Molecular Biology Reports

, Volume 46, Issue 6, pp 5785–5793 | Cite as

Superoxide-hydrogen peroxide imbalance differentially modulates the keratinocytes cell line (HaCaT) oxidative metabolism via Keap1-Nrf2 redox signaling pathway

  • Micheli Lamberti JobimEmail author
  • Verônica Farina Azzolin
  • Charles Elias Assmann
  • Vera Maria Melchiors Morsch
  • Ivana Beatrice Mânica da Cruz
  • Liliane de Freitas Bauermann
Original Article


The purpose of this study was to investigate the effect of a superoxide-hydrogen peroxide (S-HP) imbalance of the superoxide dismutase manganese dependent (SOD2) gene, generated by paraquat and porphyrin exposure, on the keratinocytes cell line (HaCaT) oxidative metabolism. Paraquat acts increasing superoxide (O2·−) levels, while porphyrin increases hydrogen peroxide (H2O2) levels, acting as VV-SOD2-like and AA-SOD2-like molecules, respectively. First of all, HaCAT cells were treated with different concentrations of paraquat and porphyrin (1; 10; 30, and 70 μM) to determine the concentration of both that causes imbalance. After defining the concentration of paraquat and porphyrin (70 μM), a time curve was performed (1, 3, 6, and 24 h) to evaluate ROS production levels. Other oxidative parameters, such as nitric oxide (NO), lipoperoxidation (TBARS) and protein carbonyl, were evaluated after 24 h of incubation, as well as genotoxic analyses, apoptosis detection, and gene expression. Our findings revealed that paraquat exposure decreased cell viability, increasing lipoperoxidation, DNA damage, and apoptosis. On the other hand, porphyrin treatment increased cell viability and proliferation, ROS and NO production, triggering protein and DNA damage. In addition, porphyrin up-regulated Keap1 and Nrf2 gene expression, while paraquat decreased Nrf2 gene expression. In this sense, we suggested that the superoxide-hydrogen peroxide imbalance differentially modulates oxidative stress on keratinocytes cell line via Keap1-Nrf2 gene expression pathway.


SOD2 Val16Ala-SOD2 SNP Paraquat Porphyrin Oxidative stress Keap1-Nrf2 pathway 



This study was supported by grants and fellowships from the following Brazilian governmental agencies: Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Bowman A, Birch-Machin MA (2016) Age-dependent decrease of mitochondrial complex II activity in human skin fibroblasts. J Invest Dermatol 136:912–919CrossRefGoogle Scholar
  2. 2.
    Zhong JL, Edwards GP, Raval C, Li H, Tyrrell RM (2010) The role of Nrf2 in ultraviolet A mediated heme oxygenase 1 induction in human skin fibroblasts. Photochem Photobiol Sci 9:18–24CrossRefGoogle Scholar
  3. 3.
    Tian FF, Zhang FF, Lai XD, Wang LJ, Yang L, Wang X, Singh G, Zhong JL (2011) Nrf2-mediated protection against UVA radiation in human skin keratinocytes. Biosci Trends 5:23–29CrossRefGoogle Scholar
  4. 4.
    Schafër M, Werner S (2015) Nrf2—a regulator of keratinocyte redox signaling. Free Radic Biol Med 88:243–252CrossRefGoogle Scholar
  5. 5.
    Deruy E, Gosselin K, Vercamer C, Martien S, Bouali F, Slomianny C, Bertout J, Bernard D, Pourtier A, Abbadie C (2010) MnSOD upregulation induces autophagic programmed cell death in senescent keratynocites. PLoS ONE 5:e12712CrossRefGoogle Scholar
  6. 6.
    Kim YS, Vallur PG, Phaeton R, Mythreye K, Hempel N (2017) Insights into the dichotomous regulation of SOD2 in cancer. Antioxidants (Basel) 6:86CrossRefGoogle Scholar
  7. 7.
    Bresciani G, Cruz IB, Paz JA, Cuevas MJ, Gonzalez-Gallego J (2013) The MnSOD Ala16Val SNP: relevance to human diseases and interaction with environmental factors. Free Radic Res 47:781–792CrossRefGoogle Scholar
  8. 8.
    Kang SW (2015) Superoxide dismutase 2 gene and cancer risk: evidence from an updated meta-analysis. Int J Clin Exp Med 8:14647–14655PubMedPubMedCentralGoogle Scholar
  9. 9.
    Li X, Shen M, Cai H, Liu K, Liu Y, Huang Z, Liang C, Deng X, Ye J, Zou Q, Li J (2016) Association between manganese superoxide dismutase (MnSOD) polymorphism and prostate cancer susceptibility: a meta-analysis. Int J Biol Markers 31:e422–e430CrossRefGoogle Scholar
  10. 10.
    Minlikeeva AN, Browne RW, Ochs-Balcom HM, Marian C, Shields PG, Trevisan M, Krishnan S, Modali R, Seddon M, Lehman T, Freudenheim JL (2016) Single-nucleotide polymorphisms and markers of oxidative stress in healthy women. PLoS ONE 11:e0156450CrossRefGoogle Scholar
  11. 11.
    Taufer M, Peres A, de Andrade VM, de Oliveira G, Sá G, do Canto ME, dos Santos AR, Bauer ME, da Cruz IB (2005) Is the Val16Ala manganese superoxide dismutase polymorphism associated with the aging process? J Gerontol A 60:432–438CrossRefGoogle Scholar
  12. 12.
    Montagner FG, Sagrillo M, Machado MM, Almeida RC, Mostardeiro CP, Duarte MM, da Cruz IB (2010) Toxicological effects of ultraviolet radiation on lymphocyte cells with different manganese superoxide dismutase Ala16Val polymorphism genotypes. Toxicol In Vitro 24:1410–1416CrossRefGoogle Scholar
  13. 13.
    Azzolin VF, Cadona FC, Machado AK, Berto M, Barbisan F, Dornelles EB, Glanzner WG, Gonçalves PB, Bica CG, da Cruz IBM (2016) Superoxide-hydrogen peroxide imbalance interferes with colorectal cancer cells viability, proliferation and oxaliplatin response. Toxicol In Vitro 32:8–15CrossRefGoogle Scholar
  14. 14.
    Berto M, Bica CG, Sá GP, Barbisan F, Azzolin VF, Rogalski F, Duarte MMF, da Cruz IBM (2015) The effect of superoxide anion and hydrogen peroxide imbalance on prostate cancer: an integrative in vivo and in vitro analysis. Med Oncol 32:251CrossRefGoogle Scholar
  15. 15.
    Schott KL, Assmann CE, Barbisan F, Azzolin VF, Bonadiman B, Duarte MMMF, Machado AK, da Cruz IBM (2017) Superoxide-hydrogen peroxide genetic imbalance modulates differentially the oxidative metabolism on human peripheral blood mononuclear cells exposed to seleno-L-methionine. Chem Biol Interact 273:18–27CrossRefGoogle Scholar
  16. 16.
    Schott KL, Assmann CE, Teixeira CF, Boligon AA, Waechter SR, Duarte FA, Ribeiro EE, da Cruz IBM (2018) Brazil nut improves the oxidative metabolism of superoxide-hydrogen peroxide chemically-imbalanced human fibroblasts in a nutrigenomic manner. Food Chem Toxicol 121:519–526CrossRefGoogle Scholar
  17. 17.
    Zhang R, Kang KA, Kim KC, Na SY, Chang WY, Kim GY, Kim HS, Hyun JW (2013) Oxidative stress causes epigenetic alteration of CDX1 expression in colorectal cancer cells. Gene 25:214–219CrossRefGoogle Scholar
  18. 18.
    Bus Gibson JE (1984) Paraquat: model for oxidant-initiated toxicity. Environ Health Perspect 55:37–46CrossRefGoogle Scholar
  19. 19.
    Esposti MD (2002) Measuring mitochondrial reactive oxygen species. Methods 26:335–340CrossRefGoogle Scholar
  20. 20.
    Choi WS, Shin PG, Lee JH, Kim GD (2012) The regulatory effect of veratric acid on NO production in LPS-stimulated RAW264.7 macrophage cells. Cell Immunol 280:164–170CrossRefGoogle Scholar
  21. 21.
    Jentzsch AM, Bachmann H, Furst P, Biesalski HK (1996) Improved analysis human of malondialdehyde in body fluids. Free Radic Biol Med 20:251–256CrossRefGoogle Scholar
  22. 22.
    Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191CrossRefGoogle Scholar
  23. 23.
    Maluf SW, Riegel M (2011) Citogenética humana, 1st edn. Artmed, Porto Alegre, pp 180–193Google Scholar
  24. 24.
    Sakai O, Uchida T, Roggia MF, Imai H, Ueta T, Amano S (2015) Role of glutathione peroxidase 4 in glutamate-induced oxytosis in the retina. PLoS ONE 10:e0130467CrossRefGoogle Scholar
  25. 25.
    Barbisan F, Motta JR, Trott A, Azzolin V, Dornelles EB, Marcon M, Algarve TD, Duarte MMF, Mostardeiro CP, Unfer T, Schott KL, Cruz IBM (2014) Methotrexate-related response on human peripheral blood mononuclear cells may be modulated by the Ala16Val-SOD2 gene polymorphism. PLoS ONE 9:1–11CrossRefGoogle Scholar
  26. 26.
    Day RM, Suzuki YJ (2006) Cell proliferation, reactive oxygen and cellular gluthatione. Dose Response 3:425–442PubMedPubMedCentralGoogle Scholar
  27. 27.
    Zhang W, Liu HT (2002) MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 12:9–18CrossRefGoogle Scholar
  28. 28.
    Kocak-Toker N, Giris M, Tulubas F, Uysal M, Aykac- Toker G (2005) Peroxynitrite induced decrease in Na+, K+-ATPase activity is restored by taurine. World J Gastroenterol 11:3554–3557CrossRefGoogle Scholar
  29. 29.
    Zhang L, Li Q, Jia S, Huang Z, Luo Y (2018) Effect of different stunning methods on antioxidant status, in vivo myofibrillar protein oxidation, and the susceptibility to oxidation of silver carp (Hypophthalmichthys molitrix) fillets during 72 h post-mortem. Food Chem 246:121–128CrossRefGoogle Scholar
  30. 30.
    Park S, Imlay JA (2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction. J Bacteriol 185:1942–1950CrossRefGoogle Scholar
  31. 31.
    Hu P, Wu T, Fan W, Chen L, Liu Y, Ni D, Bu W, Shi J (2017) Near infrared-assisted Fenton reaction for tumor-specific and mitochondrial DNA-targeted photochemotherapy. Biomaterial 141:86–95CrossRefGoogle Scholar
  32. 32.
    Lu M, Ji J, Jiang Z, You Q (2016) The Keap1–Nrf2–are pathway as a potential preventive and therapeutic target: an update. Med Res Rev 36:924–963CrossRefGoogle Scholar
  33. 33.
    Zhu H, Yan P, Wang L, Liu Y, Wen J, Zhang Q, Fan Y, Luo Y (2018) Protective properties of Huperzine A through activation Nrf2/ARE-mediated transcriptional response in X-rays radiation-induced NIH3T3 cells. J Cell Biochem 119:8359–8367CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Micheli Lamberti Jobim
    • 1
    Email author
  • Verônica Farina Azzolin
    • 1
  • Charles Elias Assmann
    • 2
  • Vera Maria Melchiors Morsch
    • 2
  • Ivana Beatrice Mânica da Cruz
    • 1
    • 3
  • Liliane de Freitas Bauermann
    • 1
  1. 1.Programa de Pós-graduação em FarmacologiaUniversidade Federal de Santa MariaSanta MariaBrazil
  2. 2.Programa de Pós-graduação em Ciências Biológicas: Bioquímica ToxicológicaUniversidade Federal de Santa MariaSanta MariaBrazil
  3. 3.Programa de Pós-graduação em GerontologiaUniversidade Federal de Santa MariaSanta MariaBrazil

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