Introduction

Keratinocytes represent the main cells of the epidermis. These cells present a high proliferative turnover and generate a dense skin barrier that protects humans against infections triggered by exogenous pathogens, such as virus and bacteria. In addition, these cells have an important functional relevance to the organism defensive responses against other several environmental stressful factors, such as radiation, heat, cold, and trauma, which generate oxidative stress states. The defensive action of keratinocytes is very important, since chronic oxidative stress exposure can trigger inflammation, skin aging, and cancer development [1].

In healthy conditions, keratinocytes present low-senescence phenotype than other cells, such as fibroblasts. Its phenotype probably is influenced by keratinocyte antioxidant defense which involves a crucial upregulation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Indeed, Nrf2 transcription factor regulates the expression of other antioxidant proteins, that effectively protect these cells against oxidative damages [2,3,4].

However, overproduction of antioxidant molecules by keratinocytes against reactive oxygen species (ROS) and reactive nitrogen species (RNS) is finely regulated to maintain the cells oxidative homeostasis. This assumption is based on a prior investigation which reported that keratinocyte senescence process could be induced by the upregulation of the superoxide dismutase manganese dependent gene (SOD2) enzyme and subsequent hydrogen peroxide (H2O2) overproduction into the mitochondria [5]. SOD2 is considered a crucial antioxidant enzyme to cells catalyzing the reaction from superoxide (O·−2) anion, which is continuously produced by mitochondrial biochemical reactions involving oxygen, into H2O2. However, an evidence has suggested that SOD2 enzyme has a dichotomous role in cancer as either tumor suppressor or promoter according to its regulation and to cell type [6]. This assumption is based on former investigations that described the association between a SOD2 single nucleotide polymorphism (SNP, rs4880) located in the 16-codon (Val16Ala-SOD2) with some types of cancer [7].

The Val16Ala-SOD2 SNP presents an important superoxide-hydrogen peroxide imbalance related mainly to its homozygous genotypes (AA and VV). Specifically, the AA-genotype has been associated to a higher risk of some cancer types, such as prostate, breast, non-Hodgkin lymphoma, lung cancer, and colorectal cancer [8,9,10]. Moreover, Taufer et al. [11] described that the AA-genotype presented higher immunosenescence markers than V-allele subjects, whereas Montagner et al. [12] described higher DNA damage in AA UV-exposed than VV UV-exposed lymphocytes suggesting that higher SOD2 levels could affect cellular aging.

Potential causal mechanisms involving AA cancer risk could be related to SOD2 efficiency [7]. In fact, SOD2 enzyme is active only into mitochondria dismutating O·−2 anion to H2O2, that subsequently is converted by glutathione peroxidase (GPx) in water and oxygen. AA-genotype presents a different SOD2-protein phenotype, which has an alpha-helix structure entering more easily into the mitochondria than VV-genotype enzymes. However, better SOD2 efficiency associated with AA-genotype generates higher H2O2 levels that easily pass through the mitochondria membranes reaching the cytoplasm and potentially increasing some ROS levels including hydroxyl radical (·OH). The basal increase of ROS levels can trigger continuous DNA damage increasing cancer risk in AA-subjects. Moreover, recent studies that established a pharmacological O·−2–H2O2 (S-HP) imbalance model using paraquat to generate high O·−2 levels and porphyrin, a SOD2-like molecule that generates high H2O2 levels showed important modulation on the proliferative state and chemotherapeutic drug resistance on colorectal and prostate cancer cell lines [13, 14]. Other recently published studies stated that the O·−2–H2O2 imbalance also modulates the oxidative metabolism of healthy cells, such as peripheral blood mononuclear cells exposed to seleno-l-methionine [15] and fibroblasts exposed to a Brazil nut extract [16].

Therefore, it is possible that the genetic O·−2–H2O2 (S-HP, respectively) imbalance triggered by the Val16Ala-SOD2 SNP could directly affect antioxidant response of keratinocytes by differential modulation of Nrf2 gene and also by differential basal oxidative metabolism markers of these cells. To test this hypothesis, the present investigation described results from an in vitro protocol using the HaCaT keratinocytes cell line submitted to a pharmacological O·−2–H2O2 imbalance to test potential differential modulation of Nrf2 gene expression and other apoptotic and oxidative markers.

Materials and methods

Chemicals and reagents

All chemicals and reagents used to perform the assays were of analytical grade and purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Reagents used to perform cell culture protocols as cell culture media, fetal bovine serum (FBS), antibiotics/antifungals, and plastics were acquired from Gibco™ Thermo Fisher Scientific (Grand Island, NY, USA) and Invitrogen Life Technologies (Carlsbad, CA, USA). Molecular biology reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA), Bio-Rad Laboratories (Hercules, CA, USA), and Qiagen (Hilden, Germany). All spectrophotometric and fluorimetric assays were performed using a 96-microplate reader SpectraMax® i3 Multimode Plate Reader (Molecular Devices, Sunnyvale, CA, USA).

Experimental study design

In this study, an in vitro protocol was performed using the HaCaT keratinocytes cell line obtained from the American Type Culture Collection (ATCC, USA). Keratinocytes represent a suitable model to investigate responses to oxidative stress due to the fact that these cells constitute some of the main cells of the human epidermis and help to protect the body against pathogens and environmental agents that cause oxidative stress, thus participating in the processes of aging and inflammation, for example [1]. Cells were submitted to a pharmacological O·−2–H2O2 imbalance to test potential differential modulation of Nrf2 gene expression and other apoptotic and oxidative markers. Cells were exposed to paraquat, an organic compound that interferes with electron transfer through the reduction in the number of donor electrons, such as NADPH in the mitochondria, increasing the superoxide anion levels; on the other hand, porphyrin is a SOD2 mimic molecule that increases the rate conversion of superoxide anion into hydrogen peroxide [17, 18], in order to generate a O·−2 and H2O2 imbalance. Therefore, oxidative parameters were evaluated according to Fig. 1.

Fig. 1
figure 1

Experimental study design

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Cell culture and treatments

Keratinocytes cell line (HaCaT) was cultured in Dulbecco’s Modified Eagle Media (DMEM) with 10% fetal bovine serum (FBS), supplemented with 1% penicillin/streptomycin and amphotericin B. Cells were cultured at 37 °C with 5% CO2 and they were expanded by obtaining the optimal amount for the experiments. Cell suspension was placed in 96-well plates (2.5 × 105 cells/well). After cell attachment, cells were treated with paraquat or porphyrin at different concentrations (1; 10; 30, and 70 μM) to determine the concentration of both that causes imbalance in HaCaT cells. After defining the concentration of paraquat and porphyrin, a time curve was performed (1, 3, 6 and 24 h) for evaluating ROS production levels. Other oxidative parameters were evaluated after 24 h of incubation.

Viability and proliferation assays

Cell viability and proliferation was evaluated after the exposure to paraquat and porphyrin for 24 and 72 h. The effect on cell viability and proliferation was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] spectrophotometric assay, as described by Montagner et al. [12]. For this assay, 2.5 × 105 cells were incubated for 1 h with MTT reagent. After, formazan salt was dissolved with dimethylsulfoxide (DMSO), and absorbance was measured at 570 nm.

Reactive oxygen species (ROS) assay

Intracellular ROS production was evaluated by levels of oxidized dichlorofluorescein (DCF) from 2′7′-dichlorofluorescein diacetate (DCFH-DA) described by Esposti et al. [19]. Keratinocytes cells were seeded in 96-well plates (5 × 105 cells/well), and after paraquat and porphyrin treatment, cells were incubated with DCFH-DA (10 μM) for 1 h at 37 °C in the dark. Fluorescence was determined at 488 nm (excitation) and 525 nm (emission).

Nitric oxide (NO) quantification

Nitric oxide (NO) production was measured by the modified Griess method previously described by Choi et al. [20]. Treated cells were incubated at room temperature with Griess reagent for 10 min and the absorbance was measured at 550 nm.

Lipoperoxidation and protein carbonylation measurement

Oxidative stress indicators were measured in keratinocytes samples. Thiobarbituric acid reactive substances (TBARS) were measured according to the modified method performed by Jentzsch et al. [21]. Protein carbonylation was determined by the Levine method with modifications.

DNA comet and micronucleus genotoxicity assays

The alkaline comet assay was performed as described by Singh et al. [22] in accordance with the general guidelines for use of the comet assay. One hundred cells (50 cells from each of the two replicate slides) were selected and analyzed. Cells were visually scored according to tail length and received scores from 0 (no migration) to 4 (maximal migration). Therefore, the damage index for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). Slides were analyzed under blind conditions by at least two individuals.

To evaluate the frequency of micronuclei (MN), which form during the cellular interphase, being of the same color and not larger than one-third of the main nucleus. It also makes it possible to evaluate the frequency of Nuclear Bridges (PN), Nuclear Buds (BUD) (also called broken eggs), and Binucleated Cells (BN) described by Maluf and Riegel [23]. In this test, 1000 cells per slide were analyzed for each of the criteria (MN, PN, BUD and BN).

Apoptosis detection

Cells in the concentration of 1 × 105 were stained by Alexa Fluor 488 Annexin V (Invitrogen) for 15 min at room temperature and washed once with PBS. Fluorescent images were obtained with a fluorescence microscope (Keyence). Percentages of Annexin V-positive apoptotic cells relative to the total number of cells were calculated according to the described by Sakai et al. [24].

Gene expression analysis by qRT-PCR

The potential differential modulation of Nrf2 and Keap1 gene expression was performed similarly to the previously described in Barbisan et al. [25]. Briefly, qRT-PCR was performed in a 20 μL reaction volume: 1 µL of the cDNA and 19 µL of QuantiFast SYBR Green PCR Kit (Qiagen, Germany). The following qRT-PCR parameters were used: 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s followed by a melt curve of 65 °C. Beta-actin was used as the housekeeping gene. Relative expression was calculated using the comparative Ct method and was expressed as the fold expression compared to the control.

Statistical analysis

The data were expressed as mean ± standard deviation (SD). The results were expressed as a percent (%) of the negative control group. Therefore, the data were submitted to one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. The statistical tests were performed using the GraphPad Prism Software. Results of p < 0.05 were considered statistically significant.

Results

Initially, we determined the effect of different paraquat and porphyrin concentrations on keratinocytes viability for 24 h (Fig. 2a) and proliferation for 72 h (Fig. 2b) at different concentrations (1, 10, 30, and 70 μM). After 24 h exposure, only the highest concentration (70 μM) of paraquat decreased cell viability, while lower concentrations of this chemical increased cell viability when compared to control levels. At the same exposure time, porphyrin at 30 μM showed cytotoxicity, and at 70 μM showed to elevate cell viability compared to the control. For 72 h of exposure, in general, paraquat and porphyrin concentrations significantly increased cell proliferation when compared to the control. From these initial results, the concentration that caused imbalance for both paraquat and porphyrin was determined at 70 μM.

Fig. 2
figure 2

HaCaT cells were treated with paraquat, porphyrin at different concentrations (1; 10; 30, and 70 μM). Viability was measured in 24 h (a) and proliferation was determinate in 72 h (b). The results were compared against the percentage of negative control (only cells). N = 3, significant differences ***p < 0.001

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After determining the concentration of 70 μM from the viability and proliferation assays, a curve was performed to evaluate ROS production levels at different exposure times (1, 3, 6, and 24 h). Results showed a significant increase of ROS production for porphyrin treatment at 70 μM after 3 h of exposure (Fig. 3).

Fig. 3
figure 3

HaCaT cells were treated with the best concentration of paraquat and porphyrin (70 μM), and a time curve was performed (1, 3, 6 and 24 h) for evaluating ROS production levels by DCFH-DA Assay. The results were compared against the percentage of negative control (only cells). N = 3, significant differences ***p < 0.001

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NO production was measured after 24 h of exposure. Results showed that porphyrin significantly increased the NO levels (Fig. 4).

Fig. 4
figure 4

HaCaT cells were treated with paraquat, porphyrin at 70 μM for 24 h for evaluating NO production levels by Griess reagent. The results were compared against the percentage of negative control (only cells). N = 3, significant differences ***p < 0.001

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Lipid and protein damage were determined by TBARS and protein carbonyl, respectively, after 24 h of exposition. Results showed that paraquat treatment increased the lipoperoxidation levels, however, porphyrin decreased this parameter compared to the control (Fig. 5a). On the other hand, porphyrin treatment increased protein carbonyl whereas paraquat did not alter the protein damage levels when compared to the control (Fig. 5b).

Fig. 5
figure 5

HaCaT cells were treated with paraquat, porphyrin at 70 μM for 24 h for evaluating lipoperoxidation levels by TBARS Assay (a) and for protein carbonyl determination (b). The results were compared against the percentage of negative control (only cells). N = 3, significant differences *p < 0.05 and ***p < 0.001

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Genotoxicity was measured by Comet and Micronucleus Assays. The results showed that paraquat and porphyrin treatment caused genotoxicity in the cells. This fact can be observed by the increase of DNA damage in the Comet Assay (Figs. 6 and 7) and micronucleus formation, mainly micronucleus and binucleate cells in relation to the control (Table 1).

Fig. 6
figure 6

HaCaT cells were treated with paraquat, porphyrin at 70 μM for 24 h for evaluating genotoxicity by Comet Assay. The results were compared against the percentage of negative control (only cells). N = 3, significant differences ***p < 0.001

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Fig. 7
figure 7

Classification of damages in the comet test according to the length of the tail

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Table 1 Summary of results for the keratinocytes micronucleus test
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Cellular apoptosis was also investigated, and the results are represented in Fig. 8. Our findings showed an increase of Annexin V marker in cells treated with paraquat, indicating high levels of apoptotic cells (Fig. 8f). On the other hand, porphyrin did not alter apoptosis levels when compared to the control.

Fig. 8
figure 8

HaCaT cells were treated with paraquat and porphyrin at 70 μM for 24 h to evaluate apoptosis by Annexin V marker. Cells were treated with porphyrin (a) and stained with Annexin V (b). Control, only cells and medium (b) and stained with Annexin V (e). Cells were exposed to paraquat (c) and stained with Annexin V (f)

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Keap1 and Nrf2 gene expression was analyzed by qRT-PCR. The results showed that porphyrin treatment increased Keap1 and Nrf2 gene expression while paraquat decreased Nrf2 gene expression when compared to the control. In this sense, the modulatory effect was more expressive after porphyrin exposure (Fig. 9).

Fig. 9
figure 9

HaCaT cells were treated with paraquat and porphyrin at 70 μM for 24 h to evaluate Keap1 and Nrf2 gene expression. N = 3, significant differences *p < 0.05 and ***p < 0.001

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Discussion

Keratinocytes are important cells of the human epidermis protecting the body against environmental stressors and pathogens, that potentially trigger oxidative stress. Chronic exposure to oxidative stress is linked to skin aging, inflammatory processes, and also cancer development [1]. SOD2 enzyme is one of the crucial enzymes in the protection against oxidative stress generated into the mitochondria. However, a specific SNP found in this enzyme, known as Val16Ala-SOD2 confers distinct enzyme efficiencies depending on the allele, especially homozygous alleles (VV and AA), that have been associated with some types of cancer [7]. Due to the generation of a superoxide-hydrogen peroxide imbalance related mainly to the homozygous genotypes, investigating how keratinocytes are affected by this oxidative imbalance is of relevance to understand the mechanisms that drive cellular aging, inflammatory diseases, and cancer development. In this sense, we investigated here the effect of the O·−2–H2O2 imbalance, generated by paraquat and porphyrin exposition, on the keratinocytes cell line (HaCaT) oxidative metabolism. Paraquat acts as VV-SOD2 like molecule, increasing O·−2, while porphyrin increases H2O2, acting as a AA-SOD2 like molecule. In general terms, our findings revealed that this imbalance differentially modulates the keratinocytes oxidative stress, mainly by modulating Keap1-Nrf2 gene expression pathway.

Our results showed that the concentration of paraquat and porphyrin that caused an imbalance in HaCaT cells (70 µM), decreased cell viability (for paraquat) and increased cell viability and proliferation (for porphyrin). Paraquat decreased viability possibly due to higher O·−2 levels production. High levels of O·−2 can generate H2O2 by dismutation reaction, catalyzed by SOD. Also, O·−2 can donate an electron to ferric ion (Fe3+) to form ferrous ion (Fe2+), that reacts with H2O2 and generates a very potent oxidant ·OH. This event is called as Fenton reaction. This molecule can generate damage in organic molecules, such as lipid, protein, and DNA [26].

On the other hand, the cellular response to H2O2 levels can directly activate in some cases specific growth signaling pathways, such as the Mitogen Activated Protein Kinases (MAPKs) [27]. This fact can explain our findings using porphyrin treatment. Our results showed that porphyrin increased cell viability and proliferation, since this molecule produces H2O2 that can activate cell growth and viability pathways.

ROS and NO production was also tested in this investigation. The results found in the DCFH-DA Assay confirm that porphyrin treatment produced high levels of ROS. Moreover, high levels of NO were detected by porphyrin exposure. However, paraquat treatment showed normal levels of ROS and NO following high lipoperoxidation levels. This result indicated that lower levels of ROS and NO were found due to the interaction of O·−2 with NO, producing peroxynitrite (ONOO−), the main molecule responsible for lipid damage. This fact was confirmed by high levels of TBARS following paraquat exposure. The study performed by Kocak-Toker et al. [28] corroborates with our findings, since these authors reported an increase in lipid damage due to ONOO− exposure. However, lipid damage was not altered following porphyrin treatment, indicating that H2O2 produced by porphyrin was directed to protein damage, since there were high levels of protein carbonyl after porphyrin treatment. The study of Zhang et al. [29] corroborates with our findings, since they reported that H2O2 is responsible to generate protein damage.

Additionally, DNA damage was determined in our study. Comet Assay as well as Micronucleus Test suggested that both treatments, with paraquat and porphyrin, caused DNA injures detected in these experimental approaches. High production of O·−2 and H2O2 can generate Fenton Reaction, that is responsible for producing ·OH, that presets high affinity to DNA and reported to cause genotoxicity [30, 31]. DNA mutations can generate cell dysfunction and induce apoptosis. This parameter also was measured in our study, and we suggested high apoptosis levels in paraquat exposure, confirming lower cell viability in the MTT Assay.

Moreover, our findings indicated that porphyrin and paraquat exposure may interfere with the Keap1-Nrf2 pathway since porphyrin increased Keap1 and Nrf2 gene expression and paraquat decreased Nrf2 expression when compared to control levels. In general terms, porphyrin seems to modulate this pathway more significantly. Previous studies reported that Keap1 stimulates Nrf2 ubiquitin-dependent proteasomal degradation. When oxidative stress conditions increase, Keap1 is inactivated, and the ubiquitination of Nrf2 stops, upregulating Nrf2 gene expression to protect cells against oxidative metabolism damage [32, 33]. Despite methodological constraints related to our study, results suggested that the O·−2–H2O2 imbalance differentially modulates the keratinocytes cell line oxidative metabolism via Keap1-Nrf2 redox signaling pathway. Porphyrin presented a more pronounced Keap1 up-regulation and a subtler Nrf2 up-regulation, while paraquat decreased the Nrf2 expression. Further investigations of other signaling pathways could help to elucidate by what other mechanisms paraquat and porphyrin could be acting in order to better understand the factors the regulate the oxidative metabolism of superoxide-hydrogen peroxide imbalanced cells.

Conclusion

In summary, we showed that the O·−2–H2O2 imbalance, generated by paraquat and porphyrin exposition, modulated the oxidative metabolism of HaCaT keratinocytes. Our findings revealed that paraquat exposure decreased cell viability, increasing lipoperoxidation and apoptosis. On the other hand, porphyrin treatment increased cell viability and proliferation, ROS and NO production, and generated protein and DNA damage. Moreover, we suggest that the O·−2–H2O2 imbalance differentially regulates the oxidative metabolism of the HaCaT keratinocytes cell line via Keap1-Nrf2 gene expression pathway. Thus, our investigation showed the relevance of the O2−–H2O2 imbalance on HaCaT cells oxidative metabolism, suggesting that possibly similar conditions could be found in AA-SOD2-like (porphyrin exposure) and VV-SOD2-like (paraquat exposure) keratinocyte cells. In general, our results suggest a differential modulation of the oxidative metabolism depending on the type of molecule (H2O2 or O·−2), and that the responses observed could be linked to the Keap1-Nrf2 redox signaling pathway.