Evaluation of Cd-induced cytotoxicity in primary human keratinocytes

Abstract

An increasing number of studies have investigated the effects of Cd on human health. Cd-induced dermatotoxicity is an important field of research, but numerous studies have focused on the effects of Cd on the human skin. Moreover, most studies have been performed using HaCaT cells but not primary keratinocytes. In this study, we provide the results describing the cytotoxic effects of Cd exposure on primary human epidermal keratinocytes obtained from different donors. The subtoxic concentration of cadmium chloride was determined via MTT assay, and transcriptomic analysis of the cells exposed to this concentration (25 µM) was performed. As in HaCaT cells, Cd exposure resulted in increased ROS levels, cell cycle arrest, and induction of apoptosis. In addition, we report that exposure to Cd affects zinc and copper homeostasis, induces metallothionein expression, and activates various signaling pathways, including Nrf2, NF-kB, TRAIL, and PI3K. Cd induces the secretion of various cytokines (IL-1, IL-6, IL-10, and PGE2) and upregulates the expression of several cytokeratins, such as KRT6B, KRT6C, KRT16, and KRT17. The results provide a better understanding of the mechanisms of cadmium-induced cytotoxicity and its effect on human epidermal skin cells.

Keywords

Cadmium keratinocytes normal human epidermal keratinocytes Transcriptomics

Introduction

Cadmium is a heavy transition metal with a broad range of industrial applications, leading to its release into the environment and severe ecological damage.^1,2 Cd does not have any biological functions and is highly toxic to all ecosystems.^3–5 Cd contamination leads to dramatic consequences for human health, including neurodegenerative diseases,⁶ several types of cancer,^1,7 and reproductive dysfunction.^8,9 Cd is poorly excreted from the organism and has a half-life of 10–35 years.¹⁰ Dermal exposure to heavy metals has received less attention than oral/inhalation exposure. However, in specific areas such as mine tailings or industrial enterprises, the risk of dermal exposure may be significant.^11,12 Notably, Wang et al. demonstrated that, in urban soil samples, Cd exhibited the highest dermal bioaccessibility relative to other heavy metals.¹³

In contact with the skin, cadmium may lead to epidermal barrier disruption accompanied by apoptosis induction and genotoxic stress.¹⁴ On the rat model, it was shown that cadmium exposure induced various skin pathologies including hyperkeratosis, acanthosis, ex-pression of pro-inflammatory cytokines and mitotic index increase.^15,16 Consequently, these changes may trigger psoriasis, atopic dermatitis and other pathologies. Previously, elevated level of Cd was observed in the skin of psoriatic patients with industrial exposure.¹⁷ Similarly, Liaw et al. showed a correlation between plasma Cd levels and psoriasis severity.¹⁸ Another clinical study revealed a significant difference in blood Cd levels between patients with psoriasis and the control group.¹⁹ These studies provide evidence supporting the significant role of Cd toxicity in the emergence of skin pathologies.

Few studies have focused on Cd cytotoxicity in keratinocytes, and most of these studies used HaCaT keratinocytes as a model cell line. In particular, Cd induces cell cycle arrest, causes genotoxic stress, and increases reactive oxygen species (ROS) formation in HaCaT keratinocytes.^20–22 HaCaT cells are a reliable alternative to normal human epidermal keratinocytes (NHEK) and are widely used in cytotoxicity studies.^23–26 However, these cells bear two gain-of-function (GOF) mutations in TP53. The novel properties of mutant p53 are realized through two mechanisms. First, mutant p53 is capable of dimerizing with other transcription factors, preventing their binding to DNA. Second, mutant p53 can activate a much larger number of targets through direct interaction with other transcription factors (NF-Y, E2F1, NF-KB, and VDR).²⁷ These cells are unable to complete differentiation, have altered growth and proliferation rates, and exhibit impaired regulation of apoptosis.^27–29 Thus, despite considered normal, HaCaT significantly differ from NHEK, which may limit the application of HaCaT cells as a model cell line for toxicological studies. Given the findings of the comparative proteomic analysis of HaCaT and NHEK, it is recommended to utilize primary cells as a model system, if feasible.³⁰ However, interpreting experimental results obtained from NHEK can be challenging due to donor-to-donor variability, the limited lifespan of the cultured cells, and differences between passages.²⁹

Transcriptomic analysis is a powerful tool to investigate gene activity under various conditions, including the impact of toxic substances.^31,32 RNA-Seq, a modern RNA sequencing technology, allows for precise identification and measurement of gene activity, including those that are weakly expressed.^33,34

The purpose of this study was to evaluate the mechanisms of Cd toxicity in NHEK. A comprehensive transcriptomic analysis was conducted to detect differentially expressed genes between control and Cd-treated NHEK. Donor-to-donor variability was levelled using cells from three independent donors.

Materials and methods

Chemicals and reagents

Cadmium chloride was purchased from Sigma-Aldrich (USA). Keratinocyte serum free medium (KSFM), GlutaMAX^TM and penicillin/streptomycin solution were purchased from Gibco (USA). Dulbecco's phosphate-buffered saline (DPBS) and 0.25% trypsin-EDTA solution were from Capricorn Scientific (Germany). Annexin V-FITC/propidium iodide (PI) apoptosis it and the Cycle Assay Kit were from Elabscience (USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent was from Dia-M (Russia). RNA isolation kit was from QIAGEN (USA). Dimethyl sulfoxide (DMSO) was purchased from Helicon (Russia). IL-1, IL-6 and IL-8 Enzyme-linked immunosorbent assay (ELISA) kits were from Vector-Best (Russia). Prostaglandin E₂ (PGE2) ELISA was from Cayman Chemical (USA).

Cell culture and treatment

NHEK were purchased from Perspectiva Research-and-Production Company (Russia). NHEK were cultured in full KSFM (1x) supplemented with 1% GlutaMAX^TM and penicillin/streptomycin (100 UI/mL and 100 μg/mL). The cells were cultured in 25 or 75 cm² tissue treated flasks (Corning, USA) in a humidified atmosphere containing 5% CO₂ at 37°C. NHEK were passaged upon reaching the confluence of approximately 50%. The medium was replaced with fresh every other day. For all experiments, the cells were harvested by trypsinization, seeded in the appropriate culture plates and grown at 37°C under humidified atmosphere containing 5% CO₂. For all experimental procedures pooled NHEK obtained from 3 donors were used in order to level donor-to donor variability. Cadmium chloride was dissolved to the desired concentration in regular culture medium. The cell morphology was observed by a Zeiss Primovert microscope (ZEISS, Germany).

MTT assay

For MTT assay the cells were seeded in 96-well tissue-treated plates (Corning, USA) at density of 5.0 × 10³ cells/well 24 h prior to cadmium treatment. The day after seeding the culture medium was replaced with medium containing cadmium. The cells were incubated for 24 h as described at 4. 2. After exposition, the culture medium was replaced with fresh containing MTT (1 µg/mL) following a 3 h incubation under culture conditions. Then, the medium was discarded and the cells were dissolved in DMSO. The absorbance was measured via iMark microplate reader (Bio-Rad, USA) at 590 nm.

Annexin V-FITC/PI apoptosis assay and cell cycle analysis

Cell cycle analysis and annexin V-FITC/PI kits were employed to detect apoptosis and cell cycle distribution. The cells were seeded in 60 mm Petri dishes at a density of 5.0 ×10⁵ cells. On the following day, the medium was replaced with containing cadmium or with fresh medium in control groups. After 24 h of exposure the cells were harvested by trypsinization, rinsed twice with DPBS solution and stained according to the manufacturer’s instructions. After staining, the cells were immediately analyzed via a ZE5 flow cytometer (Bio-Rad, USA). The raw data were analyzed using Floreada.io software (https://floreada.io/analysis).

Transcriptomic analysis

For transcriptomic analysis NHEK cells were seeded onto 100 mm Petri dishes (Corning, USA) at a density of 1.0 × 10⁶ cells per plate. The cells were cultured for 24 h, then the culture medium was replaced with containing cadmium (25 µM). After 24 h, the cells were collected by trypsinization following RNA isolation. Total RNA was isolated according to the manufacturer’s protocol and quantified using NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Transcriptome data were obtained by high throughput paired-end sequencing using Illumina NovaSeq 6000 with a read length equal to 100 bp. The TruSeq Stranded mRNA Library Prep Kit was used to prepare RNA libraries. All steps were carried out in accordance with the manufacturer’s protocol. Transcriptome profiling was performed in three replicates with a separate process of RNA extraction for each replicate. Raw sequencing data were uploaded to the NCBI SRA (https://www.ncbi.nlm.nih.gov, accessed on 14 August 2023). The accession number is PRJNA1005459 for both control and treatment cells.

ELISA

NHEK were seeded in 12-well culture plates at a density of 5.0 × 10⁴ cells/well and incubated under culture conditions. After 24 h, the culture medium was replaced with containing cadmium chloride (25 µM). The cells were treated with Cd for 24 h, after which the conditioned medium was collected for cytokine detection. Commercially available ELISA kits were used to measure the levels of IL-1, IL-6, IL-8, and PGE2 in the culture medium. The assays were performed according to the manufacturer’s protocol. Absorbance was measured using an iMark microplate reader (Bio-Rad, USA) at 450 nm.

Bioinformatic analysis

The R software environment was used for computations and visualization transcriptomic data (ver. 4.1) (R: The R Project for Statistical Computing https://www.r-project.org/). We used DESeq2 package³⁵ for differential expression analysis. For visualization we used EnhancedVolcano for volcano plot (https://github.com/kevinblighe/EnhancedVolcano, accessed on 25 July 2023) and ClusterProfiler for geneset enrichment analysis³⁶ as described in Poverennaya et al.³⁷ A source of biological knowledge, we utilized Wikipathways³⁸ and PROGENy databases.³⁹

Statistical analysis

All experiments were performed in at least 3 biological replicates. The graphs represent data as means ± SEM. Statistical analysis was carried out using one-way ANOVA with Tukey’s post-test. The analysis was performed using GraphPad Prism Version 8.0 software (GraphPad Software LLC, CA, USA). A p value of <0.05 was regarded as statistically significant.

Results

Evaluation of cadmium-induced cytotoxicity

The cytotoxic effects of cadmium chloride at different concentrations were evaluated using the MTT assay. The viability of pooled NHEK cells was analyzed after 24 h exposition to cadmium chloride at concentrations from 12.5 to 200 µM. A dose-dependent decrease in cell viability was observed within the selected range of concentrations. Significant differences compared to control were observed at concentrations of 25 µM and above (Figure 1(a)). Exposure to higher Cd concentrations (50–200 µM) resulted in severe cytotoxicity and drastically decreased cell viability. Treatment with Cd resulted in changes in cell morphology. No significant changes were observed after exposure to Cd at concentration of 12.5 µM. after treatment with 25 µM Cd, no visible changes in cell morphology were observed; however, a small number of floating cells appeared in the medium. Treatment with 50 µM Cd increased the ratio of floating cells compared to that in the control group, and some cells developed an irregular shape.

Figure 1.

Cytotoxicity of cadmium chloride in pooled NHEK after 24 h exposition at different concentrations (a) – MTT assay. Bars represent mean ± SEM. Asterisks indicate significant differences compared with the control group, **p < .01, ***p < .001 (b) – Morphology of NHEK after treatment with Cd at concentrations of 12.5–50 µM (scale bar = 50 µM).

Exposure to cadmium chloride at a concentration of 25 µM resulted in moderate cytotoxicity; thus, this concentration was selected for further experiments, including transcriptomic analysis.

Apoptosis and cell cycle assays

The cytotoxicity of cadmium chloride at the selected concentration (25 µM) was further evaluated using the Annexin V-FITC/PI assay (Figure 2(a)). After 24 h exposition, the ratio of early apoptotic (EA, Annexin V-positive, Q3) cells increased from 0.68 to 4.41%, whereas the ratio of late apoptotic cells (LA, double-positive, Q2) increased to 5.85%. The overall ratio of apoptotic cells (EA + LA) was 10.84 ± 1.19%. In line with the MTT assay results, the ratio of viable cells (unstained, Q3) decreased to 89.59%. The cell cycle distribution analysis revealed that exposure to cadmium increased the ratio of cells in the G1 phase from 41.67 ± 1.15% to 55.5 ± 0.53%. In the S phase, the differences between the control and cadmium-treated groups were not significant. The proportion of cells in G2 decreased from 22.86 ± 1.17% to 14.35 ± 2.71% (Figure 2(b)). The results showed that exposure to cadmium chloride at the selected concentration resulted in cell cycle arrest at the G0/G1 phase.

Figure 2.

Evaluation of apoptosis and cell cycle analysis after 24 h exposition with 25 µM of cadmium chloride. (a) Annexin V-FITC/Propidium iodide assay. The statistical analysis graph represents cells in early apoptosis and late apoptosis/necrosis (Q2 + Q4) (b) Flow cytometry results of cell cycle analysis. Asterisks indicate significant differences between groups. *p < .05, ***p < .001.

Transcriptomic differences

In the result of RNAseq (Illumina) analysis 13257 and 13817 genes were identified in control and Cd-treated cells, respectively (TPM >0). Transcriptome profiling was employed to assess gene expression differences between the control cells and those exposed to Cd. Analysis of the differentially expressed genes revealed 1463 genes with significant alterations. Specifically, 547 genes were downregulated, while 916 genes were upregulated upon cadmium treatment (Figure 3(a)). To gain deeper insights into the impact of Cd on cellular pathways, we conducted gene set enrichment analysis using the list of differentially expressed genes (Figure 3(B), Supplementary Table SN). Remarkably, following Cd exposure, the number of activated genes was nearly twice as high as the number of suppressed genes. Genes with differential expression were sorted in decreasing order of the range of change.

Figure 3.

Transcriptomic profiling of intact and Cd-treated NHEK (a) Volcano plot showing differentially expressed genes between control and Cd-treated cells. X-axis: log2 fold change in gene expression. Y-axis: -log10 of the p-value, emphasizing statistical significance of gene expression changes. (b) Gene Set Enrichment Analysis of differentially expressed genes between control and Cd-treated cells. FDR—false discovery rate. The geneRatio value is the proportion of genes in a specific function/pathway from the total differentially expressed genes.

In response to cadmium exposure, the most pronounced activation was observed in eight metallothioneins (MT) genes: MT1E (log₂FC = 4,84, FDR <0,0001), MT1F (log₂FC = 7,79, FDR <0,0001), MT1G (log₂FC = 12,26, FDR <0,0001), MT1H (log₂FC = 14,11, FDR <0,0001), MT1L (log₂FC = 7,79, FDR <0,0001), MT1X (log₂FC = 5,85, FDR <0,0001), MT2A (log₂FC = 1,81, FDR <0,0001) and MT1M (log₂FC = 5,85, FDR <0,0001). MT genes encode proteins that form metal-thiolate bonds with cysteine residues, facilitating metal ion chelation.⁴⁰ Additionally, in cadmium-treated cells, there was an increase in the expression of the SLC30A1 gene (log₂FC = 3,1, FDR <0,0001), which is involved in zinc transport.⁴¹

As a result of treatment with cadmium chloride, there was a significant activation of the HMOX1 gene (log₂FC = 8,71, FDR<0,0001). The induction of this gene represents an adaptive response that protects cells from oxidative damage during stress.⁴² The expression of the PPP1R15 A gene (log₂FC = 4,69, FDR <0.0001), was also increased, which is activated in response to genotoxic stress and plays an important role in cell recovery.⁴³ After cadmium exposure, the expression of certain cytokines was upregulated, such as IL1RL1 (log₂FC = 2,92, FDR = 0.01), IL13RA2 (log₂FC = 3,36, FDR <0.0001), IL36G (log₂FC = 2,6, FDR = 0.04), IL6R (log₂FC = 1,42, FDR = 0.003), IL7R (log₂FC = 3,02, FDR = 0.01), IL20RB (log₂FC = 1,78, FDR <0.0001), IL32 (log₂FC = 2,52, FDR = 0.0002), IL11 (log₂FC = 3,65, FDR <0.0001) and PTGS2 (log₂FC = 1,97, FDR <0.0001). Cytokines are signaling molecules that play a key role in regulating immune response, inflammation, and cell growth and differentiation.⁴⁴ Data analysis revealed a decrease in the expression of genes encoding laminin-332 (LM) subunits: LAMC2 (log2FC = −1,29, FDR <0.0001), LAMB1 (log2FC = −2,29, FDR <0.0001) and LAMA3 (log2FC = −1,31, FDR <0.0001). LMs play an important structural role in maintaining epithelial adhesion and also have signaling roles in proliferation processes.⁴⁵ Furthermore, the expression of six genes from the "Minichromosome Maintenance” (MCM) family was reduced: MCM10 (log₂FC = −1,87, FDR = 0.006), MCM6 (log₂FC = −1,51, FDR <0.0001), MCM4 (log₂FC = −1,77, FDR <0.0001), MCM3 (log₂FC = −1,65, FDR<0.0001), MCM7 (log₂FC = −1,63, FDR<0.0001) and MCMBP (log₂FC = −1,41, FDR = 0.008). MCM proteins play a role in DNA replication and are involved in assembling and activating the replication fork during the S-phase of the cell cycle.⁴⁶ Genes MCM3, MCM4, and MCM7 are part of the eukaryotic MCM complex, which functions as a DNA helicase necessary for genome DNA replication.⁴⁶ Additionally, the expression of the FGFBP1 (log₂FC = −2,75, FDR <0,0001) was notably reduced. The encoded protein plays a role in cell proliferation, differentiation, and migration by forming complexes with fibroblast growth factors, enhancing their biological activity on target cells. Importantly, the protein enhances the transmission of FGF2 signals during tissue regeneration, angiogenesis, and tumor growth.⁴⁷

During the enrichment analysis, the suppression of five DNA-related pathways was detected: «DNA repair pathways full network» (FDR = 1.22× $10^{‐ 6}$ ), «DNA damage and cellular response via ATR» (FDR = 6.88× $10^{‐ 6}$ ), «DNA replication» (FDR = 0.004), «DNA mismatch repair» (FDR = 0.003) and «Pyrimidine metabolism» (FDR = 0.01). These findings suggest that cadmium exposure may disrupt processes associated with DNA integrity and repair, which in turn could lead to genomic instability and increased risk of cell damage. We observed the activation of the «Zinc homeostasis» (FDR = 2× $10^{‐ 6}$ ) and «Copper homeostasis» (FDR = 0.0004) pathways in response to cadmium treatment. These pathways are activated as part of the cellular stress response induced by metals. Cadmium exposure might disturb the balance of zinc and copper ions within the cell, triggering mechanisms to restore and regulate their levels.

We also identified the activation of pathways associated with responses to oxidative stress and toxic effects, such as the «NRF2 pathway» (FDR = 0.0008), «Glucocorticoid receptor pathway» (FDR = 0.002), and «Nuclear receptors metapathway» (FDR = 2.46 × 10⁻⁵). Overall, the activation of these pathways following cadmium treatment indicates the development of an adaptive response. These pathways contribute to cell survival, reduce cellular damage, and restore cellular homeostasis.

For assessing intracellular signaling activity, we employed Pathway RespOnsive GENes (PROGENy)³⁹— a model of consensus pathway signatures—to calculate a relative activity score for 14 distinct signaling pathways (Figure 4).

Figure 4.

Signal pathways activity of differentially expressed genes between control and Cd-treated cells using PROGENy database. NES - normalized enrichment score.

The «Hypoxia» pathway (NES = 14.09) is the most active pathway in the studied perturbation. In this pathway, the gene NDRG1 (log₂FC = 2,97, FDR <0.0001) contributes significantly, being activated in cells treated with cadmium. NDRG1 is a stress-responsive protein whose expression is triggered by various stimuli, including hypoxia, oxidative stress, and exposure to heavy metals. In response to these stimuli, NDRG1 plays a role in regulating cellular proliferation, apoptosis, and angiogenesis.⁴⁸ Activation of the «p53» signaling pathway (NES = 4.23) was also notable, with the gene CDKN1A (log₂FC = 1.18, FDR <0.0001) making a substantial contribution. The expression of the JAK-STAT signaling pathway was reduced after cadmium exposure (NES = −6.73). Within this pathway, the greatest contribution comes from genes of the Interferon-Inducible (IFI) family, such as IFIT1 (log₂FC = −3,34, FDR = 0.002), IFIT3 (log₂FC = −1,83, FDR = 0.001), IFITM1 (log₂FC = −1,01, FDR <0.0001) and IFI44 (log₂FC = −2,53, FDR <0.0001). These genes play a role in regulating responses to various pathogens and cellular stress.

Suppression of the MAPK signaling pathway (NES = −2.21) was also observed. The expression of the negative regulator of this pathway, the DUSP6 gene, was increased under the influence of cadmium (log₂FC = −1.49, FDR <0.0001). This gene plays a role in controlling the duration and intensity of MAPK signal transmission.

Cadmium induces pro-inflammatory cytokines release in NHEK

In order to verify the results of transcriptomic analysis, we analyzed the levels of several pro- and anti-inflammatory cytokines in the NHEK condition medium after exposition with cadmium chloride at concentration of 25 µM using ELISA. The obtained results revealed that IL-1, IL-6, IL-10 and PGE2 were upregulated after cadmium treatment (Figure 5).

Figure 5.

Detection of cytokines in NHEK condition medium after 24 h exposition with subtoxic concentration of cadmium chloride (25 µM) via ELISA. *p < .5 compared to control group.

Discussion

This study aimed to comprehensively evaluate cytotoxicity in primary human keratinocytes after 24 h exposition to cadmium chloride with particular emphasis on transcriptomic analysis. First, the MTT assay was conducted to evaluate the toxicity of cadmium chloride in various concentration, which revealed a dose-dependent decrease in NHEK viability (Figure 1). The decrease of cell viability was statistically significant after exposure to Cd at concentrations of 25 µM and above. Based on the MTT assay, the concentration of 25 µM was selected for further experiments, including transcriptomic analysis. Annexin V/propidium iodide assay revealed, that Cd induces apoptosis in NHEK cells. In addition, cell cycle analysis revealed that exposure to Cd induced changes in cell cycle distribution, similar to the results obtained from HaCaT cell.^20,49

Cadmium can substitute other metals like zinc, copper and magnesium in the metal-binding sites of metallothioneins (MTs), resulting in their dysfunction.⁵⁰ This, in turn, may result in alterations in the homeostasis of metals, carrying out the essential biological functions.^50,51 Transcriptomic data revealed, that exposure to Cd causes significant alterations in zinc and copper homeostasis in NHEK. Also, exposition with cadmium results in significant upregulation of several MTs genes, as it has earlier been shown on different cells.^52,53 Upregulation of MTs is a protective mechanism against metal-induced cytotoxicity.^40,52,54 Cd has a high affinity to SH-groups in MTs, hence binding with MTs reduces cadmium toxicity.^55,56

Cd cytotoxicity is heavily expressed through ROS formation and oxidative stress, reflected in our transcriptomic data.^{22,49,57–59} Enrichment analysis revealed that the Nrf2 pathway, known as the oxidative stress sensor, was activated after cadmium treatment.⁴² Also, many of the Nrf2-downstream genes, associated with response to oxidative stress, were upregulated after exposure to Cd, including NQO1, NQO2, HMOX1, AKR1C1, AKR1C2, AKR1C3, SOD3).^42,60–63 ROS can also induce various cytokines in keratinocytes, confirmed by transcriptomic analysis and ELISA.^64,65 A recent study has shown that Cathepsin D preproprotein (CTSD), a protein responsible for intracellular protein degradation, was upregulated in HaCaT cells after treatment with cadmium.⁶⁶ According to our results, this gene was also upregulated in NHEK exposed to cadmium. Remarkably, two more autophagy-related genes (ATG9 and ATG12) were found to be upregulated after Cd²⁺ treatment.^66,67

It is known that keratinocytes are a major source of cytokines in the epidermis. Pro-inflammatory cytokines are actively produced in response to damage and inflammation of the epidermis. Conversely, anti-inflammatory cytokines and growth factors are important regulators of the regenerative phase, exerting pleiotropic effects and influencing dermal cells (fibroblasts, macrophages). In the analysis of transcriptomic data from Cd-treated NHEK, elevated expression levels of several cytokine genes and/or their receptors were identified. These findings were further validated by determining the levels of individual cytokines in the culture medium (Figure 5). Our results align with those obtained in a rat model, which demonstrated that cadmium induces the expression of certain cytokines in epidermal keratinocytes.¹⁵

These observations ensure coordinated behavior of both epidermal and dermal cells during the repair of damage of various origins. In our study, notably, an increase in FGFBP1 expression was observed, which enhances the transmission of the FGF2 signal—a fibroblast growth factor that exemplifies a pleiotropic cytokine of skin cells. Enhanced cytokine production by epidermal cells is also characteristic of several common chronic skin conditions, such as psoriasis, atopic dermatitis, and other diseases. In this context, the additional cytokine release (induced by cadmium) may have significant pathogenetic implications, influencing the severity of clinical manifestations, recurrence frequency, and resistance to therapy.

Our results indicate that cadmium triggers the activation of keratinocytes in response to damage. Indeed, under the influence of cadmium, the expression of KRT6B, KRT6C, KRT16, and KRT17. These keratins are important in damage regeneration.^68–70 The induction of these keratins can be seen as the development of an adaptive response to the toxic effects of cadmium, along with cytokine secretion (Figure 5). The determination of the levels of certain cytokines in the culture medium after cadmium exposure confirms the development of an adaptive response. The expression of interleukin-1 β (IL-1β) is necessary to activate the T-cells producing IL-17.⁷¹ The impact of IL-1β on keratinocytes leads to the activation of NFκB and/or MAPK (including ERK1/2 and p38)-AP1 signaling, along with IL-17, resulting in the activation of the expression of keratins 6/16/17.⁷² The secretion of IL-6 by keratinocytes is characteristic of skin injuries and activates proliferation through STAT3 activation.⁷¹ The expression of IL-10 in keratinocytes is associated with damage regeneration and prevention of fibrotic responses.⁷³ Increased expression of PGE2 is characteristic of atopic dermatitis.⁷⁴ Probably, PGE2 expression was altered to control the severity of skin injury and inflammation.

Our findings coincide with the data obtained from immortalized HaCaT keratinocytes, supporting the rationale for choosing HaCaT cells as an epidermal model to study Cd cytotoxicity and bioaccessibility.^13,29 Specifically, we confirmed that Cd exposure leads to a decrease in proliferative activity and induction of apoptosis.²⁰ For the first time, we report of signaling pathways, affected by Cd the most, and adaptive mechanisms, which include induction of MTs, redox-sensitive genes and several cytokines in NHEK. The obtained results may provide a basis for further investigation of Cd cytotoxicity and screening of potential therapeutic targets.

Conclusions

In this study, Cd cytotoxicity in NHEK cells was evaluated. In addition to apoptosis and cell cycle arrest, exposure to Cd results in significant alterations at the transcriptomic level. The most prominent differences were associated with the adaptive response, which included the induction of MTs, several cytokines, and redox-sensitive genes.

Supplemental Material

Supplemental Material - Evaluation of Cd-induced cytotoxicity in primary human keratinocytes

Supplemental Material for Evaluation of Cd-induced cytotoxicity in primary human keratinocytes by Daniil Romashin, Viktoriia Arzumanian, Ekaterina Poverennaya, Alexandra Varshaver, Nataliya Luzgina and Alexander Rusanov in Human & Experimental Toxicology.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) (No 122022800481-0).

ORCID iDs

Daniil Romashin

Alexander Rusanov

Supplemental Material

Supplemental material for this article is available online.

References

Genchi

Sinicropi

Lauria

, et al. The effects of cadmium toxicity. Int J Environ Res Publ Health 2020; 17(11): 3782.

Lane

Canty

. Cadmium exposure and consequence for the health and productivity of farmed ruminants. Res Vet Sci 2015; 101: 132–139.

Zoroddu

Aaseth

Crisponi

, et al. The essential metals for humans: a brief overview. J Inorg Biochem 2019; 195: 120–129.

Haider

Liqun

Coulter

, et al. Cadmium toxicity in plants: impacts and remediation strategies. Ecotoxicol Environ Saf 2021; 211: 111887.

Peana

Pelucelli

Chasapis

, et al. Biological effects of human exposure to environmental cadmium. Biomolecules 2022; 13(1): 36.

Ruczaj

Brzóska

. Environmental exposure of the general population to cadmium as a risk factor of the damage to the nervous system: a critical review of current data. J Appl Toxicol 2023; 43(1): 66–88.

Cui

Ahmed

Zaidi

, et al. Ins and outs of cadmium-induced carcinogenesis: mechanism and prevention. Cancer Treat Res Commun 2021; 27: 100372.

Dutta

Gorain

Choudhury

, et al. Environmental and occupational exposure of metals and female reproductive health. Environ Sci Pollut Res Int 2022; 29(41): 62067–62092.

Kumar

Sharma

. Cadmium toxicity: effects on human reproduction and fertility. Rev Environ Health 2019; 34(4): 327–338.

10.

Kubier

Wilkin

Pichler

. Cadmium in soils and groundwater: a review. Appl Geochem 2019; 108: 1–16.

11.

Chaparro Leal

Guney

Zagury

. In vitro dermal bioaccessibility of selected metals in contaminated soil and mine tailings and human health risk characterization. Chemosphere 2018; 197: 42–49.

12.

Filon

D’Agostin

Crosera

, et al. In vitro absorption of metal powders through intact and damaged human skin. Toxicol Vitro 2009; 23(4): 574–579.

13.

Wang

Gao

, et al. Dermal bioaccessibility and cytotoxicity of heavy metals in urban soils from a typical plateau city: implication for human health. Sci Total Environ 2022; 835: 155544.

14.

Dong

Wang

, et al. Study on the mechanism of cadmium chloride pollution accelerating skin tissue metabolism disorder, aging and inhibiting hair regeneration. Front Public Health 2022; 10: 1035301.

15.

Tucovic

Mirkov

Kulas

, et al. Dermatotoxicity of oral cadmium is strain-dependent and related to differences in skin stress response and inflammatory/immune activity. Environ Toxicol Pharmacol 2020; 75: 103326.

16.

Lansdown

Sampson

. Dermal toxicity and percutaneous absorption of cadmium in rats and mice. Lab Anim Sci 1996; 46(5): 549–554.

17.

Afridi

Kazi

, et al. Evaluation of cadmium, chromium, nickel, and zinc in biological samples of psoriasis patients living in Pakistani cement factory area. Biol Trace Elem Res 2011; 142(3): 284–301.

18.

Liaw

Chen

Kao

, et al. Exploring the link between cadmium and psoriasis in a nationally representative sample. Sci Rep 2017; 7(1): 1723.

19.

Wacewicz-Muczyńska

Socha

Soroczyńska

, et al. Cadmium, lead and mercury in the blood of psoriatic and vitiligo patients and their possible associations with dietary habits. Sci Total Environ 2021; 757: 143967.

20.

Cui

Xie

, et al. Mechanisms of Cd-induced cytotoxicity in normal human skin keratinocytes: implication for human health. Int J Mol Sci 2022; 23(19): 11767.

21.

Nzengue

Steiman

Guiraud

. Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines. Free Radic Res 2008; 42(2): 142–153.

22.

Nzengue

Steiman

Garrel

, et al. Oxidative stress and DNA damage induced by cadmium in the human keratinocyte HaCaT cell line: role of glutathione in the resistance to cadmium. Toxicology 2008; 243(1–2): 193–206.

23.

Sergachev

Rusanov

Trushkin

, et al. Fluorescent optical fiber sensors for cell viability monitoring. Analyst 2013; 138(14): 4066–4069.

24.

Rusanov

Luzgina

Lisitsa

. Sodium dodecyl sulfate cytotoxicity towards HaCaT keratinocytes: comparative analysis of methods for evaluation of cell viability. Bull Exp Biol Med 2017; 163(2): 284–288.

25.

Shkrigunov

Kisrieva

Samenkova

, et al. Comparative proteoinformatics revealed the essentials of SDS impact on HaCaT keratinocytes. Sci Rep 2022; 12(1): 21437.

26.

Petushkova

Rusanov

Pyatnitskiy

, et al. Proteomic characterization of HaCaT keratinocytes provides new insights into changes associated with SDS exposure. Biomed Dermatol 2020; 4(1): 4.

27.

Martynova

Pozzi

Basile

, et al. Gain-of-function p53 mutants have widespread genomic locations partially overlapping with p63. Oncotarget 2012; 3(2): 132–143.

28.

Rusanov

Romashin

Kozhin

, et al. Impact of p53 knockout on protein data set of HaCaT cells in confluent and subconfluent conditions. Data 2022; 7(3): 27.

29.

Colombo

Sangiovanni

Maggio

, et al. HaCaT cells as a reliable in vitro differentiation model to dissect the inflammatory/repair response of human keratinocytes. Mediat Inflamm 2017; 2017: 7435621.

30.

Sprenger

Weber

Zarai

, et al. Consistency of the proteome in primary human keratinocytes with respect to gender, age, and skin localization. Mol Cell Proteomics. 2013; 12(9): 2509–2521. doi:10.1074/mcp.M112.025478

23722187

31.

Kwok

Meng

, et al. Whole-transcriptome sequencing (RNA-seq) study of the ZFL zebrafish liver cell line after acute exposure to Cd2+ ions. Aquat Toxicol 2020; 228: 105628.

32.

Cen

, et al. RNA-Seq identifies key reproductive gene expression alterations in response to cadmium exposure. BioMed Res Int 2014; 2014: 529271.

33.

Mutz

Heilkenbrinker

Lönne

, et al. Transcriptome analysis using next-generation sequencing. Curr Opin Biotechnol 2013; 24(1): 22–30.

34.

Lowe

Shirley

Bleackley

, et al. Transcriptomics technologies. PLoS Comput Biol 2017; 13(5): e1005457.

35.

Love

Huber

Anders

. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15(12): 550.

36.

, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation 2021; 2(3): 100141.

37.

Poverennaya

Pyatnitskiy

Dolgalev

, et al. Exploiting multi-omics profiling and systems biology to investigate functions of TOMM34. Biology 2023; 12(2): 198.

38.

Martens

Ammar

Riutta

, et al. WikiPathways: connecting communities. Nucleic Acids Res 2021; 49(D1): D613–D621.

39.

Schubert

Klinger

Klünemann

, et al. Perturbation-response genes reveal signaling footprints in cancer gene expression. Nat Commun 2018; 9: 20.

40.

Waldron

Rutherford

Ford

, et al. Metalloproteins and metal sensing. Nature 2009; 460(7257): 823–830.

41.

Nishito

Kambe

. Zinc transporter 1 (ZNT1) expression on the cell surface is elaborately controlled by cellular zinc levels. J Biol Chem 2019; 294(43): 15686–15697.

42.

Kalthoff

Ehmer

Freiberg

, et al. Interaction between oxidative stress sensor Nrf2 and xenobiotic-activated aryl hydrocarbon receptor in the regulation of the human phase II detoxifying UDP-glucuronosyltransferase 1A10. J Biol Chem 2010; 285(9): 5993–6002.

43.

Santos

CXC

Hafstad

Beretta

, et al. Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling. EMBO J 2016; 35(3): 319–334.

44.

Suzuki

. Chronic inflammation as an immunological abnormality and effectiveness of exercise. Biomolecules 2019; 9(6): 223.

45.

Shaw

Sugden

Hamill

. Laminin polymerization and inherited disease: lessons from genetics. Front Genet 2021; 12: 707087.

46.

Han

Zhao

, et al. Integrative analysis of minichromosome maintenance proteins and their prognostic significance in melanoma. Front Oncol 2021; 11: 715173.

47.

Cheng

Lin

Young

, et al. The influence of fibroblast growth factor 2 on the senescence of human adipose‐derived mesenchymal stem cells during long‐term culture. Stem Cells Transl Med 2020; 9(4): 518–530.

48.

Fang

Kovačević

Park

, et al. Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy. Biochim Biophys Acta 2014; 1845(1): 1–19.

49.

Moon

Lee

Nam

. Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes. Hum Exp Toxicol 2019; 38(8): 992–1003.

50.

Rani

Kumar

Lal

, et al. Cellular mechanisms of cadmium-induced toxicity: a review. Int J Environ Health Res 2014; 24(4): 378–399.

51.

Brzóska

Moniuszko-Jakoniuk

. Interactions between cadmium and zinc in the organism. Food Chem Toxicol 2001; 39(10): 967–980.

52.

Kimura

Hosaka

Nakanishi

, et al. Long-term cadmium exposure enhances metallothionein-1 induction after subsequent exposure to high concentrations of cadmium in P1798 mouse lymphosarcoma cells. J Toxicol Sci 2019; 44(4): 309–316.

53.

Kaji

Yamamoto

Tsubaki

, et al. Metallothionein induction by cadmium, cytokines, thrombin and endothelin-1 in cultured vascular endothelial cells. Life Sci 1993; 53(15): 1185–1191.

54.

Nzengue

Lefebvre

Cadet

, et al. Metallothionein expression in HaCaT and C6 cell lines exposed to cadmium. J Trace Elem Med Biol 2009; 23(4): 314–323.

55.

Elinder

Nordberg

Palm

, et al. Cadmium, zinc, and copper in rabbit kidney metallothionein—relation to kidney toxicity. Environ Res 1987; 42(2): 553–562.

56.

Earley

Cubillas

Warnhoff

, et al. Cadmium hijacks the high zinc response by binding and activating the HIZR-1 nuclear receptor. Proc Natl Acad Sci USA 2021; 118(42): e2022649118.

57.

Branca

JJV

Fiorillo

Carrino

, et al. Cadmium-induced oxidative stress: focus on the central nervous system. Antioxidants 2020; 9(6): 492.

58.

Zhang

Zheng

Wang

, et al. Cadmium-induced oxidative stress promotes apoptosis and necrosis through the regulation of the miR-216a-PI3K/AKT axis in common carp lymphocytes and antagonized by selenium. Chemosphere 2020; 258: 127341.

59.

Paniagua

Diaz-Cueto

Huerta-Reyes

, et al. Cadmium exposure induces interleukin-6 production via ROS-dependent activation of the ERK1/2 but independent of JNK signaling pathway in human placental JEG-3 trophoblast cells. Reprod Toxicol 2019; 89: 28–34.

60.

Penning

. Aldo-keto reductase regulation by the Nrf2 system: implications for stress response, chemotherapy drug resistance, and carcinogenesis. Chem Res Toxicol 2017; 30(1): 162–176.

61.

Yang

Chen

Liang

, et al. Up-regulation of heme oxygenase-1 by celastrol alleviates oxidative stress and vascular calcification in chronic kidney disease. Free Radic Biol Med 2021; 172: 530–540.

62.

Ross

Siegel

. The diverse functionality of NQO1 and its roles in redox control. Redox Biol 2021; 41: 101950.

63.

Zhou

Wang

, et al. Aldo-keto reductase 1C3 mediates chemotherapy resistance in esophageal adenocarcinoma via ROS detoxification. Cancers 2021; 13(10): 2403.

64.

Choi

Park

Choi

, et al. Keratinocytes-Derived reactive oxygen species play an active role to induce type 2 inflammation of the skin: a pathogenic role of reactive oxygen species at the early phase of atopic dermatitis. Ann Dermatol 2021; 33(1): 26–36.

65.

Zhang

Zhou

, et al. Nav1.8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases. Redox Biol 2022; 55: 102427.

66.

Han

Kang

, et al. Autophagy triggers CTSD (cathepsin D) maturation and localization inside cells to promote apoptosis. Autophagy 2021; 17(5): 1170–1192.

67.

Bernard

Jin

, et al. A large-scale analysis of autophagy-related gene expression identifies new regulators of autophagy. Autophagy 2015; 11(11): 2114–2122.

68.

Jiang

Sun

Dang

, et al. TGFβ/SMAD/microRNA-486-3p signaling Axis mediates keratin 17 expression and keratinocyte hyperproliferation in psoriasis. J Invest Dermatol 2017; 137(10): 2177–2186.

69.

Jin

Wang

. Keratin 17: a critical player in the pathogenesis of psoriasis. Med Res Rev 2014; 34(2): 438–454.

70.

Wong

Coulombe

. Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair. J Cell Biol 2003; 163(2): 327–337.

71.

MacLeod

Mansbridge

. The innate immune system in acute and chronic wounds. Adv Wound Care 2016; 5(2): 65–78.

72.

Zhang

Yin

Zhang

. Keratin 6, 16 and 17—critical barrier alarmin molecules in skin wounds and psoriasis. Cells 2019; 8(8): 807.

73.

Singampalli

Balaji

Wang

, et al. The role of an IL-10/hyaluronan Axis in dermal wound healing. Front Cell Dev Biol 2020; 8: 636.

74.

Fogh

Herlin

Kragballe

. Eicosanoids in skin of patients with atopic dermatitis: prostaglandin E2 and leukotriene B4 are present in biologically active concentrations. J Allergy Clin Immunol 1989; 83(2 Pt 1): 450–455.

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