The oxidation of p-phenylenediamine, an ingredient used for permanent hair dyeing purposes, leads to the formation of hydroxyl radicals: Oxidative stress and DNA damage in human immortalized keratinocytes

Toxicol Lett. 2015 Dec 15;239(3):194-204. doi: 10.1016/j.toxlet.2015.09.026. Epub 2015 Oct 9.


The hair-dyeing ingredient, p-phenylenediamine (PPD), was previously reported to be mutagenic, possibly by inducing oxidative stress. However, the exact mechanism of PPD in inducing oxidative stress upon skin exposure during hair-dyeing in human keratinocytes remains unknown. The aim of our studies was therefore to investigate the toxicity of PPD and its by-products in human immortalized keratinocytes (HaCaT) after auto-oxidation and after reaction with hydrogen peroxide (H2O2). We found that the PPD half maximal effective cytotoxic concentration (EC50) to HaCaT is 39.37 and 35.63 μg/mL after 24 and 48 h, respectively, without addition of H2O2 to induce oxidation. When PPD (10 or 100 μg/mL) is combined with 10.5 μg/mL of H2O2, intracellular ROS production by HaCaT after 1 h was significantly increased and enhanced levels of DNA damage were observed after 4 h of exposure. After 24 h incubations, 20 μg/mL of PPD increased the level of DNA oxidation in HaCaT. Also, we found that the in vitro reaction between PPD and H2O2, even below the maximum allowance by cosmetic industries, released hydroxyl radicals which can damage DNA. Taken together, we conclude that PPD alone and when combined with H2O2 increases the formation of reactive oxygen species in human keratinocytes, leading to oxidative stress and subsequent DNA damage. These alterations suggest that the mechanism by which PPD exposure, alone or combined with H2O2, damages keratinocytes by the formation of the high reactive HO∙ radicals.

Keywords: DNA damage; HaCaT; Hair dyes; Oxidative stress; p-Phenylenediamine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Chromatography, Liquid
  • DNA Damage / drug effects
  • Dose-Response Relationship, Drug
  • Hair Dyes / analysis*
  • Humans
  • Hydrogen Peroxide / metabolism
  • Hydroxyl Radical / metabolism*
  • Keratinocytes / drug effects*
  • Keratinocytes / metabolism
  • Malondialdehyde / metabolism
  • Oxidative Stress / drug effects*
  • Phenylenediamines / toxicity*
  • Reactive Oxygen Species / metabolism
  • Skin / cytology
  • Tandem Mass Spectrometry


  • Hair Dyes
  • Phenylenediamines
  • Reactive Oxygen Species
  • Hydroxyl Radical
  • Malondialdehyde
  • Hydrogen Peroxide
  • 4-phenylenediamine