SummaryAlthough p-phenylenediamine (PPD) has been recognized as an extreme sensitizer for many years, the exact mechanism of sensitization has not been elucidated yet. Penetration and the ability to bind to proteins are the first two hurdles that an allergen has to overcome to be able to sensitize. This review is an overview of studies regarding PPD penetration through skin (analogues) and studies on the amino acids that are targeted by PPD. To complete this review, the auto-oxidation and N -acetylation steps involved in PPD metabolism are described. In summary, under normal hair dyeing exposure conditions, <1% of the applied PPD dose penetrates the skin. The majority (>80%) of PPD that penetrates will be converted into the detoxification products monoacetyl-PPD and diacetyl-PPD by the N -acetyltransferase enzymes. The small amount of PPD that does not become N -acetylated is susceptible to auto-oxidation reactions, yielding proteinreactive PPD derivatives. These derivatives may bind to specific amino acids, and some of the formed adducts might be the complexes responsible for sensitization. However, true in vivo evidence is lacking, and further research to unravel the definite mechanism of sensitization is needed.Key words: haptenation; penetration; p-phenylenediamine; protein binding; sensitization.p-Phenylenediamine (PPD) is an aromatic amine known for its extreme sensitizing potency, and may cause severe allergic contact dermatitis. It is predominantly used as a precursor in oxidative hair dyes, but it can also be found in so-called 'temporary henna tattoos', despite the prohibition of this application in Europe and the United States (1, 2). Hair dyeing -in both consumer and occupational settings -has been shown to be a common cause of PPD sensitization (3). Whereas allergic contact dermatitis in consumers gives rise to erythema and oedema (sometimes severe) of the face, eyelids, scalp, and neck, the hands are most affected in hairdressers (4).Although allergic contact dermatitis caused by PPD has been studied extensively, the exact pathways of sensitization and elicitation are not yet completely unravelled. Nevertheless, as is generally known, in order to induce a T cell-mediated immune response, a chemical needs to undergo several steps. Briefly, a chemical has to penetrate the stratum corneum to reach the living epidermis. Here, the low molecular weight chemical, the hapten, will haptenate (i.e. be bound to) cutaneous protein. The hapten-protein complex will be formed inside the cell or be taken up and subsequently processed by antigen-presenting cells of the epidermis or dermis. These cells become activated and migrate to regional lymph nodes, where they may encounter and activate allergen-specific naïve T cells. Expanded progeny of these allergen-specific T cells, which are then turned into effector and memory T cells, will recirculate and migrate to the skin. On re-exposure to the initial allergen, the abovementioned process will recur, but now memory T cells can immediately release cytokines and chemo...

N-acetyltransferase 1 (NAT1)-mediated N-acetylation in keratinocytes is an important detoxification pathway for the hair dye ingredient para-phenylenediamine (PPD). Because NAT1 can be regulated by various exogenous compounds, including some NAT1 substrates themselves, we investigated NAT1 expression in keratinocytes and the interactions between PPD and NAT1. NAT1 activity was found to be cell-cycle phasedependent. Maximum NAT1 activities (mean: 49.7 nmol/mg/ min) were estimated when HaCaT keratinocytes were arrested in G 0 /G 1 phase, whereas nonsynchronized cells showed the lowest activities (mean: 28.9 nmol/mg/min). It is noteworthy that we also found an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. This evidence suggests an association between NAT1 and proliferation in keratinocytes. Regarding the interaction between NAT1 and PPD, we found that keratinocytes N-acetylate PPD; however, this N-acetylation was saturated with increasing PPD concentrations. HaCaT cultured in medium supplemented with PPD (10 -200 M) for 24 h showed a significant concentration-dependent decrease (17-50%) in NAT1 activity. PPD also induced down-regulation of NAT1 activity in human primary keratinocytes. Western blot studies using a NAT1-specific antibody in HaCaT showed that the loss of enzyme activity was associated with a decline in the amount of NAT1 protein, whereas no changes in the amounts of NAT1 P1 (NATb)-dependent mRNA were found by quantitative reverse transcription-polymerase chain reaction analysis, suggesting the involvement of a substrate-dependent mechanism of NAT1 down-regulation. In conclusion, these data show that overall N-acetylation capacity of keratinocytes and consequently detoxification capacities of human skin is modulated by the presence of NAT1 substrates and endogenously by the cell proliferation status of keratinocytes.

Since animal testing should be avoided whenever possible, the development of in vitro tests for predicting the effect of chemicals becomes a major field. This rise of in vitro test systems led to an increased requirement for well-characterized continuously growing cell lines. Monitoring of the cells during test and routine culture is necessary to gain relevant and reproducible results. In the present study, the influence of passaging under constant culture conditions on the human keratinocyte cell line HaCaT was investigated. Data demonstrated that growth rate rose with increasing passages. Doubling times of the cells were decreased to 24 ± 0.6 h in the late passages (12-16), in comparison to 36.2 ± 1.5 h in the early passages (2-8). These data were confirmed by a fall in mRNA expression levels of keratin 1 and transglutaminase 1 within the passages. Furthermore, the activities of the xenobiotic metabolizing phase II enzyme N-acetyltransferase 1 (NAT1) were higher in the late passages compared to the early passages. These results are contrary to an expected decrease in enzyme activity and proliferation rate induced by replicative senescence or cell aging. Data also indicate that routine culture might result in significant changes in proliferation and phase II metabolism. These findings reinforce the necessity of a strict characterization and knowledge of regulation of in vitro systems, as well as the need for new biomarkers, in order to use cells for the development and evaluation of reproducible in vitro test systems.

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