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Comet assay used to investigate genetox potential of aromatic amine hair dye precursors

Researchers from Switzerland and the USA have been working in collaboration investigating aromatic amines. The three aromatic amines chosen for this study are widely used as precursors in oxidative hair dye formulations.  The compounds chosen were: p-phenylenediamine (PPD), 2,5-diaminotoluene (DAT) and 4-amino-2-hydroxytoluene (AHT).

Acetylation is a reaction that introduces an acetyl functional group into a chemical compound. N-acetylation has been described as a detoxification reaction for aromatic amines.  However, within the publication, the researchers describe how there is limited data available showing that this metabolic conversion step changes the aromatic amines genotoxicity potential.

In the hope of gaining a deeper mechanistic understanding of this chemical class, the researchers compared the genotoxicity profiles of the parent compounds and their N-acetylated metabolites.

About 60% of women and 5–10% of men in Western countries are using hair dyes.  Oxidative hair dyes, which are the most widely used, work as a bicomponent system: primary intermediates, usually monocyclic p-phenylenediamines (PPD), are mixed with couplers, usually meta-substituted aminophenols, and hydrogen peroxide in an alkaline cream formulation.

While the starting materials easily diffuse into the hair, the large coloured hair dye molecules formed by the polymerisation of primary intermediates and couplers are trapped in the hair matrix, thereby creating a permanent colour. The primary intermediates and couplers are also known as hair dye precursors.

The comet assay was performed with V79, V79NAT1*4 (a N-actyltransferase proficient cell line) and HaCaT (Human Keratinocyte) cells.  The cells were treated with the compounds (DAT, PPD, AHT and some of their acetylated derivatives, at various concentrations) for either three or 48 hours.  For full information of the testing conditions, incubation times and compounds used, you should refer to the original publication.

Dried comet slides were stained with SYBR Gold and evaluated using Comet Assay IV from Perceptive Instruments, UK. Coding of slides was done by a third person using random numbers and opaque tape and the slides were scanned in a meandering way to ensure that no cell was scored twice.  Fifty cells per slide (150 per dose group) were evaluated, and all Comet Assay IV parameters were recorded, but only tail intensity (TI%) and Olive tail moment (OTM) were chosen for evaluation.

The arithmetic mean of the TI% and OTM data from 50 cells per slide was calculated and used as statistical/experimental unit. Mean and SD values from the three slides were given for each concentration level investigated.

PPD showed a strong genotoxic effect in V79 cells after 3-hour exposure at concentrations of 100 µg/ml and higher.
In V79NAT1*4 cells, the genotoxic effect was negligible up to the highest tested concentration, however one of the doses tested showed an anomalous result, which was not regarded as biologically meaningful by the investigators.
In HaCaT cells, a weak biologically relevant increase was detected at the highest concentrations tested.

The lowest concentration of DAT investigated elicited a strong DNA damaging effect in standard V79 cells following 3-hour incubation.
In the V79NAT1*4 cells, DNA damage was only observed at the three highest tested DAT concentrations.
HaCaT cells were even less susceptible to DAT.

A genotoxic potential of AHT was observed after 48 hours of incubation with V79 cells at the lowest concentration.
DNA damage in both V79NAT1*4 and HaCaT cells started at substantially higher concentrations. 

While PPD, DAT and AHT all show genotoxic potential when the comet assay was performed on V79 cells, their N-acetylated metabolites completely lacked genotoxic potential. 

The researchers collated comet assay, micronucleus test, S. typhimurium reversion assay, chromosome aberration data from this study to conclude that N-acetylation is a detoxification reaction for the three aromatic amine hair dye precursor molecules investigated. All observed N-acetylated metabolites completely lack genotoxic potential as shown in the S. typhimurium reversion assay, micronucleus test and comet assay performed with V79 cells.

This case study is based upon:
N-acetylation of three aromatic amine hair dye precursor molecules eliminates their genotoxic potential
Mutagenesis. 2014 Jan;29(1):37-48. Epub 2013 Nov 25.
Zeller A, Pfuhler S.