in the drinking water for 32 weeks, the concentrations of urinary metabolites were 10.9 mg/L DMAV, 5.7 mg/L TMAO, 0.3 mg/L AsBe, 1.3 mg/L AsIII i , and 1.2 mg/L unidentified metabolites.

MMA AsVi Unknown TMAO DMA0

10

20

30

40

50 ppm100 ppm

Cancer Letters 134 (1998) 29±36

pp

m

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Conclusion

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The high doses required to induce rat bladder tumors and the unique metabolism and kinetics of DMAV. Dimethylarsinic acid (DMAV) is carcinogenic to the rat urinary bladder, but not in mice.DMA can act as cocarcinogen with the BBN to cause hyperplasia to rat bladder. The trivalent form of arsenic exhibits greater genotoxic effects than the pentavalent counterparts as it could be easily taken up by the cells.

Major notification

Objective: With my research project I propose to address, Can arsenic as DMA and BBN damage the tissue more in male or

female? Which protein or factors are involved in this mechanism?

Animal and treatment: Male and female C57BL/6 homozygous total 60 mice in 3 groups,

each group contains 10 mice in both male & female. For treatment, control, BBN (300 ppm), BBN & DMA (100/150

ppm) 8/10 weeks for whole groups feeding with water twice in a week.

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My Proposal

Control BBN BBN & DMA BBN & DMA (HC)

Male 8/10 8/10 8/10 8/10

Female

8/10 8/10 8/10 8/10

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