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Lead toxicity has been associated with its ability to interact and damage DNA. However, its molecular mechanisms of action are not fully understood. In vitro studies in our laboratory indicated that lead nitrate (PbNO3) induces cytotoxicity and oxidative stress to human liver carcinoma (HepG2) cells in a dose-dependent manner. In this research, we hypothesized that n-acetyl-cysteine (NAC), a known antioxidant compound, affords protection against lead-induced cell death associated with genotoxic damage. To test this hypothesis, HepG2 cells were treated either with a physiologic dose of NAC, NAC plus PbNO3, or PbNO3 alone, respectively, followed by incubation in humidified 5% CO2 incubator at 37°C for 48 hr. The cell viability was determined by trypan blue exclusion test. The degree of DNA damage was detected by micro gel electrophoresis (comet) assay. Our results showed that lead exposure induces a substantial cytotoxicity as well as a significant genotoxicity to HepG2 cells. However, co-treatment with a physiologic dose (500μM) of NAC slightly increases cell viability, and significantly reduces (p < 0.05) the degree of DNA damage. Hence, NAC treatment may be a promising therapeutic candidate for chemoprevention against lead toxicity, based on its ability to scavenge free radicals.
Lead is one of the most toxic heavy metals that have been reported to interact with DNA, but the molecular mechanism of this interaction is not fully understood. The genotoxic potential of lead has been examined in several studies. 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 been 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 (7, 8). Although NAC has a long history of therapeutic application, little data are available on the role of NAC in the prevention of cytogenotoxic effects caused by exposure to lead in vitro. Hence, the present study was designed to evaluate the protective role of NAC against lead-induced cytogenotoxic effects to human liver carcinoma (HepG2) cells.
Reference solution (1000 ± 10 ppm) of lead nitrate (PbNO3) (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).
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 humidified 5% CO2 incubator at 37°C for 48 hr according to previous experiments in our laboratory (9, 10).
To assess the cell viability, 1 × 104 cells were plated in each well of 96-well plates. The plates were placed in a humidified 5% CO2 incubator at 37°C to allow cells to attach to the substratum for 2 to 3 days. From a recently published paper, we reported that PbNO3 is cytotoxic to HepG2 cells, showing a 48 hr-LD50 of 37.5 ± 9.2μg/mL (10). Hence, to examine the effect of NAC on PbN03-induced cytogenotoxic effects, cells were treated either with a physiologic dose NAC, NAC plus PbNO3, or PbNO3 alone, followed by incubation in a humidified 5% CO2 incubator for 48 hr. The cells incubated in culture medium alone served as a control for cell viability (untreated wells). The cell viability was assessed by the trypan blue exclusion test (Life Technologies) using a hemocytometer to manually count the cells. Briefly, ten μl of a 0.5% solution of the dye was added to 100μl of treated cells (1.0 × 105/ml). The suspension was then applied to a hemocytometer. Both viable and nonviable cells were counted. A minimum of 200 cells were counted for each data point in a total of eight microscopic fields.
Cells were counted (10,000 cells/well) and aliquots of 100μL of the cell suspension were placed in each well of 96 plates, treated with 100μl aliquot of either media, NAC, NAC plus PbN03, or PbN03 alone, respectively, and incubated in a humidified 5% CO2 incubator at 37°C for 48 hr. After incubation, the cells were centrifuged, washed with phosphate buffered saline (PBS) free calcium and magnesium, and re-suspended in 100μL PBS. In a 2mL tube, 50μL 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).
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 present study indicates that NAC treatment increases cell viability and affords protection of DNA damage in HepG2 cells exposed to PbNO3 (Table 1). The treatment of these cells with 30μg/mL of PbNO3 resulted in a significant decrease of cell viability accompanied by a markedly increase in DNA damage compared to the control cells. Interestingly, co-treatment of cells with a physiologic dose (500μM) of NAC and 30μg/mL of PbNO3 resulted in a slight increase in cell viability and minimal DNA damage compared to PbNO3 alone. Together, our results indicate that PbN03 represents a potential cytogenotoxic agent in vitro. However, NAC treatment attenuates the cytogenotoxic effects mediated by PbNO3 exposure in human liver carcinoma (HepG2) cells.
In this report, we present the novel use of NAC for the prevention of PbNO3-induced cytogenotoxic effects to human liver carcinoma (HepG2) cells. Recent studies in our laboratory showed that low to high-level of arsenic trioxide exposure induced cytogenotoxic effects to human leukemia (HL-60) cells in a dose-dependent manner (11). Similar to our previous findings, exposure of HepG2 cells to 30μg/mL of PbNO3 caused substantial level of cell death associated with a high degree of DNA damage, manifested by an increase in percentage of DNA in the tail and olive tail moment. Interestingly, co-treatment with a physiologic dose (500μM) of NAC markedly lowered the cytogenotoxic effects of PbNO3 in vitro. Consistent with our finding, Dick and his colleagues reported that NAC pretreatment prevents TNF-α production in primary alveolar macrophages treated with ultrafine nickel particles (12). Other studies indicated that NAC protects macrophage cell line (THP-1) against diesel exhaust particle chemicals (13). Yang and his co-workers also reported that NAC lowers DNA damage produced by water-soluble cigarette smoke in human lymphoid cells containing Epstein–Barr virus episomes (14). In vitro studies suggest that the toxicity and increase in lipid peroxidation induced by lead in cancer cell lines can be ameliorated by antioxidants such as NAC (15). Based on this in vitro study, we speculate that NAC treatment may be a promising therapeutic candidate for chemoprevention against lead toxicity, probably due to NAC ability to scavenge free radicals. The observed preventive effect of NAC in the present study suggests that treatment with physiologic doses of this antioxidant could reduce the cytogenotoxic effects induced by heavy metals.
This research was financially supported in part by a grant from the National Institutes of Health (Grant No. 2G12RR013459-11) through the RCMI Center for Environmental Health, 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.