Mechanisms Mediating the Combined Toxicity of Paraquat and Maneb in SH-SY5Y Neuroblastoma Cells

Abstract

Epidemiological and experimental studies have demonstrated that combined exposure to the pesticides paraquat (PQ) and maneb (MB) increases the risk of developing Parkinson's disease. However, the mechanisms mediating the toxicity induced by combined exposure to these pesticides are not well understood. The aim of this study was to investigate the mechanism(s) of neurotoxicity induced by exposure to the pesticides PQ and MB isolated or in association (PQ + MB) in SH-SY5Y neuroblastoma cells. PQ + MB exposure for 24 and 48 h decreased cell viability and disrupted cell membrane integrity. In addition, PQ + MB exposure for 12 h decreased the mitochondrial membrane potential. PQ alone increased reactive oxygen species (ROS) and superoxide anion generation and decreased the activity of mitochondrial complexes I and II at 12 h of exposure. MB alone increased ROS generation and depleted intracellular glutathione (GSH) within 6 h of exposure. In contrast, MB exposure for 12 h increased the GSH levels, the glutamate cysteine ligase (GCL, the rate-limiting enzyme in the GSH synthesis pathway) activity, and increased nuclear Nrf2 staining. Pretreatment with buthionine sulfoximine (BSO, a GCL inhibitor) abolished the MB-mediated GSH increase, indicating that MB increases GSH synthesis by upregulating GCL, probably by the activation of the Nrf2/ARE pathway. BSO pretreatment, which did not modify cell viability per se, rendered cells more sensitive to MB-induced toxicity. In contrast, treatment with the antioxidant N-acetylcysteine protected cells from MB-induced toxicity. These findings show that the combined exposure of SH-SY5Y cells to PQ and MB induced a cytotoxic effect higher than that observed when cells were subjected to individual exposures. Such a higher effect seems to be related to additive toxic events resulting from PQ and MB exposures. Thus, our study contributes to a better understanding of the toxicity of PQ and MB in combined exposures.

Figure 1

PQ and MB cytotoxicity in neuroblastoma SH-SY5Y cells. SH-SY5Y cells were incubated with vehicle (PBS or DMSO), PQ (10–3000 μM), or with MB (1–30 μM) for 24 h. Cell viability was evaluated by the reduction of MTT (A and B) and by the LDH release assay (C and D). Results of MTT assays are expressed as the percentage of MTT reduction with respect to control values. Results of LDH release assays were expressed as percent of LDH released, where the 100% value represents control cells treated with 2% Triton X-100 for 15 min. Data are represented as mean ± SEM (n = 8). *p < 0.05, p < 0.01, and ***p < 0.001 indicate statistical difference from control by one-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 2

Combined exposure to low concentrations of PQ and MB induces cytotoxicity in neuroblastoma SH-SY5Y cells. Cells were exposed to 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 24 (A and B) and 48 h (C and D). Cell viability was evaluated by the reduction of MTT (A and C) and by the LDH release assay (B and D). Results of MTT assays are expressed as the percentage of MTT reduction with respect to control values (n = 5–6). Results of LDH release assays were expressed as percent of LDH released, where the 100% value represents control cells treated with 2% Triton X-100 for 15 min (n = 5–7). Data are represented as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical difference from control; #p < 0.05, ##p < 0.01, and ###p < 0.001 indicate statistical difference from PQ + MB by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 3

PQ and MB exposure induces reactive species production in neuroblastoma SH-SY5Y cells. Cells were incubated with vehicle (PBS or DMSO) or with 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 6 h (A and C) or 12 h (B and D). Results of reactive species production (A and B, n = 8) and superoxide anion generation (C and D, n = 5–8) were expressed as the percentage of control. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical difference from control by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 4

Combined exposure to low concentrations of PQ and MB decreases the MMP in neuroblastoma SH-SY5Y cells. Cells were incubated with vehicle (PBS or DMSO) or with 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 6 (A) or 12 h (B). The MMP was determined from the ratio of fluorescence intensity from JC-1 aggregates (red) and monomeric (green) fluorescence. Results were expressed as the percentage of control. Data are represented as mean ± SEM (n = 5–7). ***p < 0.001 indicates statistical difference from control; ##p < 0.01 indicates statistical difference from PQ + MB by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 5

Effects of PQ and MB on mitochondrial complex I and complex II activities in neuroblastoma SH-SY5Y cells. Cells were incubated with vehicle (PBS or DMSO) or with 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 12 h. NADH dehydrogenases (complex I) activity (A) was measured by the rate of NADH-dependent ferricyanide reduction in 340 nm and calculated as nmol/min/mg protein (n = 4). Complex II activity (B) was measured by following the decrease in absorbance due to the reduction of 2,6-DCIP at 600 nm, calculated as nanomole of 2,6-DCIP reduced/min/mg of protein and (n = 5). Results were expressed as the percentage of control. Data are represented as mean ± SEM and & indicate a significant (p < 0.05) main effect of PQ by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 6

Effects of PQ and MB on GSH levels in neuroblastoma SH-SY5Y cells. Cells were incubated with vehicle (PBS or DMSO) or with 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 6 h (A) and 12 h (B). GSH levels were determined as nonprotein thiols (NPSH), whose levels in the control condition were 6.46 ± 0.55 nmol of NPSH/mg protein. Results were expressed as the percentage of control. Data are represented as mean ± SEM (n = 6–7). *p < 0.05 and ***p < 0.001 indicate statistical difference from control by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 7

Effects of MB on GSH levels, GCL activity, and Nrf2 nuclear translocation in neuroblastoma SH-SY5Y cells. Cells were incubated with vehicle (DMSO) or with 10 μM MB for 12 h (A and B). GSH levels were determined as nmol GSH/mg of protein, and the results were expressed as percentage of control (n = 4). GCL activity was expressed as a percentage of control, whose activity was 6.77 ± 0.78 nmol of NADH oxidized/min/mg protein (n = 4). Data are represented as mean ± SEM *p < 0.05 indicates statistical difference from control using the Student’s t-test (A and B). Cells were incubated with 25 μM BSO for 10 h and then were exposed to 10 μM MB for 12 h (C). GSH levels were determined as nonprotein thiols (NPSH), and the results were expressed as the percentage of control (n = 4). Data are represented as mean ± SEM. ***p < 0.001 indicates statistical difference from control; ###p < 0.001 indicates statistical difference from MB + BSO by two-way ANOVA, followed by Tukey’s HSD posthoc test. Cells were incubated with vehicle, with 10 μM TBQ (positive control), or with MB for 6 h (D). The nuclear translocation of Nrf2 was evaluated by immunofluorescence (green). Cell nuclei were stained with Hoechst 33258 (blue). A representative merge of the green and blue fluorescence is shown. (Magnification 200×).

Figure 8

Effects of PQ and/or MB and BSO on cell viability in neuroblastoma SH-SY5Y cells. Cells were incubated with BSO (25 μM) for 24 h and then exposed to 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 48 h. Cell viability was evaluated by the reduction of MTT, and the results are expressed as the percentage of MTT reduction with respect to control values. Data are represented as mean ± SEM (n = 7). *p < 0.05 and ***p < 0.001 indicate statistical difference from control; ##p < 0.01 and ###p < 0.001 indicate statistical difference from PQ + MB and from MB, respectively, by two-way ANOVA, followed by Tukey’s HSD posthoc test.

Figure 9

NAC treatment protects from MB-induced cytotoxicity in neuroblastoma SH-SY5Y cells. Cells were pretreated with NAC (500 μM) for 1 h and then exposed to 100 μM PQ and 10 μM MB alone or in combination (PQ + MB) for 48 h. Cell viability was evaluated by the reduction of MTT, and the results are expressed as the percentage of MTT reduction with respect to control values. Data are represented as mean ± SEM (n = 3). **p < 0.01 and ***p < 0.001 indicate statistical difference from control; #p < 0.05 and ##p < 0.01 indicate statistical difference from PQ + MB and from MB, respectively, by two-way ANOVA, followed by Tukey’s HSD posthoc test.