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The aim of this in vitro study was to evaluate the long-term effect of bleaching on human enamel. Four groups of enamel specimens were prepared (n = 20): group 1: bleaching with Opalescence Boost [40% hydrogen peroxide (H 2 O 2 ), 3 × 20 min/week]; group 2: control group (the specimens were stored in human saliva); group 3: beaching with Vivastyle Paint on Plus (6% H 2 O 2 , 2 × 10 min/day), and group 4: bleaching with Opalescence PF 16% [16% carbamide peroxide (CP), 6 h/day]. After each bleaching session the specimens were stored in human saliva. Knoop microhardness and surface roughness were measured: before bleaching, after 2-week and after 8-week bleaching. After 2-week treatment, surface roughness was significantly increased in all experimental groups (p < 0.05), while among them no significant difference was found (p > 0.05). The roughness changes exerted after 8-week bleaching were not significantly higher than the ones after 2 weeks (p > 0.05). After 8-week treatment, the increase in roughness caused by 16% CP was significantly higher (p < 0.05) than the one caused by 40% H 2 O 2 . Microhardness increased in all groups including control; however, only 40% H 2 O 2 increased the microhardness significantly (p < 0.05). The effect of bleaching on enamel was not shown to be dependent on the method or the H 2 O 2 concentration. Bleaching with CP 16% resulted in higher roughness than bleaching with H 2 O 2 , while 40% H 2 O 2 caused the higher microhardness increase. The present study showed that in-office bleaching with 40% H 2 O 2 seems to be at least as safe as home bleaching as far as their effects on human enamel are concerned.

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

The findings that many primitive human hematopoietic cells give rise to daughter cells that adopt different cell fates and/or show different proliferation kinetics suggest that hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) can divide asymmetrically. However, definitive experimental demonstration is lacking due to the current absence of asymmetrically segregating marker molecules within the primitive hematopoietic cell compartment. Thus, it remains an open question as to whether HSCs/HPCs have the capability to divide asymmetrically, or whether the differences that have been observed are established by extrinsic mechanisms that act on postmitotic progenitors. Here, we have identified 4 proteins (CD53, CD62L/L-selectin, CD63/lamp-3, and CD71/transferrin receptor) that segregate differentially in about 20% of primitive human hematopoietic cells that divide in stroma-free cultures. Therefore, this indicates for the first time that HSCs/HPCs have the capability to divide asymmetrically. Remarkably, these proteins, in combination with the surrogate stem-cell marker CD133, help to discriminate the more primitive human cultivated HSCs/HPCs. Since 3 of these proteins, the transferrin receptor and the tetraspanins CD53 and CD63, are endosomal-associated proteins, they may provide a link between the endosomal compartment and the process of asymmetric cell division within the HSC/HPC compartment.