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The Isothiocyanate Sulforaphane Depends on the Nrf2/γ-GCL/GSH Axis to Prevent Mitochondrial Dysfunction in Cells Exposed to Methylglyoxal

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Abstract

Methylglyoxal (MG) is a reactive dicarbonyl presenting both endogenous (e.g. glycolysis) and exogenous (e.g. food cooking) sources. MG induces neurotoxicity, at least in part, by affecting mitochondrial function, including a decline in the oxidative phosphorylation (OXPHOS) system activity, bioenergetics failure, and redox disturbances. Sulforaphane (SFN) is an isothiocyanate found mainly in cruciferous vegetables and exerts antioxidant and anti-inflammatory effects in mammalian cells. SFN also decreases mitochondrial vulnerability to several chemical stressors. SFN is a potent activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master regulator of the mammalian redox biology. Here, we have investigated whether and how SFN would be able to prevent the MG-induced mitochondrial collapse in the human neuroblastoma SH-SY5Y cells. The cells were exposed to SFN at 5 µM for 24 h prior to the administration of MG at 500 µM for additional 24 h. We found that SFN prevented the MG-induced OXPHOS dysfunction and mitochondrial redox impairment. SFN stimulated the activity of the enzyme γ-glutamylcysteine ligase (γ-GCL), leading to increased synthesis of glutathione (GSH). Inhibition of γ-GCL with buthionine sulfoximine (BSO) or silencing of Nrf2 using small interfering RNA (siRNA) against this transcription factor reduced the levels of GSH and abolished the mitochondrial protection promoted by SFN in the MG-treated cells. Thus, SFN protected mitochondria of the MG-challenged cells by a mechanism involving the Nrf2/γ-GCL/GSH axis.

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Acknowledgements

MRO receives a “Bolsa de Produtividade em Pesquisa 2 - PQ2” fellow from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) (protocol number 301273/2018-9). This work was partially supported by CNPq (protocol numbers 400216/2016-7). RCBG receives a fellow from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

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Authors and Affiliations

  1. Departamento de Ciências da Natureza, Universidade Federal Fluminense (UFF), Campus Universitário de Rio das Ostras, Rio de Janeiro, Brazil

    Flávia Bittencourt Brasil

  2. Grupo de Estudos em Terapia Mitocondrial, Departamento de Bioquímica “Tuiskon Dick”, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Rua Ramiro Barcelos, 2600 (Anexo), Porto Alegre, RS, CEP 90035-000, Brazil

    Rênata Cristina Bertolini Gobbo & Marcos Roberto de Oliveira

  3. Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Departamento de Bioquímica “Tuiskon Dick”, Instituto de Ciências Básicas da Saúde (ICBS), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

    Rênata Cristina Bertolini Gobbo, Fernanda dos Santos Petry & Marcos Roberto de Oliveira

  4. Programa de Pós-Graduação em Ciências da Saúde (PPGCS), Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brazil

    Fhelipe Jolner Souza de Almeida

  5. Grupo de Estudos em Neuroquímica e Neurobiologia de Moléculas Bioativas, Universidade Federal de Mato Grosso (UFMT), Av. Fernando Corrêa da Costa, 2367, Cuiaba, MT, CEP 78060-900, Brazil

    Fhelipe Jolner Souza de Almeida, Matheus Dargesso Luckachaki & Marcos Roberto de Oliveira

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  1. Flávia Bittencourt Brasil
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11064_2020_3204_MOESM1_ESM.pdf

Figure S1 Mitochondrial isolation quality control. The activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) was evaluated in the mitochondrial fraction obtained from the SH-SY5Y cells to check the purity of the samples.

11064_2020_3204_MOESM2_ESM.pdf

Figure S2 Sulforaphane (SFN) prevented the release of cytochrome c to the cytosol (A), the upregulation in the activity of the caspases-9 (B) and -3 (C), and the increase in the levels of cleaved PARP (D) and of DNA fragmentation (E) in methylglyoxal (MG)-treated SH-SY5Y cells. Cells were treated with SFN at 5 µM for 24 h before the administration of MG at 500 µM for additional 24 h. Data are presented as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.01 vs the control group, #p < 0.01 vs MG-treated cells.

11064_2020_3204_MOESM3_ESM.pdf

Figure S3 Buthionine sulfoximine (BSO) suppressed the effect of a pretreatment with sulforaphane (SFN) on the levels of cytosolic cytochrome c and of malondialdehyde (MDA, a molecular marker of lipid peroxidation) in the membranes of mitochondria obtained from SH-SY5Y cells exposed to methylglyoxal (MG). Buthionine sulfoximine (BSO) at 400 µM was administrated to the cells for 24 h in the presence or absence of SFN at 5 µM. Then, the cells were exposed to MG for further 24 h. Data are presented as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, *p < 0.01 vs the control group, #p < 0.01 vs MG-treated cells, º p < 0.01 vs SFN + MG-treated cells.

11064_2020_3204_MOESM4_ESM.pdf

Figure S4 The effects of silencing Nrf2 on the activity of this transcription factor. Data are presented as the mean ± SEM of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, a p < 0.01 vs the control cells transfected with negative control (NC) siRNA; b p < 0.01 vs the SFN-treated cells transfected with NC siRNA

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Brasil, F.B., Gobbo, R.C.B., de Almeida, F.J.S. et al. The Isothiocyanate Sulforaphane Depends on the Nrf2/γ-GCL/GSH Axis to Prevent Mitochondrial Dysfunction in Cells Exposed to Methylglyoxal. Neurochem Res 46, 740–754 (2021). https://doi.org/10.1007/s11064-020-03204-x

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