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Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1

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Abstract

The main objectives of this work were to evaluate the effects of hydrogen sulfide on oxidative stress and cytotoxicity parameters in HepG2 cells and to assess the extent to which cytochrome P450 2E1 (CYP2E1) activity modulates the effects of hydrogen sulfide on oxidative stress and cytotoxicity. Sodium hydrosulfide (NaHS) caused time- and concentration-dependent cytotoxicity in both non-P450-expressing HepG2 cells (C34 cells) and CYP2E1-overexpressing HepG2 cells (E47 cells); however, NaHS-dependent cytotoxicity was higher in E47 than C34 cells. Cytotoxicity by NaHS in C34 and E47 cells was mainly necrotic in nature and associated with an early decrease in mitochondrial membrane potential. NaHS caused increased oxidation of lipophilic (C11-BODIPY581/591) and hydrophilic (DCFH-DA) probes only in E47 cells, at a time point prior to overt cytotoxicity. Trolox, an amphipathic antioxidant, partially inhibited both the cytotoxicity and the increased oxidative stress detected in E47 cells exposed to NaHS. Cell-permeable iron chelators and CYP2E1 inhibitors significantly inhibited the oxidation of C11-BODIPY581/591 in E47 cells in the presence of NaHS. NaHS produced lipid peroxidation and cytotoxicity in E47 cells supplemented with a representative polyunsaturated fatty acid (docosahexaenoic acid) but not in C34 cells; these effects were inhibited by α-tocopherol, a lipophilic antioxidant. These data suggest that CYP2E1 enhances H2S-dependent cytotoxicity in HepG2 cells through the generation of iron-dependent oxidative stress and lipid peroxidation.

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Abbreviations

C11-BODIPY581/591 :

4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid

C34:

HepG2 cell line derived after transfection with pCI-neo vector

CYP2E1:

Cytochrome P450 2E1

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DFO:

Deferoxamine mesylate

DIP:

α,α′-Dipyridyl

E47:

HepG2 cell line derived after transfection with pCI-neo vector containing the human CYP2E1 cDNA

MEM:

Minimal essential medium

MEMexps :

MEM supplemented 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 10 mM HEPES at pH 7.4

PITC:

Phenylisothiocyanate

PI:

Propidium iodide

Rhodamine 123:

R123

ROS:

Reactive oxygen species

TBARS:

Thiobarbituric acid reactive substances

Trolox:

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

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Acknowledgments

The authors are grateful to Dr. Arthur I. Cederbaum (Mount Sinai School of Medicine, New York, USA) for his gift of C34 and E47 cells and for critical reading of the manuscript. The authors are grateful to Dr. Thomas Goodwin, Dr. Liz Gron and Dr. Randall Kopper (Hendrix College, Conway, AR) for critical reading of the manuscript. This work was supported by Cottrell College Science Award #7854 from Research Corporation for Science Advancement, and NIH grant number P20 RR-16460 from the IDeA Networks of Biomedical Research Excellence Program of the National Center for Research Resources.

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  1. Chemistry Department, Hendrix College, 1600 Washington Avenue, Conway, AR, 72032, USA

    Andres A. Caro, Sarah Thompson & Jonathan Tackett

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  1. Andres A. Caro
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  2. Sarah Thompson
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Correspondence to Andres A. Caro.

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Caro, A.A., Thompson, S. & Tackett, J. Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1. Cell Biol Toxicol 27, 439–453 (2011). https://doi.org/10.1007/s10565-011-9198-2

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  • DOI: https://doi.org/10.1007/s10565-011-9198-2

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