Molecular and genomic approach for understanding the gene-environment interaction between Nrf2 deficiency and carcinogenic nickel-induced DNA damage

Abstract

Nickel (Ⅱ) is a toxic and carcinogenic metal which induces a redox imbalance following oxidative stress. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a redox factor that regulates oxidation/reduction status and consequently mediates cytoprotective responses against exposure to environmental toxicants. In this study, we investigated the protective roles of the Nrf2 gene against oxidative stress and DNA damage induced by nickel at sub-lethal doses. Under nickel exposure conditions, we detected significantly increased intracellular ROS generation, in addition to higher amounts of DNA damage using comet assay and γ-H2AX immunofluorescence staining in Nrf2 lacking cells, as compared to Nrf2 wild-type cells. In addition, we attempted to identify potential nickel and Nrf2-responsive targets and the relevant pathway. The genomic expression data were analyzed using microarray for the selection of synergistic effect-related genes by Nrf2 knockdown under nickel treatment. In particular, altered expressions of 6 upregulated genes (CAV1, FOSL2, MICA, PIM2, RUNX1 and SLC7A6) and 4 downregulated genes (APLP1, CLSPN, PCAF and PRAME) were confirmed by qRT-PCR. Additionally, using bioinformatics tool, we found that these genes functioned principally in a variety of molecular processes, including oxidative stress response, necrosis, DNA repair and cell survival. Thus, we describe the potential biomarkers regarded as molecular candidates for Nrf2-related cellular protection against nickel exposure. In conclusion, these findings indicate that Nrf2 is an important factor with a protective role in the suppression of mutagenicity and carcinogenicity by environmental nickel exposure in terms of gene-environment interaction.

Figure 1

The measurement of oxidative stress in nickel-treated Nrf2 lacking RKO cells. (A) Intracellular ROS were detected using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCHDA). The levels of ROS were increased in Nrf2 knockdown RKO cells under 20 μM nickel exposure, in comparison to wild-type cells. (B) The average of fluorescence intensity in the H₂DCHDA-stained cells was quantified. The asterisks indicate significant difference at P-value <0.05.

Figure 2

The detection of DNA strand break in nickel-treated Nrf2 lacking RKO cells. (A) The alkaline comet assay was conducted to assess DNA damage in wild-type and Nrf2 knockdown cells under nickel treatment. Nickel exposure in the Nrf2 knockdown cells induced significantly high levels of DNA strand breaks as compared to the wild-type cells. (B) Comet tail length was measured in arbitrary units. (C) The DNA strand breaks were detected by γ-H2AX immunofluorescence staining in wild-type and Nrf2 knockdown cells under nickel exposure. In Nrf2 knockdown cells, the higher amount of foci were found in response to nickel. (D) Fluorescence density of foci was analyzed with a microscope. The asterisks indicate significant difference at P-value <0.05.

Figure 3

Comparison of gene expression patterns obtained from DNA microarray and qRT-PCR in Nrf2 knockdown under nickel exposure. Bars indicate the magnitude of gene expression changes in DNA microarray and qRT-PCR, respectively. Y-axis denotes the fold expression change of the respective gene. (A and B) The expressions of the upregulated and downregulated genes, respectively.

Figure 4

Pathway analysis of toxicogenomic data. Magnified red circles indicate upregulated genes and blue circles indicate downregulated genes in our microarray data. Genes validated by qRT-PCR are indicated by asterisks. The bioinformatics analysis of our microarray data using Pathway Studio software (version 7.1) show nickel and Nrf2 responsive relationships in terms of cell processes and diseases.

Figure 5

Scheme showing synergistic genotoxic effects between Nrf2 knockdown and nickel exposure. Nickel can stimulate the intracellular increase of ROS generation and DNA strand break in Nrf2 knockdown cells, as compared to Nrf2 wild-type cells. In addition, we suggest the potential biomarkers and molecular pathway for Nrf2-related cellular protection against nickel exposure. Our results emphasize the significance of the study of interaction between genes and environments.