Skin sensitization resulting in allergic contact dermatitis is a common occupational health issue. In this study, the effect of mixing two skin sensitizers on the skin sensitization response was investigated. Skin sensitizers are generally classified into T helper type 1 (Th1) or T helper type 2 (Th2), depending on the induced cytokine profile. Dinitrochlorobenzene (DNCB) and oxazolone (Oxa) are Th1 skin sensitizers and phthalic anhydride (PA) and toluene diisocyanate (TDI) are Th2 skin sensitizers. We investigated the effect on skin sensitization response to mixtures of three pairs of these sensitizers: DNCB and Oxa, DNCB and PA, and PA and TDI, using guinea pig maximization test and mouse ear swelling test. In guinea pigs sensitized with the mixture of DNCB and Oxa or PA and TDI, there were changes of skin sensitization response to DNCB and Oxa, and that to PA. On the other hand, there was no mixture effect in guinea pigs sensitized with the mixture of DNCB and PA. The skin sensitization responses were decreased in mice sensitized with the mixtures of DNCB and Oxa or PA and TDI, whereas the mixture effect was not observed in mice sensitized with the mixture of DNCB and PA. The present findings revealed that mixture effect on the skin sensitization response was observed after simultaneous exposure to two skin sensitizers, and the effect was determined by combinations of mixed skin sensitizers.
Amino acid derivative reactivity
assay (ADRA) is an in
chemico assay for assessing the skin sensitization potential
of chemicals by evaluating the reactivity of nucleophilic reagents
that mimic skin proteins. N-(2-(1-Naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), used as nucleophilic reagents, are small-molecule
derivatives of two different amino acids, each with a naphthalene
ring attached. The rate of decrease in the amount of NAC or NAL in
the reaction solution is evaluated in this assay as an indicator of
the test substance’s skin sensitization ability. However, the
products formed between the nucleophilic reagent and the test substance,
which play an important role in vivo, are not directly
identified. Therefore, six highly reactive chemicals, including the
proficiency substances listed in the OECD Test Guidelinessquaric
acid diethyl ester, 2-methyl-2H-isothiazol-3-one
(MI), p-benzoquinone, palmitoyl chloride, diphenylcyclopropenone
(DPCP), and imidazolidinyl urea (IU)were used to determine
each formed product. Samples were prepared according to the standard
ADRA method, and the formed products were predicted on the basis of
the reaction mechanism. Excluding DPCP, the estimated structures were
validated using mass spectrometry and nuclear magnetic resonance spectrometry
on the synthesized samples. In this manner, the products of each nucleophile
were confirmed for all examined test substances. The estimated structure
products were obtained through a series of reactions initiated by
the nucleophilic attack of NAC’s thiol group or NAL’s
amino group on the test substance’s electron-deficient carbonyl
carbon. However, contrary to expectations, disulfide-linked-type ring-opened
products were detected in the case of MI, and products with free formaldehyde
in solution were detected in the case of IU. In summary, all skin
sensitizers tested herein reacted with NAC and/or NAL to give products.
This supports the theoretical validity of ADRA, which provides an
indirect evaluation of the formed products based on a decrease in
nucleophilic reagents.
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