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Effects of N-acetyl-l-cysteine on target sites of hydroxylated fullerene-induced cytotoxicity in isolated rat hepatocytes

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Abstract

The effects of N-acetyl-l-cysteine (NAC) on cytotoxicity caused by a hydroxylated fullerene [C60(OH)24], which is known a nanomaterial and/or a water-soluble fullerene derivative, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C60(OH)24 at a concentration of 0.1 mM caused time (0–3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, and reduced glutathione (GSH) and protein thiol levels, as well as the accumulation of glutathione disulfide and malondialdehyde (MDA), indicating lipid peroxidation. Despite this, C60(OH)24-induced cytotoxicity was effectively prevented by NAC pretreatment ranging in concentrations from 1 to 5 mM. Further, the loss of mitochondrial membrane potential (MMP) and generation of oxygen radical species in hepatocytes incubated with C60(OH)24 were inhibited by pretreatment with NAC, which caused increases in cellular and/or mitochondrial levels of GSH, accompanied by increased levels of cysteine via enzymatic deacetylation of NAC. On the other hand, severe depletion of cellular GSH levels caused by diethyl maleate at a concentration of 1.25 mM led to the enhancement of C60(OH)24-induced cell death accompanied by a rapid loss of ATP. Taken collectively, these results indicate that pretreatment with NAC ameliorates (a) mitochondrial dysfunction linked to the depletion of ATP, MMP, and mitochondrial GSH level and (b) induction of oxidative stress assessed by reactive oxygen species generation, losses of intracellular GSH and protein thiol levels, and MDA formation caused by C60(OH)24, suggesting that the onset of toxic effects is at least partially attributable to a thiol redox-state imbalance as well as mitochondrial dysfunction related to oxidative phosphorylation.

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Abbreviations

DEM:

Diethyl maleate

DCHF-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DNP-NAC:

2,4-Dinitrophenyl S-conjugate of N-acetyl-l-cysteine

DMSO:

Dimethyl sulfoxide

GSH:

Glutathione

GSSG:

Glutathione disulfide

HEPES:

N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)

MDA:

Malondialdehyde

MPT:

Mitochondrial permeability transition

NAC:

N-acetyl-l-cysteine

ROS:

Reactive oxygen species

MMP:

Mitochondrial membrane potential

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Authors and Affiliations

  1. Division of Toxicology, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-0073, Japan

    Yoshio Nakagawa, Akiko Inomata, Akio Ogata & Dai Nakae

  2. Division of Environmental Health, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-0073, Japan

    Toshinari Suzuki

  3. Division of Food Additives, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-0073, Japan

    Kazuo Nakajima

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  1. Yoshio Nakagawa
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  2. Toshinari Suzuki
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  3. Kazuo Nakajima
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  4. Akiko Inomata
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  5. Akio Ogata
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Correspondence to Yoshio Nakagawa.

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Nakagawa, Y., Suzuki, T., Nakajima, K. et al. Effects of N-acetyl-l-cysteine on target sites of hydroxylated fullerene-induced cytotoxicity in isolated rat hepatocytes. Arch Toxicol 88, 115–126 (2014). https://doi.org/10.1007/s00204-013-1096-3

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