Penetration and haptenation of p-phenylenediamine.

Although 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 protein-reactive 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.

Cyanamide-mediated Inhibition of N-acetyltransferase 1.

Cyanamide has been used for decades for medical intentions in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. Its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolized in vivo mainly via coenzyme A dependent N-acetylation by N-acetyltransferases. Although described to be a substrate for N-acetyltransferases (NATs), cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for N-acetyltransferases. Therefore, a more detailed investigation of its interrelations with N-acetyltransferases was performed. We analyzed the impact of cyanamide on NAT1 activities of human monocytes (monocytic THP-1 cells) using the classical substrate p-aminobenzoic acid. We found that a 24h treatment with physiologically relevant concentrations of cyanamide decreased the NAT1 activity significantly. Based on this observation we performed additional experiments using recombinant human NAT1 and NAT2 to achieve further insights. In detail a significant dose- and time-dependent inhibition of NAT1 activity was observed for 100 and 1000µM cyanamide using recombinant human NAT1*4. However, cyanamide did not inhibit recombinant NAT2*4. Experiments testing cyanamide as substrate did not provide evidence that cyanamide is metabolized via coenzyme A dependent N-acetylation in vitro by human NAT1 or NAT2, THP-1 or human liver cytosol. Therefore we can conclude that the observed enzyme inhibition (around 50% and 25% after treatment with 0.5 and 0.25mM CA, respectively) is not based on substrate-dependent down-regulation of NAT1. Further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of NAT1, leading to its rapid inhibition (significant inhibition after 30min and 2h for 1000 and 100µM CA, respectively). Addition of the reduction agent dithiothreitol (DTT) did not modify the effect, indicating that oxidative processes that can be reversed by 5mM DTT are not likely involved in the inhibition. Taken together our results show that cyanamide is able to inhibit NAT1 most likely via interaction with the active site cysteine residue. Thereby cyanamide might modulate NAT1 dependent detoxification and activation of arylamines.

Variable NAT1 enzyme activity in long-term cultured human HaCaT 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.

Characterization of N-acetyltransferase 1 activity in human keratinocytes and modulation by para-phenylenediamine.

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 phase-dependent. 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 microM) 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.