Implication of microRNA regulation in para-phenylenediamine-induced cell death and senescence in normal human hair dermal papilla cells

Abstract

Para-phenylenediamine (PPD) is a major component of hair coloring and black henna products. Although it has been largely demonstrated that PPD induces allergic reactions and increases the risk of tumors in the kidney, liver, thyroid gland and urinary bladder, the effect on dermal papilla cells remains to be elucidated. Therefore, the current study evaluated the effects of PPD on growth, cell death and senescence using cell-based assays and microRNA (miRNA) microarray in normal human hair dermal papilla cells (nHHDPCs). Cell viability and cell cycle analyses demonstrated that PPD exhibited a significant cytotoxic effect on nHHDPCs through inducing cell death and G2 phase cell cycle arrest in a dose-dependent manner. It was additionally observed that treatment of nHHDPCs with PPD induced cellular senescence by promoting cellular oxidative stress. In addition, the results of the current study indicated that these PPD-mediated effects were involved in the alteration of miRNA expression profiles. Treatment of nHHDPCs with PPD altered the expression levels of 74 miRNAs by ≥ 2-fold (16 upregulated and 58 downregulated miRNAs). Further bioinformatics analysis determined that these identified miRNA target genes were likely to be involved in cell growth, cell cycle arrest, cell death, senescence and the induction of oxidative stress. In conclusion, the observations of the current study suggested that PPD was able to induce several cytotoxic effects through alteration of miRNA expression levels in nHHDPCs.

Figure 1

Effect of PPD on viability of nHHDPCs. nHHDPCs (5×10³) were seeded into 96-well plates and treated with various concentrations of PPD (0, 100, 200, 300, 400, 500 and 600 μM) for 24 h. Cell viability was measured using the water-soluble tetrazolium salt assay. Values are presented as the mean ± standard error of the mean of the percentage of control optical density of experiments performed in triplicate. ^*P<0.05 vs. 0 μM PPD. PPD, para-phenylenediamine; nHHDPCs, normal human hair dermal papilla cells.

Figure 2

PPD induced cell death and G₂ arrest in nHHDPCs. nHHDPCs (2×10⁶ cells) were seeded into 60-mm culture dishes, treated with PPD (0, 200, 400 and 600 μM) for 24 h, collected and stained with PI. (A) The fluorescence-intensity distribution of the stained cells was analyzed by flow cytometry. (B) The percentage of sub-G₁ cells and the ratio of G₁/G₂ cells were then quantified. Sub-G₁, G₁, S and G₂/M phases were separated using gates M1, M2, M3 and M4, respectively. ^*P<0.05 vs. 0 μM PPD. PPD, para-phenylenediamine; nHHDPCs, normal human hair dermal papilla cells; PI, propidium iodide.

Figure 3

PPD increases intracellular ROS production and senescence in nHHDPCs. (A) nHHDPCs (2×10⁶ cells) were seeded into 60-mm culture dishes and treated with PPD (0 and 400 μM) for 24 h. Cells were then collected and stained with DCF-DA solution. The fluorescence-intensity distribution of DCF-DA-stained cells was analyzed using flow cytometry. Alterations in the intracellular levels of ROS were determined using the M1 gate. (B) Senescence was measured using the senescence associated-β-galactosidase assay. nHHDPCs (2×10⁶) were seeded in 60-mm culture dishes and treated with PPD (0 and 400 μM) for 48 h, fixed and then reacted with X-gal. ^*P<0.05 vs. 0 μM PPD. PPD, para-phenylenediamine; ROS, reactive oxygen species; nHHDPCs, normal human hair dermal papilla cells; DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate.