Roles of oxidative stress, apoptosis, and inflammation in metal-induced dysfunction of beta pancreatic cells isolated from CD1 mice

Abstract

The diabetogenic effects of metals including lead (Pb), mercury (Hg), cadmium (Cd), and molybdenum (Mo) have been reported with poorly identified underlying mechanisms. The current study assessed the effect of metals on the roles of oxidative stress, apoptosis, and inflammation in beta pancreatic cells isolated from CD-1 mice, via different biochemical assays. Data showed that the tested metals were cytotoxic to the isolated cells with impaired glucose stimulated insulin secretion (GSIS). This was associated with increased reactive oxygen species (ROS) production, lipid peroxidation, antioxidant enzymes activities, active proapoptotic caspase-3 (cas-3), inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels in the intoxicated cells. Furthermore, antioxidant-reduced glutathione (GSH-R), cas-3 inhibitor z-VAD-FMK, IL-6 inhibitor bazedoxifene (BZ), and TNF-α inhibitor etanercept (ET) were found to significantly decrease metal-induced cytotoxicity with improved GSIS in metals' intoxicated cells. In conclusion, oxidative stress, apoptosis, and inflammation can play roles in metals-induced diabetogenic effect.

Keywords: Cadmium; Inflammation; Lead; Mercury; Molybdenum; Oxidative stress; Pancreatic beta cells.

Fig. 1

MTT and LDH assays showed the cytotoxic effects of Pb, Hg, Cd, and Mo on pancreatic beta cells isolated from CD-1 mice. The cytotoxic effects of these metals were tested at concentrations of 0.1, 1, 10, and 100 µM and time points of 3, 6, 12, 24, and 48 h post-exposure. Cell viability was expressed as percent from controls assuming their corresponding controls viability is 100%. Experiments were conducted in triplicates with at least 3 wells for each tested metal’s concentration per experiment. Data are expressed as means ± SEMs.

Fig. 2

The effects of Pb, Hg, Cd, and Mo at their MTT-estimated IC50s (50 µM, 70 µM, 30 µM, and 120 µM, respectively) and at a lower concentration of 10 µM on cas-3 activities (2A) and the expression of genes coding for cas-3 (2B), anti-apoptotic Bcl-2 (2C), and pro-apoptotic Bax (2D) in pancreatic beta cells isolated from CD-1 mice. 2E and 2F show the effects of the tested metals at their estimated IC50s on cas-3, anti-apoptotic Bcl-2, and pro-apoptotic Bax protein levels, as measured by western blotting. The density of the bands was quantified and normalized in relation to the total actin bands as a reference protein using GelQuant.NET software provided by biochemlabsolutions.com. All experiments were conducted in triplicates. One-way ANOVA p-values are shown herein. A Dunnett post-test was used to compare the metal-exposed cells to the non-exposed control cells. Data are expressed as means ± SDs. Significance is shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Fig. 3

The effects of Pb, Hg, Cd, and Mo at their MTT-estimated IC50s (50 µM, 70 µM, 30 µM, and 120 µM, respectively) and at a concentration of 10 µM on the ROS production (3A), lipid peroxidation (TBARS; 3B), and Nrf2 gene expression (3C) of pancreatic beta cells isolated from CD-1 mice. Each experiment was repeated for at least 3 times. One-way ANOVA p-values are shown herein. A Dunnett post-test was used to compare the metal-exposed cells to the non-exposed control cells. Data are expressed as means ± SDs. Significance is shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Fig. 4

The effects of Pb, Hg, Cd, and Mo at their MTT-estimated IC50s (50 µM, 70 µM, 30 µM, and 120 µM, respectively) and at a concentration of 10 µM on the CAT activities (4A), SOD activities (4B), and expression of genes coding for CAT (4C), SOD1 (4D), and SOD2 (4E) in pancreatic beta cells isolated from CD-1 mice. 4F and 4G show the effects of the tested metals at their estimated IC50s on CAT, SOD1, and SOD2 protein levels, as measured by western blotting. The density of the bands was quantified and normalized in relation to the total actin bands as a reference protein using GelQuant.NET software provided by biochemlabsolutions.com. All experiments were conducted in triplicates. One-way ANOVA p–values are shown herein. A Dunnett post-test was used to compare the metal-exposed cells to the non-exposed control cells. Data are expressed as means ± SDs. Significance is shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Fig. 5

The effects of Pb, Hg, Cd, and Mo at their MTT-estimated IC50s (50 µM, 70 µM, 30 µM, and 120 µM, respectively) and at a concentration of 10 µM on IL-6 (5A), TNF-α (5B), and the expression of genes coding for IL-6 (5C) and TNF-α (5D) in pancreatic beta cells isolated from CD-1 mice. 5E and 5F show the effects of the tested metals at their estimated IC50s on IL-6 and TNF-α protein levels, as measured by western blotting. The density of the bands was quantified and normalized in relation to the total actin bands as a reference protein using GelQuant.NET software provided by biochemlabsolutions.com. All experiments were conducted in triplicates. One-way ANOVA p-values are shown herein. A Dunnett post-test was used to compare the metal-exposed cells to the non-exposed control cells. Data are expressed as means ± SDs. Significance is shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Fig. 6

The effects of Pb, Hg, Cd, and Mo at their MTT-estimated IC50s (50 µM, 70 µM, 30 µM, and 120 µM, respectively) and at a concentration of 10 µM on the GSIS of pancreatic beta cells isolated from CD-1 mice (6A–6D), 24 hrs post exposure. Also, the protective effects of GSH-R (10 µM), cas-3 inhibitor z-VAD-FMK (200 µM), IL–6 inhibitor BZ (10 µM), and TNF-α inhibitor ET (150 µg mL⁻¹) were evaluated. GSH-R, cas-3 inhibitor z-VAD-FMK, IL–6 inhibitor BZ, and TNF-α inhibitor ET were shown to have significant protective effects on the GSIS of metal-treated samples to differing extents (A–D). These compounds also significantly reduced the cytotoxic effects of the tested metals on the isolated beta pancreatic cells (E–H). MTT data were shown after subtraction of the blank’s readings from all wells values. All experiments were conducted in triplicates. One-way ANOVA p-values are shown herein. A Tukey post-test was used to compare the metal-exposed cells to the non-exposed control cells. Data are expressed as means ± SDs. For comparison between the metal-treated samples and other samples treated with metals in association with the protective compounds, significance is shown as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. For comparison between the non-treated control cells and the experimental groups, significance is shown as ^$ for p < 0.05, ^$$ for p < 0.01, and ^$$$ for p < 0.001.