Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes

doi:10.1016/j.taap.2004.05.004

Toxicology and Applied Pharmacology

Volume 201, Issue 2, 1 December 2004, Pages 166-177

https://doi.org/10.1016/j.taap.2004.05.004 Get rights and content

Abstract

Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, including methylarsenicals (MAs) and dimethylarsenicals (DMAs). To assess interindividual variation in the capacity of liver to metabolize iAs, we examined the metabolic fate of arsenite (iAs^III) in normal primary human hepatocytes obtained from eight donors and cultured under standard conditions. Methylation rates, yields, and distribution of arsenicals were determined for hepatocytes exposed to 0.3–30 nmol of iAs^III/mg of protein for 24 h. Although the accumulation of arsenic (As) by cells was a linear function of the initial concentration of iAs^III in culture, the concentration of As retained in cells varied several fold among donors. DMAs was the major methylated metabolite found in cultures exposed to low concentrations of iAs^III; at higher concentrations, MAs was always predominant. Maximal rates for methylation of iAs^III were usually attained at 3 or 9 nmol of iAs^III/mg of protein and varied about 7-fold among donors. For most donors, the methylation rate decreased at the highest iAs^III concentrations. MAs was the major methylated metabolite retained in cells regardless of exposure level. DMAs was the major methylated metabolite found in medium. The interindividual differences in rates for iAs^III methylation were not strictly associated with variations in basal mRNA levels for cyt19, an As-methyltransferase. Analysis of the coding sequence of cyt19 identified one heterozygote with Met287Thr mutation in a single allele. Thus, genetic polymorphism of cyt19 along with other cellular factors is likely responsible for interindividual differences in the capacity of primary human hepatocytes to retain and metabolize iAs^III.

Section snippets

Primary cultures of human hepatocytes

Hepatocytes were isolated as previously described Hamilton et al., 2001, LeCluyse et al., 2000 from normal hepatic tissue obtained from the Tissue Procurement and Analysis Core Facility at the University of North Carolina at Chapel Hill. Samples of normal liver were collected with patient consent during resections performed at North Carolina Memorial Hospital of the University of North Carolina at Chapel Hill or were obtained from non-transplantable donor livers. Study protocols for tissue

Functional characterization of cultured primary human hepatocytes

In preliminary studies, we examined the metabolic integrity and redox status of freshly isolated cells suspended in supplemented Williams' medium E (7.5 × 10⁵ viable cells per ml) and of adherent cells cultured for up to 5 days in the same medium (7.5 × 10⁵ viable cells per well in 1 ml medium). The effect of time in culture on the capacity of cells to metabolize iAs was also examined. Freshly plated monolayers contained a relatively large number of condensed apoptotic cells. These cells

Discussion

The work reported here examines qualitative and quantitative aspects of the variation of iAs metabolism in human liver using cultured primary hepatocytes from eight donors. Primary cultures of human hepatocytes have been widely used to study xenobiotic metabolism in vitro LeCluyse, 2001, Meunier et al., 2000. Under optimized culture conditions, primary hepatocytes retain differentiated structure and exhibit metabolic properties of normal liver. For example, in cultured primary human

Acknowledgements

S.B.W. is a postdoctoral fellow in the Curriculum in Toxicology, University of North Carolina at Chapel Hill and is supported by Training Grant T901915 of the U.S. Environmental Protection Agency-University of North Carolina Toxicology Research Program. M.S. is supported by NIH grant ES09941, US EPA Cooperative Agreement CR829522, and a Clinical Nutrition Research Center Grant DK 56350. This manuscript has been reviewed in accordance with the policy of the National Health and Environmental

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Arsenic, a metalloid and naturally occurring element, is one of the most abundant elements in the earth's crust. Water is contaminated by arsenic through natural sources (underground water, minerals and geothermal processes) and anthropogenic sources such as mining, industrial processes, and the production and use of pesticides. Humans are exposed to arsenic mainly by drinking contaminated water, and secondarily through inhalation and skin contact. Arsenic exposure is associated with the development of vascular disease, including stroke, ischemic heart disease and peripheral vascular disease. Also, arsenic increases the risk of tumors of bladder, lungs, kidneys and liver, according to the International Agency for Research on Cancer and the Food and Drug Administration. Once ingested, an estimated 70–90% of inorganic arsenic is absorbed by the gastrointestinal tract and widely distributed through the blood to different organs, primarily to the liver, kidneys, lungs and bladder and secondarily to muscle and nerve tissue. Arsenic accumulates in the organs, especially in the liver. Its excretion mostly takes place through urination. The toxicokinetics of arsenic depends on the duration of exposure, pathway of ingestion, physicochemical characteristics of the compound, and affected biological species. The present review outlines of arsenic toxic effects focusing on different cancer types whit highest prevalence's by exposure to this metalloid and signaling pathways of carcinogenesis.

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¹: These authors contributed equally to the research described in this paper.

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Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes

Abstract

Section snippets

Primary cultures of human hepatocytes

Functional characterization of cultured primary human hepatocytes

Discussion

Acknowledgements

Methods Enzymol

Methods Enzymol

Toxicology

Chem.-Biol. Interact

J. Biol. Chem

Toxicol. Appl. Pharmacol

J. Biol. Chem

Eur. J. Pharm. Sci

J. Biol. Chem

Environ. Res

Anal. Biochem

Biochem. Pharmacol

Toxicol. Appl. Pharmacol

Toxicol. Appl. Pharmacol

J. Chromatogr., B

Biochem. Biophys. Res. Commun

Toxicol. Appl. Pharmacol

Toxicol. Lett

The influence of liver disease on the methylation of arsenite in humans

Arch. Toxicol

Aquaglyceroporin AQP9: solute permeation and metabolic control of expression in liver

Proc. Natl. Acad. Sci. U.S.A

The reaction of methylarsenicals with thiols: some biological implications

J. Inorg. Biochem

Transfer of arsenite from glutathione to dithiols: a model of interaction

Chem. Res. Toxicol

Pharmacokinetic modeling of arsenite uptake and metabolism in hepatocytes—Mechanistic insights and implications for further experiments

J. Pharmacokinet. Pharmacodyn

Abnormal methylation capacity in human liver cirrhosis

Int. J. Pharm. Res