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. Author manuscript; available in PMC: 2015 Nov 6.
Published in final edited form as: Met Ions Biol Med. 2008;10:419–424.

N-Acetyl-cysteine Protection Against Lead-Induced Oxidative Stress and Genotoxicity in Human Liver Carcinoma (HepG2) Cells

Clement G Yedjou 1, Daren Waters 1, Paul B Tchounwou 1,*
PMCID: PMC4636190  NIHMSID: NIHMS113144  PMID: 26549975

Abstract

The human liver carcinoma (HepG2) cells as well as other cell lines are particularly susceptible to oxidative damage, and it is therefore important to find agents that protect against this process. N-acetyl-cysteine (NAC) is the acetylated form of L-cysteine. It has an impressive list of protective effects including: antioxidant activity, decrease of the biologically effective dose of carcinogens, anti-inflammatory activity, immunological effects, inhibition of progression to malignancy and metastasis, and protection from the adverse effects of chemopreventive and chemotherapeutic agents. Previous studies in our laboratory have shown that lead nitrate induces cytotoxicity and oxidative stress to HepG2 cells in a dose-dependent manner. In this research, we hypothesized that the antioxidant, n-acetyl-l-cysteine attenuates oxidative stress and genotoxicity, and thereby provides cellular protection against lead toxicity. To this hypothesis, we performed the thiobarbituric acid test for lipid peroxidation and the microgel electrophoresis (comet) assay for genotoxicity. The results generated from the thiobarbituric acid test showed a significant reduction of lipid peroxidation by-product (malondialdehyde) in HepG2 cells co-exposed to NAC and lead nitrate compared to lead nitrate alone. Incubation of HepG2 cells with increasing concentrations of NAC decreased the amount of MDA formation progressively in lead nitrate-treated HepG2 cells. Data obtained from the comet assay indicated a strong dose-response relationship with regard to lead nitrate-induced genotoxic damage in HepG2 cells. However, the addition of NAC in vitro showed a significant reduction (p < 0.05) in the comet tail length, percentage of DNA cleavage, comet tail moment, as well as comet tail arm respectively in cells co-treated with NAC and lead nitrate. Findings from these studies demonstrated that NAC inhibits malondialdehyde (MDA) production and genotoxicity in lead nitrate-treated HepG2 cells in a dose-dependent manner. Under this in vitro condition, NAC was found to be effective in reducing MDA formation, cellular injury, and genotoxic damage in HepG2 cells exposed to lead nitrate.

INTRODUCTION

Lead is one of the most toxic heavy metals to living beings. In vitro and in vivo studies indicate that lead compounds are not directly genotoxic, but may cause genetic damage through various indirect mechanisms. These include inhibition of DNA synthesis and repair, oxidative damage, and interaction with DNA-binding proteins and tumor suppressor proteins (1, 2). Tests for genotoxicity have indicated that lead compounds cause chromosomal damage, induce chromosomal aberrations, micronuclei, and increased SCE (3, 4). N-Acetylcysteine (NAC), a potent antioxidant has used clinically for decades for the treatment of many diseases. It plays an important role in the production of glutathione, which provides intracellular defense against oxidative stress (5), and it participates in the detoxification of many molecules (6). During the last decade, numerous in vitro and in vivo studies have suggested that NAC has beneficial medicinal properties including inhibition of carcinogenesis, tumorigenesis, and mutagenesis, as well as the inhibition of tumor growth and metastasis (79). In light of the long history of therapeutic application of NAC, we suggest that use of this compound may be of interest in conditions where certain heavy metal-mediated forms of cell death and/or apoptosis via oxidative stress contribute significantly to disease.

Although NAC is an excellent scavenger of free radicals and chelator of heavy metal (10, 11), it remains unclear whether this compound affords cellular protection to HepG2 cells against lead genotoxicity. Hence, the present study was designed to elucidate whether exposure to NAC could modulates oxidative stress associated with lead nitrate genotoxicity in human hepatic carcinoma (HepG2) cells.

II. MATERIALS AND METHODS

Chemicals and Test Media

Reference solution (1000 ± 10 ppm) of lead nitrate (CAS No. 10099-74-8, Lot No. 981735-24) with a purity of 100% was purchased from Fisher Scientific (Fair Lawn, New Jersey). Dulbecco’s modified eagle’s medium (DMEM) was purchased from Life Technologies (Grand Island, New York). Fetal bovine serum (FBS), n-aceltyl-l-cysteine, phosphate buffered saline (PBS) were obtained from Sigma Chemical Company (St. Louis, MO).

Tissue Culture

Human liver carcinoma (HepG2) cells were grown in 96-well format plastic plates in DMEM supplemented with 10% FBS, and 1% penicillin-streptomycin. Cells were maintained in a 5% CO2 incubator at 37°C for 48 hours according to previous experiment in our laboratory (12, 14).

Lipid peroxidation Assay

Malondialdehyde (MDA) is formed during lipid peroxidation. The concentration of MDA was measured by using a lipid peroxidation assay kit (Calbiochem-Novabiochem, San Diego, CA). Briefly, 2 × 106 HepG2 cells/mL (untreated as a control) and (co-treated with NAC at 0.125, 0.25, 0.5 mM and 0.5 mM NAC plus 30 μg/mL lead nitrate) were cultured in a total volume of 10 ml growth medium for 48 hrs. After the incubation period, cells were collected in 15 mL tube, followed by low-speed centrifugation. The cell pellets were re-suspended in 0.5 ml of Tris-HCl, pH 7.4, and lysed using a sonicator (W-220; Ultrasonic, Farmingdale, NY) under the conditions of duty cycle 25% and output control 40% for 5 sec on ice. The protein concentration of the cell suspension was determined using a protein assay kit (BioRad, Hercules, C.A.). A 200μl aliquot of the culture medium or 2 mg of cell lysate protein was assayed for MDA according to the lipid peroxidation assay kit protocol (Calbiochem-Novabiochem, San Diego, CA).The absorbance of the sample was monitored at 586 nm, and the concentration of MDA was determined from a standard curve.

Cell Treatment for Comet/Genotoxicity Assay

Cells were counted (10,000 cells/well) and re-suspended in media with 10% FBS. Aliquots of 100 μL of the cell suspension were placed in each well of 96 plates, treated with 100μl aliquot of either media, NAC, NAC plus lead nitrate, or lead nitrate alone, respectively and incubated in a 5% CO2 at 37°C for 48 hrs. After incubation, the cells were centrifuged, washed with PBS free calcium and magnesium, and re-suspended in 100 μL PBS. In a 2 mL tube, 50 μL of the cells suspension and 500 μL of melted LMAgarose were mixed and 75 μL pipetted onto a pre-warmed cometslide. The slides were placed flat in the dark at 4°C for 10 minutes to allow the mixture to solidify and then immersed in prechilled lysis solution at 4°C for 40 minutes. Slides were removed from lysis solution, tapped, and immersed in Alkaline Solution for 40 minutes at room temperature in the dark. Slides were washed twice for 5 min with Tris-Borate-EDTA (TBE). Slides were electrophoresed at low voltage (300 mA, 25V, 4°C) for 20 minutes. Slides were placed in 70% ethanol for 5 min, removed, tapped, and air dried for overnight. Slides were stained with SYBR Green stain designed for the Comet Assay, and allowed to air dry at room temperature for six hours. SYBR Green stained cometslides were viewed with an Olympus fluorescence microscope and analyzed using LAI’s Comet Assay Analysis System software (Loates Associates, Inc. Westminster, MD).

Statistical Analysis

Data were presented as means ± SDs. Statistical analysis was done using one way analysis of variance (ANOVA Dunnett’s test) for multiple samples and Student’s t-test for comparing paired sample sets. P-values less than 0.05 were considered statistically significant. The MDA levels and comet tail length (μm) were presented graphically in the form of histograms, using Microsoft Excel computer program.

RESULTS

Lipid peroxidation Assay

The treatment of HepG2 cells with lead nitrate resulted in a marked increase of MDA production, an indication of oxidative stress and cell injury. In contrary, incubation of HepG2 cells with increasing concentrations of NAC decreased the amount of MDA production in dose-dependent manner (Fig 1). The MDA production level was greatly reduced when cells were treated with 0.5 mM NAC and 30μg/mL of lead nitrate compared to lead nitrate alone. Findings from these studies suggest that the antioxidant property of NAC in vitro inhibits the generation of reactive oxygen species (ROSs) that contribute to lead-induced toxicity.

Figure 1.

Figure 1

Protective effect of NAC on lead nitrate induced oxidative stress in HepG2 cells.

Cell Treatment for Comet/Genotoxicity Assay

Representative images of HepG2 Comet cells either with NAC, NAC plus lead nitrate or lead nitrate alone are presented in (Figure 2 and 4). The treatment of HepG2 cells with 30 μg/mL of lead nitrate resulted in a significant increase (p < 0.05) in the mean of comet tail length (μm) compared to the control (Fig 3). Interestingly, co-treatment of these cells with 500 μM NAC resulted in a significant decrease (p<0.05) in the mean values of comet tail length of HepG2 cells compared to lead alone. Similar trend was observed in the mean value of the tail moment, arm tail, and percentages of DNA cleavage.

Figure 2.

Figure 2

Representative SYBR Green Comet assay images using LAI’s Analysis System software (Loates Associates, Inc. Westminster, MD), 10x objective

Figure 4.

Figure 4

Micrographs of Typical Comets using the Leica Microsystems, 10x HCX FL plan objective, FITC filter cube.

Figure 3.

Figure 3

Comet assay of HepG2 cells showing the comet tail length

*Significantly different (p < 0.05) from the control, according to the Dunnett’s test.

DISCUSSION

Lipid peroxidation Assay

Using the lipid peroxidation assay, we observed a maximum cell protection from oxidative stress at 0.5 mM NAC. In contrary, the treatment of cells with lead nitrate alone resulted in a marked increase of MDA production in HepG2 cells. Recent studies demonstrated that the toxic effects of heavy metals (lead) are particularly mediated through oxidative stress and the release of lipid peroxidation by-products (11, 14). The antioxidant activities of NAC have been examined by various methods in vitro and in vivo. Using the lipid peroxidation assay protocol, we found that NAC attenuates oxidative stress associated with lead nitrate toxicity in HepG2 cells. This suggests that antioxidants such as NAC may play an important role in the treatment of lead poisoning, and hence, may serve as excellent scavengers of free radicals and chelator of heavy metals.

Cell Treatment for Comet/Genotoxicity Assay

The comet assay is a very sensitive method that has been used in a number of studies for the evaluation of genome damage caused by chemical and physical agents (15). Recent studies in our laboratory show that low to high-level of arsenic trioxide exposure in vitro can induce significant cytogenetic damage in human leukemia (HL-60) cells (13). Similar to our previous findings, exposure of HepG2 cells to 30 μg/mL resulted in a high degree of DNA damage. To the best of our knowledge, a comet assay has not yet been described in human liver carcinoma (HepG2) cells, so that the results of this study may be the first results to show a significant increase in the comet tail length in lead nitrate-treated cells compared to the control group. These results confirm that lead nitrate can produce DNA damage detectable by this method. Interestingly, co-treatment of cells with 500 μM NAC markedly lowered the genotoxic effect associated with lead nitrate in vitro compared to lead nitrate alone.

V. CONCLUSION

In the present study, NAC co-treatment decreased malondialdehyde, a by-product of lipid peroxidation. Consistent with these findings, NAC also markedly lowered the genotoxic damage associated with lead nitrate exposure in HepG2 cells. This finding suggests that NAC protects cells against oxidative stress/DNA damage associated with exposure to lead nitrate in vitro. Overall, findings from these studies suggest that NAC attenuates lead nitrate-induced oxidative stress and genetic damage in HepG2 cells probably by inhibiting the generation of reactive oxygen species that contribute to cell killing. To our knowledge, NAC showed inhibitory activity to lead nitrate in the in vitro studies. We believe that its protective effect on oxidative damage in HepG2 cells exposed to lead nitrate might be related to both its ability to scavenge free radicals and to chelate metal ions.

Acknowledgments

This research was financially supported in part by a grant from the National Institutes of Health (Grant No. 1G12RR13459), through the RCMI Center for Environmental Health, in part by a grant from the NIMH-COR Honor High School Program (Grant No: MH57113), and in part by a grant from the U.S. Department of the Army (Cooperative Agreement No. W912H2-04-2-0002) through the CMCM Program at Jackson State University.

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