Chronic arsenic exposure increases TGFalpha concentration in bladder urothelial cells of Mexican populations environmentally exposed to inorganic arsenic

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Abstract

Inorganic arsenic (iAs) is a well-established carcinogen and human exposure has been associated with a variety of cancers including those of skin, lung, and bladder. High expression of transforming growth factor alpha (TGF-α) has associated with local relapses in early stages of urinary bladder cancer. iAs exposures are at least in part determined by the rate of formation and composition of iAs metabolites (MAsIII, MAsV, DMAsIII, DMAsV). This study examines the relationship between TGF-α concentration in exfoliated bladder urothelial cells (BUC) separated from urine and urinary arsenic species in 72 resident women (18–51 years old) from areas exposed to different concentrations of iAs in drinking water (2–378 ppb) in central Mexico. Urinary arsenic species, including trivalent methylated metabolites were measured by hydride generation atomic absorption spectrometry method. The concentration of TGF-α in BUC was measured using an ELISA assay. Results show a statistically significant positive correlation between TGF-α concentration in BUC and each of the six arsenic species present in urine. The multivariate linear regression analyses show that the increment of TGF-α levels in BUC was importantly associated with the presence of arsenic species after adjusting by age, and presence of urinary infection. People from areas with high arsenic exposure had a significantly higher TGF-α concentration in BUC than people from areas of low arsenic exposure (128.8 vs. 64.4 pg/mg protein; p < 0.05). Notably, exfoliated cells isolated from individuals with skin lesions contained significantly greater amount of TGF-α than cells from individuals without skin lesions: 157.7 vs. 64.9 pg/mg protein (p = 0.003). These results suggest that TGF-α in exfoliated BUC may serve as a susceptibility marker of adverse health effects on epithelial tissue in arsenic-endemic areas.

Introduction

Inorganic arsenic (iAs) is naturally occurring and ubiquitously present in the environment. iAs in drinking water is the main source of human exposure. Epidemiologic data have shown that chronic exposure of humans to iAs compounds is associated with cardiovascular diseases, liver and kidney injury, diabetes mellitus, neurologic disorders and an increased incidence of liver, lung, skin and bladder cancer (ATSDR, 1999).

The relationship between iAs exposure and bladder cancer has been well documented (National Research Council, 1999). However, the mechanism by which iAs induces cancer has not been thoroughly characterized. Laboratory studies have shown that the metabolic conversions of iAsIII and iAsV in biological systems yield metabolites for which toxic and carcinogenic potentials differ significantly from those of the parent compounds. iAs is enzymatically methylated in human tissues to mono- and dimethylated metabolites that contain trivalent arsenic (AsIII) or pentavalent arsenic (AsV). A recently identified arsenic methyltransferase (AS3MT) is the key enzyme in this pathway (Waters et al., 2004). Several studies have shown that methylated trivalent arsenicals are more acutely toxic than iAsIII and are more potent than iAsIII as enzyme inhibitors, cytotoxins, and genotoxins (Styblo et al., 2000, Mass et al., 2001, Gomez et al., 2005). In addition, exposures to low concentrations of either MAsIII or DMAsIII induce cell proliferation and production of growth promoting cytokines in normal human keratinocytes (Vega et al., 2001). Moreover, exposure of iAs and its metabolites has been linked to activation of AP-1 and NF-kappaB (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways (Drobna et al., 2003, Hughes and Kitchin, 2006).

Increased transforming growth factor alpha (TGF-α) production has previously been reported in normal human keratinocytes treated with micromolar concentration of iAsIII (Germolec et al., 1996, Germolec et al., 1997) and in the skin of mice exposed to iAsIII in drinking water (Germolec et al., 1998).

In a population study in Bangladesh, urinary levels of TGF-α correlated with both urinary total As (TAs) and the occurrence of iAs-associated skin lesions (Do et al., 2001). In addition, the appearance of high TGF-α expression has been associated with local relapses in early stages of bladder cancer (Thogersen et al., 2001).

Therefore, in the present study, we analyzed the relationship among TGF-α concentration in exfoliated bladder urothelial cell (BUC) separated from urine and urinary arsenic species in people environmentally exposed to iAs in an arsenic-endemic area of Mexico. Furthermore, we investigated the association between the TGF-α levels and the presence of arsenic-related skin lesions.

Section snippets

Chemicals and reagents

Arsenic acid disodium salt (Na2HAsVO4), sodium m-arsenite (NaAsIIIO2), both > 99% pure, and dimethylarsinic acid [DMAsV; as (CH3)2AsVO(OH); 98% pure] were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methylarsonic acid (MAsV) disodium salt [CH3AsVO(ONa)2; 99% pure] was obtained from Ventron (Danvers, MA, USA). The trivalent methylated arsenicals methyloxoarsine (MAsIIIO; CH3AsIIIO) and iododimethylarsine of DMAsIII [DMAsIII; (CH3)2AsIII] were synthesized by W.R. Cullen (University of

Results

The study population characteristics, exposure variables, and effect markers are presented in Table 1. In this population (n = 72) all the subjects were women between 18 and 51 years old. A statistically significant positive correlation was found between the log-transformed TGF-α concentration in exfoliated cells and TAs concentration in urine collected from Zimapan residents (Fig. 1; r = 0.44, p < 0.0001). The average TGF-α concentration was significantly higher in individuals with high iAs exposure

Discussion

A variety of studies indicate that the TGF-α plays an important role in the modulation of differentiation and proliferation cells. Moreover, altered cellular responsiveness to growth factors is one of the factors involved in carcinogenesis. Epidemiological studies had shown that humans develop tumors in urinary bladder after chronic drinking water iAs exposure (Chiou et al., 1995, Hopenhayn-Rich et al., 1996, Steinmaus et al., 2006).

The goal of this study was to characterize the urinary pattern

Acknowledgments

This study was supported by Mexican Council for Science and Technology (Conacyt), grant 50097-M. We greatly appreciate the personnel from the Sanitary Jurisdiction in the Municipality of Zimapán, Hidalgo, Mexico for their help in the field study. The authors acknowledge Becton Dickinson for their generous donation of sterile containers for urine. The technical assistance of Eliud A. Garcia-Montalvo, Araceli Hernandez-Zavala, Luz C. Sánchez-Peña, Rachel Patterson and Angel Barrera-Hernández is

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    Portions of the data included in this paper were presented in poster session at the 45th Annual Conference of The Society of Toxicology, March 2006, San Diego, CA.

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