Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

Abstract

Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium. Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG₂) cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet) assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay). The result of MTT assay indicated that cadmium chloride induces toxicity to HepG₂ cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05) increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG₂ cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05) was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG₂) cells.

Keywords: DNA damage; HepG2 cells; apoptosis; cadmium chloride; cytotoxicity; oxidative stress.

Figure 1

Cytotoxic effect of cadmium chloride on human liver carcinoma (HepG₂) cells. Cells were cultured with increasing concentrations (1, 2, 3, 4, and 5 μg/mL) of cadmium chloride for 48 h as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean ± SD of three experiments with six replicates per concentration. * Significantly different (p < 0.05) from the control, according to the Dunnett’s test.

Figure 2

Cadmium chloride-induced lipid peroxidation in human liver carcinoma (HepG₂) cells. Cells were incubated for 48 h with increasing concentrations of cadmium chloride (1, 2, 3, 4, and 5 μg/mL). Lipid hydroperoxide levels were determined as described in Materials and Methods. * Significantly different (p < 0.05) from the control, according to the Dunnett’s test. Data are representative of three independent experiments.

Figure 3

Cadmium chloride induced DNA damage in human liver carcinoma (HepG₂) cells. Cells were treated for 48 hours with medium (A) supplemented with solvent or 1 (B); 2 (C); 3 (D); 4 (E); and 5 (F) μg/mL cadmium chloride. Representative comet images were analyzed using LAI’s Comet Assay Analysis System software (Loates Associates, Inc. Westminster, MD, USA).

Figure 4

Comet assay of HepG₂ cells showing the percentages of DNA cleavage (Left) and comet tail lengths (Right) as a function of cadmium chloride concentrations. Each point represents mean ± SD of three independent experiments. * Significantly different (p < 0.05) from the control, according to the Dunnett’s test.

Figure 5

Representative flow cytometry analysis data from annexin V/PI assay. The histograms show a comparison of the distribution of annexin V/PI negative cells (M1) and annexin V/PI positive cells (M2) after 48 h exposure to cadmium chloride. A = control; B = 1 μg/mL; C = 2 μg/mL; D = 3 μg/mL; E = 4 μg/mL; and F = 5 μg/mL.

Figure 6

Annexin V and PI positive cells. Cells were exposed to different concentrations of cadmium chloride as described in the Materials and Methods. * Significantly different (p < 0.05) from the control, according to the Dunnett’s test.