Mouse Model of Hydroquinone Hypersensitivity via Innate and Acquired Immunity and its Promotion by Combined Reagents.

We established a mouse model of contact hypersensitivity (CHS) to hydroquinone (HQ), a widespread chemical in our environment. HQ was painted onto flanks; then, HQ was challenged by painting onto ear pinnas on days 7 and 14. The CHS after the second challenge was markedly greater than that after the first challenge. Both challenges increased thymic stromal lymphopoietin and T helper type 2 cytokines in ear pinnas, whereas IFN-γ (typical T helper type 1 cytokine) was decreased, despite an increase in IL-18 (typical IFN-γ inducer). In nude mice (T cell-reduced), although a first challenge induced CHS, a second challenge did not augment it. In severe combined immunodeficient, severe combined immunodeficient-beige, and IL-1-deficient mice, CHS was not induced. However, CHS was inducible in severe combined immunodeficient-beige mice after transfer of natural killer cells from HQ-sensitized normal mice. Tretinoin (used for enhancing the skin-whitening effect of HQ) and resin monomers (used to prevent polymerization of HQ) lowered the HQ concentration needed to establish sensitization to HQ. The augmented CHS after a second challenge was reduced by JNJ7777120, dexamethasone, suplatast tosilate (T helper type 2-cytokine inhibitor), and anti-thymic stromal lymphopoietin antibody. These results suggest that (i) thymic stromal lymphopoietin, IL-1, and T and/or natural killer cells are important in establishing and augmenting CHS to HQ and (ii) inflammatory chemicals may promote CHS to HQ as adjuvants.

Development of LLNA:DAE: a new local lymph node assay that includes the elicitation phase, discriminates borderline-positive chemicals, and is useful for cross-sensitization testing.

We developed a new local lymph node assay (LLNA) that includes the elicitation phase termed LLNA:DAE for discrimination of borderline-positive chemicals as classified by the LLNA modified by Daicel based on ATP content (LLNA:DA) and for cross-sensitization testing. Although the LLNA:DA method could help identify skin sensitizers, some skin irritants classified as non-sensitizers by the LLNA were classified as borderline positive. In addition, the evaluation for the cross-sensitization potential between chemicals was impossible. In the LLNA:DAE procedure, test group of mice received four applications of chemicals on the dorsum of the right ear for induction and one application on the dorsum of the left ear for elicitation. Control group of mice received one chemical application on the dorsum of the left ear. We evaluated the sensitizing potential by comparing the weights of the lymph nodes from the left ears between the two groups. The results of using the LLNA:DAE method to examine 24 chemicals, which contained borderline-positive chemicals, were consistent with those from the LLNA method, except for nickel chloride (NiCl2). Two chemical pairs, 2,4-dinitrochlorobenzene (DNCB) with 2,4-dinitrofluorobenzene (DNFB) and hydroquinone (HQ) with p-benzoquinone (p-BQ), showed clear cross-sensitization with each other, while another chemical pair, DNFB with hexylcinnamic aldehyde (HCA) did not. Taken together, our results suggest that the LLNA:DAE method is useful for discriminating borderline-positive chemicals and for determining chemical cross-sensitization.

Experimental study on cross-reactivity of alpha-arbutin toward p-phenylenediamine and hydroquinone in guinea pigs.

Hydroquinone (HQ) has been used as a skin-lightening cosmetic ingredient, while it has been known that HQ shows sensitizing potential and cross-reactivity toward a strong sensitizer, p-phenylenediamine (PPD). alpha-Arbutin, a glycoside of HQ (4-hydroxyphenyl alpha-D-glucopyranoside), is used worldwide as a skin-lightening agent. The aim of this study was to evaluate the cross-reactivity of alpha-arbutin toward PPD and HQ. All tests were performed using the guinea pig maximization test. In experiments on the cross-reactivity of alpha-arbutin or HQ to PPD, six animals in each group were induced with PPD at 0.1% by i.d. injection and at 1.0% by topical application. The animals were challenged with alpha-arbutin, HQ or PPD (as a positive control) at concentrations of 0.01%, 0.05% and 0.1%. In experiments on the cross-reactivity of alpha-arbutin to HQ, four animals in each group were induced with HQ at 2% by i.d. injection and at 1% by topical application. The animals were challenged with alpha-arbutin or HQ (as a positive control) at concentrations of 0.2%, 2% and 20%. The cross-reactivity toward PPD was observed with HQ (4/6) only at 0.1% challenge. However, alpha-arbutin showed no apparent cross-reactivity to either PPD or HQ even at their highest challenge concentrations. Potent sensitization was observed with PPD (6/6) even at 0.01% challenge and with HQ (3/4) at 0.2%. In conclusion, glycosylation of HQ remarkably reduced the sensitization potency of HQ and the cross-reactivity of HQ to PPD.

Specific immune responses in workers exposed to benzene.

UNLABELLED: The aim of this study was to elaborate a method for detection of specific IgG antibodies (Abs) to the haptenes p-benzoquinone (p-BQ) and hydroquinone (HQ) for assessment of specific humoral immune responses. Plasma and urine, collected from petrochemical plant workers have been analyzed. The workers were divided into three professional groups in ascending order of benzene exposure. The concentration of benzene in the air was determined by gas chromatography with mass-spectrometry and trans,trans-muconic acid (biomarker of benzene exposure) in urine-by liquid chromatography with UV-detection. Specific IgG Abs to haptenes p-BQ and HQ in plasma were determined with newly developed ELISA. The relationships "exposure-effect," revealed increased levels of specific IgG to haptens correlating with the benzene exposure. The "exposure-response" relationships demonstrated that workers with value of OD over X+2SD were 62% low exposure group, 68% in group with level of exposure on Threshold Limit Value (TLV) and 91% in the highest exposure group. The data obtained show that there is a good correlation between antibody production and the biomarker of exposure t,t-muconic acid. CONCLUSION: The newly developed method is applicable for assessment of specific humoral immune responses in workers exposed to benzene. There was a good correlation between benzene exposure and formation of antibodies against benzene metabolites.

Activation of the human RANTES gene promoter in a macrophage cell line by lipopolysaccharide is dependent on stress-activated protein kinases and the IkappaB kinase cascade: implications for exacerbation of allergic inflammation by environmental pollutants.

Macrophages are targeted by environmental pollutants and play a role in allergic inflammation. We explored the molecular basis for induction of RANTES (regulated upon activation, normal T-cells expressed and secreted) mRNA by lipopolysaccharide (LPS) and the redox-active quinone, tert-butylhydroxyquinone (tBHQ). We demonstrate that transcriptional activation of the human RANTES promoter by LPS is dependent on specific AP-1 and NF-kappaB response elements, which are regulated by c-Jun N-terminal kinase (JNK) and NF-kappaB kinase cascades, respectively. The transcriptional activation of the TRE3/4 site is mediated through the transcriptional activation of c-Jun by JNK. A c-Jun mutant which lacks a transcriptional activation domain interfered in the activation of the RANTES promoter. Similarly, kinase-inactive NF-kappaB inducing kinase interfered in the activation of the RANTES promoter. While activation of the RANTES promoter could also be blocked by the downstream kinase-inactive IkappaB kinases, only IKKalpha appears to be LPS-inducible. tBHQ also exerted subtle effects on the human RANTES promoter and induced mRNA expression in parallel with generating NF-kappaB shift complexes.

Detection of benzoquinone adducts to rat liver protein sulfhydryl groups using specific antibodies.

Benzoquinone is an electrophilic metabolite of bromobenzene and other simple aromatic compounds of toxicological interest including benzene, phenol, hydroquinone, and acetaminophen. In reacting with proteins benzoquinone shows great selectivity for Michael addition to sulfhydryl groups and formation of S-(2,5-dihydroxyphenyl) protein adducts. To facilitate the specific detection and eventual isolation and identification of such adducted proteins, we prepared an antiserum capable of recognizing hydroquinone moieties by immunizing rabbits with keyhole limpet hemocyanin modified with 3-[2,5-dihydroxyphenyl)thio]propanoyl groups as haptens. The antiserum had a high titer and showed high specificity for hapten in competitive ELISA with hapten analogues. In Western blot experiments the antiserum detected not only synthetically haptenized control proteins but also several proteins from rat liver microsomes that had been incubated in vitro with [14C]bromobenzene. This binding was completely blocked by free hapten, showing that it was hapten-specific. Each of the microsomal protein bands detected in the Western blots also contained radioactivity, but not all radioactive protein bands reacted with antibody. This antiserum should prove useful in exploring the role of protein arylation by benzoquinone in cytotoxic responses to its metabolic precursors.

Sensitization of mice to paraphenylenediamine and structurally-related compounds: adjuvant effects of vitamin A supplementation.

Allergic contact dermatitis from moderate and weak contact sensitizers is generally studied with guinea pigs, since they are readily sensitized to contact allergens. Mice, by contrast, are poor responders to weak contact allergens. However, the variety of in vitro murine systems as well as murine specific reagents make mice the preferable species. With the use of vitamin A supplementation, 2 protocols were developed which sensitized CBA/J female mice to paraphenylenediamine. Mice were sensitized by 5 daily topical applications to shaven dorsal skin. Alternately, mice were sensitized by 2 intraperitoneal injections of antigen pulsed spleen cells. Sensitization to paraphenylenediamine was determined by ear swelling following topical application. Vitamin A supplementation was found to be essential for optimum response. Lymph node and spleen cells from sensitized mice were capable of proliferating to paraphenylenediamine in vitro. With the use of vitamin A supplementation and intraperitoneal injection, CBA/J mice were also sensitized to a number of compounds structurally related to paraphenylenediamine, including the ortho- and meta-derivatives of paraphenylenediamine, as well as hydroquinone and resorcinol. These new protocols, combined with vitamin A supplementation, expand the use of mice to study moderate sensitizers with minimal animal utilization.

Study on cross-reactivity to the para group.

In 80 patients, positive to at least one hapten of the para group (para-phenylenediamine, diaminodiphenylmethane, benzocaine, PPD mix), patch tests were carried out with freshly prepared solutions of para-phenylenediamine (PPD) and of 3 selected aromatic compounds related structurally to PPD (para-aminophenol, ortho-aminophenol, hydroquinone). The number of positive reactions correlated with the rate of decomposition of the substances as evaluated by high-pressure liquid chromatography. PPD, which was almost decomposed after 24 h, gave the highest number of positive reactions, followed by ortho-aminophenol and by para-aminophenol, while hydroquinone, which was oxidized to the extent of 35%, did not give any reactions. To evaluate if a different rate of oxidation can modify the patch test response, in the same patients and in 10 normal volunteers, tests were carried out with PPD solutions containing the oxidizing agent silver oxide (0.1%). By this procedure a significant increase in the number of positive responses was observed. The results suggest that the rate of decomposition and therefore the amount of quinone(s) generated, might be the key to eliciting patch test responses to oxidizable aromatic haptens.

Contact allergy to Phacelia spp. (Hydrophyllaceae).

Six persons involved with cultivation trials of Phacelia spp. developed a contact allergy to certain plants of this species. Geranyl(hydro)quinone and related substances, including the geranylhydroquinone geroquinol (INN), which has been introduced as a radioprotective agent, are potent allergens and probably are more often the cause of allergic eczema than is apparent from the literature.

Investigation of the prohapten concept. Cross reactions between 1,4-substituted benzene derivatives in the guinea pig.

It has been proposed that the cross-reactions seen clinically between hydroquinone and para-phenylenediamine (PPD) arise from the formation of a common hapten, benzoquinone, in vivo, and that these chemicals therefore represent "prohaptens". A series of 1,4-substituted benzene derivatives has been used to examine this prohapten concept in the guinea pig model. Using both topical and intradermal routes of application, it is demonstrated that in the guinea pig 1,4-substituted benzene derivatives capable of oxidation to benzoquinone, including hydroquinone and PPD, show only restricted evidence of cross-reactions. These results support the prohapten concept. However taken in combination with data on cross-reactivity with 1,2- and 1,3-substituted benzenes, rather than giving rise to a single common hapten, they can be more readily interpreted as the formation of a spectrum of antigenic determinants in vivo, some of which are shared in common.

A potent contact allergen of Phacelia (Hydrophyllaceae).

The major contact allergen of Phacelia crenulata (Hydrophyllaceae) has been identified as geranylhydroquinone. A maximization test of geranylhydroquinone showed this to be a potent sensitizer comparable in degree to poison oak/ivy urushiol. Comparative patch testing on humans with urushiol established that the phacelia allergen does not cross-react with poison oak or ivy.