Cytotoxicity and chromosomal aberrations induced by acrylamide in V79 cells: Role of glutathione modulators

Nuno G. Oliveira, Marta Pingarilho, Célia Martins, Ana Sofia Fernandes, Sofie Vaz, Vanda Martins, José Rueff, Jorge Francisco Gaspar

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Abstract

Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the Conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, Studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH. were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In Summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario. (C) 2009 Elsevier B.V. All rights reserved.
Original languageEnglish
Pages (from-to)87-92
Number of pages5
JournalMutation Research-Genetic Toxicology And Environmental Mutagenesis
Volume676
Issue number1-2
DOIs
Publication statusPublished - 2009

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Acrylamide
Chromosome Aberrations
Glutathione
Buthionine Sulfoximine
Esters
Cell-Free System
Poisons
Acetylcysteine
Fluorescent Dyes
Carcinogens
Heating
Culture Media
Oxidative Stress
Homeostasis
Carbohydrates

Keywords

  • Acrylamide
  • Chromosomal aberrations
  • Glutathione
  • Cytotoxicity

Cite this

@article{c3b195616b61428f8e67611b3a40256c,
title = "Cytotoxicity and chromosomal aberrations induced by acrylamide in V79 cells: Role of glutathione modulators",
abstract = "Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the Conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, Studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH. were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In Summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario. (C) 2009 Elsevier B.V. All rights reserved.",
keywords = "CULTURES, HEPG2 CELLS, GLYCIDAMIDE, Glutathione, MAMMALIAN-CELLS, V-79 CELLS, MONOETHYL ESTER, GENOTOXICITY, Acrylamide, CytotoxicityHUMAN LYMPHOID-CELLS, METABOLISM, Chromosomal aberrations, DEPLETION, Acrylamide, Chromosomal aberrations, Glutathione, Cytotoxicity",
author = "Oliveira, {Nuno G.} and Marta Pingarilho and C{\'e}lia Martins and Fernandes, {Ana Sofia} and Sofie Vaz and Vanda Martins and Jos{\'e} Rueff and Gaspar, {Jorge Francisco}",
year = "2009",
doi = "10.1016/j.mrgentox.2009.04.009",
language = "English",
volume = "676",
pages = "87--92",
journal = "Mutation Research-Genetic Toxicology And Environmental Mutagenesis",
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publisher = "Elsevier Science B.V., Inc",
number = "1-2",

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TY - JOUR

T1 - Cytotoxicity and chromosomal aberrations induced by acrylamide in V79 cells: Role of glutathione modulators

AU - Oliveira, Nuno G.

AU - Pingarilho, Marta

AU - Martins, Célia

AU - Fernandes, Ana Sofia

AU - Vaz, Sofie

AU - Martins, Vanda

AU - Rueff, José

AU - Gaspar, Jorge Francisco

PY - 2009

Y1 - 2009

N2 - Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the Conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, Studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH. were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In Summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario. (C) 2009 Elsevier B.V. All rights reserved.

AB - Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the Conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, Studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH. were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In Summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario. (C) 2009 Elsevier B.V. All rights reserved.

KW - CULTURES

KW - HEPG2 CELLS

KW - GLYCIDAMIDE

KW - Glutathione

KW - MAMMALIAN-CELLS

KW - V-79 CELLS

KW - MONOETHYL ESTER

KW - GENOTOXICITY

KW - Acrylamide

KW - CytotoxicityHUMAN LYMPHOID-CELLS

KW - METABOLISM

KW - Chromosomal aberrations

KW - DEPLETION

KW - Acrylamide

KW - Chromosomal aberrations

KW - Glutathione

KW - Cytotoxicity

U2 - 10.1016/j.mrgentox.2009.04.009

DO - 10.1016/j.mrgentox.2009.04.009

M3 - Article

VL - 676

SP - 87

EP - 92

JO - Mutation Research-Genetic Toxicology And Environmental Mutagenesis

JF - Mutation Research-Genetic Toxicology And Environmental Mutagenesis

SN - 1383-5718

IS - 1-2

ER -