Skin sensitization to fragrance hydroperoxides: interplay between dendritic cells, keratinocytes and free radicals.

BACKGROUND: Skin sensitization to hydroperoxides (R-OOHs) of the commonly used fragrance terpenes limonene, linalool and citronellol is frequently reported. R-OOHs are believed to initiate the process leading to sensitization and allergic contact dermatitis through mechanisms involving radical intermediates. Thus, radical intermediates, keratinocytes and dendritic cells (DCs) may act in concert to initiate the process. OBJECTIVES: To evaluate individual DC activation profiles by R-OOHs in the context of keratinocytes with regard to frequency, specificity and magnitude of upregulation. METHODS: We used 2D and 3D cocultures with keratinocytes/reconstructed human epidermis (RHE) and DCs to evaluate cell surface levels of the costimulatory molecules CD86, CD80 and the adhesion molecule CD54 on cocultured DCs. Analysis of radical formation from limonene hydroperoxides in RHE was performed using electron paramagnetic resonance combined with the spin trapping technique. RESULTS: R-OOHs induce donor-dependent DC activation. Major differences were found between the limonene-OOHs. Limonene-1-OOH was stronger with respect to both frequency and magnitude of response. Using a 3D coculture model, no DC activation was detected after topical application of 0·2% limonene-OOHs (20 µg cm-2 ), while 1·2% limonene-1-OOH or 2% limonene-2-OOH induced DC activation. Furthermore, we demonstrated differences in the carbon and oxygen radicals formed from the limonene-OOHs using RHE, mimicking what may happen in vivo. CONCLUSIONS: We report clear individual differences in DC maturation induced by the most important hydroperoxides. Response rates and magnitude of response both indicate that very small structural alterations in the hydroperoxides are translated into specific DC responses. In addition, we provide more insight into the amounts of hydroperoxides that can activate DCs and induce sensitization.

Continuous usage of a hair dye product containing 2-methoxymethyl-para-phenylenediamine by hair-dye-allergic individuals.

BACKGROUND: Despite a positive patch test reaction to para-phenylenediamine (PPD) and/or toluene-2,5-diamine (PTD), many people attempt to continue dyeing their hair with products containing PPD or its derivatives. OBJECTIVES: Investigation of elicitation reactions among PPD/PTD-allergic individuals to hair dye products containing the less sensitizing PPD derivative 2-methoxymethyl (ME)-PPD. METHODS: Elicitation reactions were studied in 43 PPD/PTD-allergic individuals by a 45-min pretest with an ME-PPD-containing hair dye on their forearm. Upon a negative result this was followed by exposure to subsequent hair colour treatment(s). RESULTS: Overall, 38 of 43 PPD/PTD-allergic individuals did not develop an elicitation reaction during the pretest with ME-PPD-containing hair dye products, and were eligible for subsequent hair colour treatments. Of these 38 PPD/PTD-allergic individuals, 29 tolerated subsequent hair dyeing with ME-PPD-containing hair dye products, while seven showed mild and two showed moderate/marked allergic reactions upon the first hair colour treatment. CONCLUSIONS: Hair dye products with the less sensitizing ME-PPD were tolerated by 29 of 43 (67%) PPD/PTD-allergic individuals throughout continued hair dyeing with an average of nine treatments per year. Five individuals reacted upon pretesting, while only mild-to-moderate/marked skin reactions occurred upon hair dyeing in nine individuals who were not identified by the pretest. To our knowledge this is the first study among PPD/PTD-allergic individuals indicating that a negative 45-min pretest with a hair dye product helps to avoid severe allergic reactions.

Cross-elicitation responses to 2-methoxymethyl-p-phenylenediamine under hair dye use conditions in p-phenylenediamine-allergic individuals.

BACKGROUND: The factors influencing elicitation responses in individuals allergic to p-phenylenediamine (PPD) in hair dyes are not well understood. OBJECTIVES: Investigation of the elicitation response to the new, less-sensitizing PPD alternative 2-methoxymethyl-p-phenylenediamine (ME-PPD) under simulated hair dye use conditions. METHODS: The cross-elicitation response to ME-PPD (2% in a hair dye test product for 30 min on forearm then rinsing) was analysed at days 2 and 3 in 30 PPD-allergic individuals with diagnostic patch test grades +, ++ or +++ according to the classification of the International Contact Dermatitis Research Group. RESULTS: Cross-reactivity to the ME-PPD-containing hair dye test product was elicited in nine of 30 subjects (30%), while 70% were negative. Cross-reactivity was elicited in two of four cases with grade +++, three of 10 with grade ++ and four of 16 with grade +. Under identical conditions, PPD was previously found to elicit a response in 21 of 27 PPD-allergic individuals. In 18 of these 21 individuals, either the strength of the cross-elicitation response to ME-PPD was decreased or no response occurred. CONCLUSIONS: Under simulated hair dye use conditions, a significantly lower degree of cross-elicitation to ME-PPD (30%) was observed than previously reported for PPD (32 of 38, 84%). Additionally, a decreased cross-elicitation strength was observed across all three patch test grades, likely reflecting the reduced skin-sensitization properties of ME-PPD. Consequently, careful dermatological evaluation is required to assess cross-reactivity to ME-PPD in patients allergic to hair dyes.

Assessment of the elicitation response in subjects weakly sensitized to p-phenylenediamine.

BACKGROUND: A 30-min application of a hair dye product containing 2% p-phenylenediamine (PPD) to subjects diagnostically graded +, showed that 12 of 18 reacted; eight of 18 with a true + and four of 18 with a doubtful (?+) response, whereas six of 18 did not react at all. In vitro skin-binding experiments showed that for diagnostic patch test conditions the measured exposure level (MEL) is more than 10-fold higher than the MEL for hair dyeing conditions. OBJECTIVE: To further analyse the limited elicitation response of the diagnostically + graded subjects to a PPD hair dye product, under standardized test conditions mimicking product usage, by varying exposure time and dose. METHODS: A hair dye model formulation containing 2% PPD, applied for 30, 45 and 60 min and a diagnostic PPD TRUE test(®) were applied to assess elicitation responses to increasing PPD exposure levels. Grading was performed according to International Contact Dermatitis Research Group guidelines. RESULTS: Six subjects were available for this follow-up study. One of six subjects responded with a + elicitation response to the hair dye model applied for 60 min. Four of the five remaining subjects elicited a + response to the PPD TRUE test(®) applied subsequently, while one of five responded doubtfully. CONCLUSIONS: Increasing the PPD exposure time twofold--resulting in a 5-6% increase of sensitivity of this hair dye model test--or further extending the exposure time 48-fold, was found sufficient to increase the MEL above the thresholds needed to elicit individuals with a + diagnostic PPD patch test who did not react to typical hair dye use conditions with a MEL of about 6·8 µg cm⁻². This analysis confirms that consideration of the MEL is a useful tool to better characterize thresholds of elicitation than consideration of the applied dose alone.

Elicitation of the immune response to p-phenylenediamine in allergic patients: the role of dose and exposure time.

BACKGROUND: Usage of hair dye products containing p-phenylenediamine (PPD) is a concern for PPD-allergic individuals. OBJECTIVES: The present study investigates the role of dose and exposure time on elicitation of allergic contact dermatitis under conditions of permanent hair dyeing. METHODS: Elicitation responses after application of a typical hair dye product containing 2% PPD for 30 min followed by rinsing were analysed in 38 PPD-allergic individuals with a documented history of hair dye-related allergy. Skin binding experiments in vitro were performed to distinguish the dose available for elicitation from the dose applied. RESULTS: A positive reaction was elicited in 20 of 20 patients with grades ++ to +++ and 12 of 18 with grade + according to the classification of the International Contact Dermatitis Research Group. Under conditions of diagnostic patch testing (48 h exposure), the dose available for elicitation is more than 10-fold higher compared with the dose available for hair dyeing (30-min exposure, rinsing of product). CONCLUSIONS: This investigation demonstrates that under simulated hair dye use conditions the actual exposure to PPD is more than an order of magnitude lower than under diagnostic patch testing, although sufficient to elicit a clearly noticeable reaction in 84% of PPD patch test-positive individuals.

Para-phenylenediamine and allergic sensitization: risk modification by N-acetyltransferase 1 and 2 genotypes.

BACKGROUND: Para-phenylenediamine (PPD) is a common contact sensitizer causing allergic contact dermatitis, a major skin problem. As PPD may need activation to become immunogenic, the balance between activation and/or detoxification processes may influence an individual's susceptibility. PPD is acetylated and the metabolites do not activate dendritic-like cells and T cells of PPD-sensitized individuals. OBJECTIVES: To investigate whether PPD can be acetylated in vitro by the two N-acetyltransferases 1 (NAT1) and 2 (NAT2). Based on the assumption that N-acetylation by NAT1 or NAT2 is a detoxification reaction with respect to sensitization, we examined whether NAT1 and NAT2 genotypes are different between PPD-sensitized individuals and matched controls. METHODS: Genotyping for NAT1 and NAT2 polymorphisms was performed in 147 PPD-sensitized individuals and 200 age- and gender-matched controls. Results Both PPD and monoacetyl-PPD were N-acetylated in vitro by recombinant human NAT1 and to a lesser extent by NAT2. Genotyping for NAT1*3, NAT1*4, NAT1*10, NAT1*11 and NAT1*14 showed that genotypes containing the rapid acetylator NAT1*10 allele were under-represented in PPD-sensitized cases (adjusted odds ratio 0.72, 95% confidence interval 0.45-1.16). For NAT2, NAT2*4, NAT2*5AB, NAT2*5C, NAT2*6A and NAT2*7B alleles were genotyped. Individuals homozygous for the rapid acetylator allele NAT2*4 were under-represented in cases compared with controls (4.3% vs. 9.4%), but this trend was not significant. CONCLUSIONS: With respect to data indicating that NAT1 but not NAT2 is present in human skin, we conclude that NAT1 genotypes containing the rapid acetylator NAT1*10 allele are potentially associated with reduced susceptibility to PPD sensitization.

[Sensitisation to p-Phenylenediamine. Effects of metabolism and individual susceptibility]. / Sensibilisierung auf p-Phenylendiamin. Einfluss von Metabolisierung und individuellen Risikofaktoren.

Due to its constant exposure to small molecular weight compounds, the skin is a major target for allergic reactions. Para-phenylenediamine (PPD) is a common and strong contact allergen. Recent studies have revealed new aspects of steps involved in sensitization and allergic contact dermatitis to PPD, giving insight into the cutaneous metabolism of small molecular compounds and its effect on activation and deactivation of immunogenic substances. Molecular epidemiological studies have suggested that polymorphisms in genes encoding cytokines (e.g. TNF-alpha) or metabolizing enzymes (e.g. N-acetyltransferase) may have influence on these mechanisms and the individual susceptibility for sensitization.

Association between TNFA-308 G/A polymorphism and sensitization to para-phenylenediamine: a case-control study.

BACKGROUND: Para-phenylenediamine (PPD) and related chemicals are common contact sensitizers, frequently causing allergic contact dermatitis (ACD). The cytokine tumor necrosis factor-alpha (TNF-alpha) plays a key role in contact sensitization. METHODS: In this case-control study, we evaluated the distribution of variations in the regulatory region of the gene for TNF-alpha (TNFA-308 G/A) in 181 Caucasian individuals with a history of ACD and sensitization to PPD and 161 individuals with no history of sensitization to PPD. RESULTS: The frequency of GA or AA TNFA genotypes was significantly higher in individuals sensitized to PPD than in age- and gender-matched controls giving an odds ratio (OR) of 2.16 (95% confidence interval, CI: 1.35-3.47; P = 0.0016). This relation was even more pronounced when restricting cases to females over 45 years (OR = 3.71; 95% CI: 1.65-8.31; P = 0.0017) vs younger females (less than or equal to 45 years; OR = 2.41; 95% CI: 1.03-5.65; P = 0.044) or males (OR = 1.05; 95% CI: 0.449-2.47; P = 1.0). In addition, a logistic regression model revealed a significant effect for TNFA-308 AA and AG vs GG genotype (point estimate = 2.152; 95% Wald CI: 1.332-3.477). CONCLUSIONS: These findings suggest a possible role for the TNFA-308 genetic polymorphism as a susceptibility factor for chemically induced ACD.

p53 and K-ras mutations in lung cancers from former and never-smoking women.

Somatic p53 mutations are common in lung cancer. Active cigarette smoking is positively correlated with the total frequency of p53 mutations and G:C to T:A transversions on the nontranscribed (DNA coding) strand. Mutational hotspots within the p53 gene, e.g., codon 157, have been identified for tobacco-related lung cancer, whereas these same mutations are found rarely in other cancers. Such data implicate specific p53 mutations as molecular markers of smoking. Because limited data exist concerning the p53 mutation frequency and spectra in ex-smokers and nonsmokers, we have analyzed p53 and K-ras mutations in 126 lung cancers from a population-based case-control study of nonsmoking (n = 117) or ex-smoking (n = 9) women from Missouri with quantitative assessments of exposure to environmental tobacco smoke. Mutations in the p53 gene were found in lung cancers from lifetime nonsmokers (19%) and ex-smokers (67%; odds ratio, 9.08; 95% confidence interval, 2.06-39.98). All deletions were found in tumors from patients who were either ex-smokers or nonsmokers exposed to passive smoking. The G:C to A:T transitions (11 of 28; 39%) were the most frequent p53 mutations found and clustered in tumors from lifetime nonsmokers without passive smoke exposure. The incidence of K-ras codon 12 or 13 mutations was 11% (14 of 115 analyzed) with no difference between long-term ex-smokers and nonsmokers. These and other results indicate that p53 mutations occur more commonly in smokers and ex-smokers than in never-smokers. Such comparisons provide additional evidence of genetic damage caused by tobacco smoke during lung carcinogenesis.

Characterization of T cell responses to fragrances.

Fragrances are worldwide a major cause of allergic contact dermatitis (ACD), a delayed-type hypersensitivity reaction mediated by T lymphocytes. We investigated T cell responses to fragrances using peripheral blood mononuclear cells (PBMC) and T cells from skin lesions of fragrance-allergic patients. The components of a fragrance mixture (eugenol, isoeugenol, geraniol, oak moss, alpha-amyl cinnamic aldehyde, cinnamic aldehyde, cinnamic alcohol, and hydroxycitronellal) that is commonly used in the patch test were studied in vitro in the lymphocyte transformation test (LTT). PBMC from fragrance-allergic patients (n = 32) showed significant stimulations to all eight fragrances. The calculated stimulation indices (SI) varied between 2.1 and 21.8. The influence of metabolic enzymes on T cell stimulation was studied for two fragrances. Interestingly, stimulation of eugenol and isoeugenol was increased in the presence of antigen-modified human liver microsomes (CYP450) or recombinant CYP1A1 in five of seven cases. Furthermore, we established 18 T cell clones (TCC) from a skin lesion reacting specifically to eugenol. FACS analysis revealed that the majority (n = 15, 83%) of TCC were CD3(+), CD4(+), and HLA-DR(+). Seventeen percent (n = 3) of the clones were CD8(+). TCC (n = 4) released significant amounts of IL-2 and IFN-gamma but no IL-4 and IL-5. In addition, CD4(+) TCC (n = 3) showed antigen-induced cytotoxic activities against autologous B cells. In summary, we demonstrated for the first time that fragrance-specific CD4(+) and CD8(+) T lymphocytes are present in fragrance-allergic individuals. In addition, our results suggest that CYPs can be involved in the formation of the nominative antigen.

Lack of association between the -260 C-->T promoter polymorphism of the endotoxin receptor CD14 gene and the CD14 density of unstimulated human monocytes and soluble CD14 plasma levels.

OBJECTIVE: CD14 is a receptor for endotoxin and binds components of Gram-positive and Gram-negative bacteria. CD14-bearing monocytes respond to stimulation with the increased synthesis and release of cytokines. The recently described -260 C-->T promoter polymorphism of the CD14 gene has been found to be related to a risk of myocardial infarction. This study evaluated the role of this polymorphism in the expression of monocyte and soluble CD14. Moreover, the effect of the CD14 -260 genotypes for the ex vivo TNF-alpha response to endotoxin was analyzed in whole blood. PATIENTS AND PARTICIPANTS: Ninety-five healthy blood donors were studied. MEASUREMENTS AND RESULTS: CD14 -260 genotyping was performed by means of a real-time PCR with fluorescence labeled hybridization probes. CD14 expression on human monocytes (mCD14) was assessed by fluorescence-activated cell sorting analysis with anti-CD14 monoclonal antibodies. Plasma levels of soluble CD14 (sCD14) were measured by ELISA. The TNF-alpha synthesis was determined by chemiluminescence in whole blood after endotoxin stimulation. There were no differences in mCD14 density, sCD14 levels, or the tumor necrosis factor-alpha concentrations between individuals with the three different CD14 -260 genotypes CC, CT, and TT. CONCLUSIONS: The CD14 -260 polymorphism does not affect the CD14 expression of unstimulated circulating monocytes or soluble CD14 plasma levels.

N-Acetylation of paraphenylenediamine in human skin and keratinocytes.

Skin is the major target of allergic reactions to paraphenylenediamine (PPD). Such small molecules require activation to become immunogenic. The balance between activation and/or detoxification processes is critical for immunogenic potentials of compounds. Therefore, we investigated N-acetylation (NAT) capacities of human skin for PPD to gain a better understanding of its mechanisms of action. PPD is acetylated to monoacetyl-PPD (MAPPD), which in turn is acetylated to N,N'-diacetyl-PPD (DAPPD). This was found using cytosolic fractions from human skin (n = 9) and cultured normal human epidermal keratinocytes (n = 7). The cutaneous activities for MAPPD formation ranged from 0.41 to 3.68 nmol/mg/min (9-fold variation) and DAPPD formation from 0.65 to 3.25 nmol/mg protein/min (5-fold), respectively. Similar results were obtained with keratinocytes. NAT activities toward both substrates, PPD and MAPPD, were correlated in keratinocytes (r = 0.930), suggesting that the reactions were catalyzed by the same enzyme. Formation of MAPPD and DAPPD was competitively inhibited in the presence of p-aminobenzoic acid (300 microM), a typical NAT1 substrate, but not by sulfamethazine. These kinetic characteristics suggest that the acetylation of PPD in human skin and keratinocytes is predominantly attributable to the polymorphic NAT1, although both mRNAs (NAT1 and NAT2) are synthesized in human skin and keratinocytes. The metabolism of PPD by NAT1 in human skin and keratinocytes as well as the virtual absence of NAT2 activity may have important toxicological implications. In the case of PPD, our results emphasize that N-acetylation status may be a susceptibility factor for the development of an allergy to PPD.

Environmental tobacco smoke, genetic susceptibility, and risk of lung cancer in never-smoking women.

BACKGROUND: Exposure to environmental tobacco smoke (ETS) is considered to be a major lung cancer risk factor for never smokers. We investigated the hypothesis that never-smoking women who are exposed to ETS and develop lung cancer are a genetically susceptible population. METHODS: Archival tumor tissues were analyzed from 106 never-smoking women enrolled in a case-control study of ETS (and other personal and environmental factors) and lung cancer risk. We analyzed germline polymorphisms in genes that have been associated with cancer susceptibility and whose products activate (cytochrome P450 1A1 [CYP1A1]) and detoxify (glutathione S-transferases M1 [GSTM1] and T1 [GSTT1]) chemical carcinogens found in tobacco smoke. RESULTS: When compared with never smokers who had no ETS exposure and developed lung cancer (n = 55), never smokers with exposure to ETS who developed lung cancer (n = 51) were more likely to be deficient in GSTM1 activity (i.e., were GSTM1 null) because of a genetic polymorphism in the GSTM1 gene (odds ratio = 2.6; 95% confidence interval = 1.1-6.1). A statistically significant rising trend in risk occurred with increasing ETS exposure (two-sided P =. 02), reaching a more than sixfold excess risk in those exposed to 55 pack-years of ETS (ETS pack-year = ETS produced by an active smoker, within a confined space such as a room, who smokes one pack of cigarettes a day for a year). No evidence was found of associations between GSTT1 deficiency or the CYP1A1 valine variant and lung cancer risk due to ETS exposure. CONCLUSIONS: A common genetic polymorphism divides the population of never smokers into two groups of approximately equal size, one (homozygous carriers of the GSTM1 null allele) that has a statistically significant greater risk of lung cancer from ETS than the other (heterozygous or homozygous carriers of the wild-type GSTM1 allele).

Identification of N-acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes.

Three polymorphic sites in the N-acetyltransferase 2 (NAT2) gene were detected using rapid cycle DNA amplification with allele-specific fluorescent probes and melting curve analysis. Two fluorogenic adjacent hybridization probes were designed to NAT2*5A (C(481)T), NAT2*6A (G(590)A), and NAT2*7A (G(857)A). During amplification, probe hybridization is observed as fluorescence resonance energy transfer. The fluorescence increases every cycle as the product accumulates during amplification. A single base mismatch resulted in a melting temperature shift (T(m)) of 5 to 6 degrees C, allowing for the easy distinction of a wild-type allele from the mutant allele. The protocol is rapid, requiring 40 min for the completion of 45 cycles including the melting curves. It is also a simple and flexible method, since DNA templates prepared from different sources, including DNA from serum and paraffin-embedded tissue sections, could be used without adverse effects. Fluorescence genotyping of all three polymorphisms in a total of 155 DNA samples correlated perfectly with our previously validated genotyping by restriction enzyme digestion (PCR-RFLP). This new facile approach allows for the easy detection of NAT2 polymorphisms in hundreds of samples in only a day.

Laboratory methods for the determination of genetic polymorphisms in humans.

The determination of genetic polymorphisms for susceptibility to human disease has been rapidly increasing since the introduction of the polymerase chain reaction (PCR). In most laboratories the ability exists to conduct studies on more than 10,000 persons, and the prospect of even larger investigations is approaching. Many methods can be used for genotyping individuals but some are more common and less expensive than others. Newer methods will allow for automation. As the number of studies on genetic polymorphisms increases it is to be expected that more pitfalls will be encountered. While larger studies will reduce the importance of misclassification, quality control methods will have to be applied to the processing of large numbers of samples.