Proliferative responses in the local lymph node assay associated with concomitant exposure to 1,4-phenylenediamine and methyldibromo glutaronitrile: evidence for synergy?

BACKGROUND: A key consideration when undertaking risk assessments should be the potential for synergy between contact allergens. Previously, this concept has only been investigated during elicitation in contact allergic individuals. OBJECTIVE: To determine whether there exists evidence for synergy between contact allergens during the induction phase of skin sensitization using the mouse local lymph node assay (LLNA) as a model system. METHOD: Proliferative responses in draining lymph nodes were assessed with increasing concentrations of 1,4-phenylenediamine (PPD), methyldibromo glutaronitrile (MDBGN), and a combination of PPD and MDBGN. RESULT: Data from each of two independent experiments show that lymph node cell proliferation associated with combined exposure to PPD and MDBGN was, in general, only modestly increased relative to that predicted from a simple summation of their individual responses. CONCLUSIONS: Although the increase in response is very modest, it does imply a relationship between this combination of sensitizers that may not be simply additive in terms of their ability to stimulate proliferative responses in draining lymph nodes. The reproducibility of this observation should be confirmed in future studies with additional pairs of contact allergens to ascertain whether or not this represents evidence of synergy.

The impact of vehicle on the relative potency of skin-sensitizing chemicals in the local lymph node assay.

The identification and characterization of chemicals that possess skin-sensitizing potential are typically performed using predictive tests. However, human exposure to skin-sensitizing chemicals often occurs via a matrix (vehicle) that differs from that used in these tests. It is thus important to account for the potential impact of vehicle differences when undertaking quantitative risk assessment for skin sensitization. This is achieved through the application of a specific sensitization assessment factor (SAF), scaled between 1 and 10, when identifying an acceptable exposure level. The objective of the analysis described herein is to determine the impact of vehicle differences on local lymph node assay (LLNA) EC3 values (concentrations of test chemical required to provoke a 3-fold increase in lymph node cell proliferation). Initially, the inherent variability of the LLNA was investigated by examining the reproducibility of EC3 values for 14 chemicals that have been tested more than once in the same vehicle (4:1 acetone:olive oil, AOO). This analysis reveals that the variability in EC3 value for these chemicals following multiple assessments is <5-fold. Next, data from the literature and previously unpublished studies were compiled for 18 chemicals that had been assessed in the LLNA using at least 2 of 15 different vehicles. These data demonstrate that often the variability in EC3 values observed for a given chemical in different vehicles is no greater than the 5-fold inherent variability observed when assessing a chemical in the same vehicle on multiple occasions. However, there are examples where EC3 values for a chemical differ by a factor of more than 10 between different vehicles. These observations were often associated with an apparent underestimation of potency (higher EC3 values) with predominantly aqueous vehicles or propylene glycol. These data underscore the need to consider vehicle effects in the context of skin-sensitization risk assessments.

Preservatives and skin sensitization quantitative risk assessment.

BACKGROUND: Preservatives are an unfortunately common cause of allergic contact dermatitis (ACD). Often, this is in association with exposure to cosmetics or medicaments. Recently, a quantitative risk assessment (QRA) approach to the quantitation of safe exposure levels for sensitizers has been promulgated as a more effective tool for the identification of acceptable levels of potential sensitizers in consumer products. OBJECTIVE: To assess this QRA approach, which facilitates the prediction of acceptable exposure levels to skin sensitizers in consumer products, levels that are normally below the threshold for the induction of skin sensitization. METHODS: Retrospective QRA analysis on four preservatives in five consumer product types. RESULTS: The analysis shows that functional levels of preservatives may be somewhat above an ideal exposure level for some product types, an outcome that is consistent with the clinical picture. CONCLUSION: QRA represents a new tool that in the future should be used in combination with the assessment of microbiologic protection needs of specific product types to limit the problem of preservative ACD.

Proactive surveillance of contact allergies: an important component of the risk management strategy for skin sensitizers.

Risk assessment serves to ensure that dermal exposure to skin sensitizers does not result in the acquisition of allergic skin disease. Traditionally, the approach adopted was one of comparative analysis, involving benchmarking against other allergens of known potency. More recently, efforts have been made to embrace a quantitative risk assessment (QRA) approach. However, the accuracy of any risk assessment is reflected in the extent to which it meets the fundamental objective stated above. Thus, clinical experience is of key importance. There exists the possibility for the originators of risk assessments relating to chemicals that possess skin-sensitizing potential to work directly with the clinical community to proactively obtain this experience through specific surveillance programmes. This forms the focus of this review article. The current status of the QRA approach is considered initially. A recently published example of one such surveillance programme that was undertaken as a collaborative initiative between industry and the clinical community is then reviewed. Finally, a possible strategy for the future is presented, in which it is suggested that surveillance strategies might be deployed in certain situations as an adjunct to the initial risk assessment. It is hoped that such a framework might further improve the efficacy of future approaches to skin sensitization risk assessment.

Oral tolerance to contact allergens: a common occurrence? A review.

Experimental and clinical oral tolerance to contact allergens has been reported sporadically, most notably in respect of nickel, and is generally assumed to be an uncommon phenomenon. There has recently been increased understanding of the immunological mechanisms inducing and maintaining oral tolerance. There are several contact allergens, including fragrance, antioxidant, and preservative chemicals, to which subjects are exposed through both cutaneous and oral routes. We examine the possibility that oral tolerance to contact allergens may be more common than previously thought. Animal models of oral tolerance to contact allergens indicate that cutaneous exposure to small, subsensitizing doses of contact allergens might negate any subsequent attempts to induce tolerance by oral administration. Extrapolating these observations to common human practises raises the possibility that application of contact allergens (fragrances, preservatives and antioxidants) in consumer products used by children could prevent or inhibit the later acquisition of specific tolerance resulting from 'natural' dietary exposure after weaning. Existing data on formaldehyde may conflict with this theory, though this could be explained by allergen specificity. We propose that further work in this area is needed.

The skin sensitization potential of resorcinol: experience with the local lymph node assay.

Resorcinol is a simple aromatic chemical (1,3-benzenediol) that has found widespread use, particularly as a coupler in hair dyes. Clinical experience clearly shows that resorcinol is a (albeit uncommon) skin sensitizer. By contrast, predictive methods, both animal and human, have previously failed to identify resorcinol as such. Here, we describe the outcome of a recent local lymph node assay performed in accordance with Organisation for Economic Co-operation and Development guideline 429, which correctly identified resorcinol as a skin sensitizer. Clear evidence of a dose response was apparent, and an EC3 value of approximately 6% was calculated. This suggests that the skin-sensitizing potency of resorcinol is approximately 2 orders of magnitude lower than that of p-phenylenediamine but similar to that of hexyl cinnamic aldehyde. These data show the importance of adherence to test guidelines and aligns the clinical experience with resorcinol with that obtained in predictive animal methods.

The biocide polyhexamethylene biguanide remains an uncommon contact allergen.

Polyhexamethylene biguanide (PHMB) is used as a preservative in cosmetics and personal care products. Previous studies had shown a frequency of sensitization to PHMB of 0.5% and 0.4% in unselected dermatitis patients. The objective of this study was to determine the current frequency of sensitization to PHMB. From July 2005 until December 2005, a total of 1975 consecutive patients were patch tested with PHMB. 10 patients (0.5%) had positive patch test reactions to PHMB 2.5% aq. (9+ and 1++) and 16 patients (0.8%) to PHMB 5.0% aq. (12 +, and 4 ++). The test reaction pattern (reaction index and positivity ratio) of both preparations and a limited concordance between results from both concentrations (Cohen's kappa = 0.35) are probably indicative of a substantial number of false positive reactions. Potential causal exposures were assessed by a case by case analysis and by referring to surrogate markers of exposure in terms of concomitant reactions. Occupational exposures were identified as a probable cause of sensitization. Further risk factors included leg dermatitis and old age. The frequency of sensitization remains low. It is very unlikely that exposure to cosmetics or personal care products may have played a role in the few cases sensitized.

Evidence that two alkyl ester quaternary ammonium compounds lack substantial human skin-sensitizing potential.

BACKGROUND: Alkyl ester quaternary ammonium compounds (ester quats) are used extensively in fabric rinse conditioners. It is important to document in the literature the outcome of historical studies that were performed to assess the risk of adverse skin effects associated with their use. OBJECTIVES: (1) To document the outcomes of historical studies performed to evaluate the skin sensitizing potential of two ester quats (the di-[hardened tallow fatty acid] ester of 2,3-dihydroxypropyl-trimethyl ammonium chloride [HEQ] and the dialkyl ester of triethanol ammonium methyl sulfate [TEA-Quat]) and (2) to demonstrate that these ester quats lack marked skin-sensitizing potential in humans, such that they do not present a risk of contact allergy for consumers who use fabric rinse conditioners. METHODS: Each material was assessed in the human maximization test in a panel of 25 volunteers. Diagnostic patch testing was also performed with each material in a population of 239 patients undergoing routine patch testing for suspected allergic contact dermatitis. These data are also considered in the context of an exposure-based quantitative risk assessment. RESULTS: Neither HEQ nor TEA-Quat was found to cause skin sensitization under the conditions of the human maximization test. No evidence of contact allergy to the materials was found among the relatively small population assessed by diagnostic patch testing. CONCLUSIONS: This study provides evidence that HEQ and TEA-Quat lack substantial skin-sensitizing potential in humans. Taken together with similar data for other ester quats, it suggests that compounds in this class are unlikely to be significant human contact allergens.

Elicitation response characteristics to permanent hair dye in paraphenylenediamine-allergic volunteers.

BACKGROUND: Elicitation response characteristics to complete permanent hair dye products in paraphenylenediamine (PPD)-allergic volunteers have not previously been explored in detail. OBJECTIVES: To assess the elicitation response characteristics observed in PPD-allergic volunteers upon patch testing with complete hair dyes. METHODS: PPD-allergic volunteers were assigned to 1 of 3 groups depending upon whether they elicited + (group 1), ++ (group 2) or +++ (group 3) reactions following the standard diagnostic procedure. Each group was subsequently patch tested with 2 complete hair dyes (A and B) for 30 min, 1 hr and 24 hr. Patch sites were examined 1 day, 2 days and 3 days after patch removal. RESULTS: Exposure to either hair dye for 30 min or 1 hr was insufficient to yield positive patch test reactions in all of the PPD-allergic patients in groups 1 or 2. Application of either hair dye for 24 hr was sufficient to yield positive reactions in all of the individuals within groups 2 and 3. CONCLUSIONS: The frequency of positive patch test reactions observed following 24-hr exposure to complete permanent hair dyes is comparable to that observed following 48-hr exposure to 1% PPD/petrolatum in those individuals whose degree of sensitization is such that they typically present ++ or +++ reactions diagnostically.

The impact of exposure variables on the induction of skin sensitization.

Whereas many investigations of the variables associated with the elicitation of allergic contact dermatitis have been undertaken, to the point where we can begin to predict the likelihood of elicitation occurring in a given situation, the same is not true for the induction of skin sensitization. Studies have demonstrated that increasing dose has an impact; in an experimental setting, a number of variables received attention some decades ago. However, in the work reported here, the relative importance of the frequency and the duration of exposure is highlighted. In an investigation using a human repeated insult patch test, it was demonstrated that reduction of the exposure duration from 48 hr to 5 min decreased the rate of sensitization to 1% p-phenylenediamine (PPD) from 54% to 3%. However, in an extended clinical study, it was observed that infrequent but longer duration and higher concentration exposure to PPD was significantly less likely to induce sensitization compared to more frequent, short duration, and lower concentration exposure. Detailed statistical analysis of the results indicated that the most important factor driving the induction of skin sensitization was the number of exposures.

A future approach to measuring relative skin sensitising potency: a proposal.

Current approaches to skin sensitisation risk assessment are dependent upon the availability of information regarding two fundamental parameters. Firstly, data relating to the relative skin sensitising potency of the chemical, and secondly, information regarding likely conditions of human exposure. During the past two decades, much has been achieved in terms of refining methods capable of informing these parameters. For example, the development of the local lymph node assay (LLNA) has made it possible to predict skin sensitising hazard, and to determine relative skin sensitising potency, in a way that was not possible previously. Taken together with accurate information about predicted exposure, such potency data can be used to facilitate the derivation of effective risk assessments. However, although the LLNA provides an integrated assessment of skin sensitising activity, it does require the use of experimental animals and there is growing enthusiasm for designing robust alternative approaches that will reduce or obviate that need. Progress is being made in defining alternative experimental strategies that avoid animal use, but it is clear that accurate characterisation of skin sensitisation hazards will require the effective integration of various sources of information. For this reason, we exemplify here one possible approach that, in theory, provides a framework for not only the identification of skin sensitising chemicals, but also the estimation of relative sensitising potency. This paradigm depends upon development of an understanding of the various biological, biochemical and chemical factors that impact on the allergenic properties of chemicals and the acquisition of skin sensitisation, and an ability to measure these in vitro.

Glutathione transferases.

This review describes the three mammalian glutathione transferase (GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases inactivate endogenous alpha,beta-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through metabolism of 15d-PGJ2, GST may enhance gene expression driven by nuclear factor-kappaB (NF-kappaB). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and increased risk of myocardial infarction and stroke. Targeted disruption of murine genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective, whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu, and Pi classes, an observation suggesting they are part of an adaptive mechanism that responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative stress.

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.

Expression of the murine glutathione S-transferase alpha3 (GSTA3) subunit is markedly induced during adipocyte differentiation: activation of the GSTA3 gene promoter by the pro-adipogenic eicosanoid 15-deoxy-Delta12,14-prostaglandin J2.

The expression of class alpha, micro, and pi glutathione S-transferases (GSTs) has been examined during the adipose conversion of mouse 3T3-L1 cells. The GSTA4, GSTM1, and GSTP1/2 subunits are expressed constitutively in confluent 3T3-L1 cells, and their levels remain essentially unchanged during adipocyte differentiation. By contrast, the GSTA3 subunit is virtually undetectable in confluent 3T3-L1 cells under basal conditions, but its expression is markedly induced during adipose conversion. Inhibition of the 3T3-L1 adipogenic program demonstrated that GSTA3 expression is associated specifically with acquisition of the adipocytic phenotype. Reporter gene assays demonstrated that the mouse GSTA3 5(')-upstream region is transcriptionally activated by 15-deoxy-Delta(12,14)-prostaglandin J(2) through an antioxidant response element, suggesting that this pro-adipogenic eicosanoid may be involved in regulating GSTA3 expression during adipogenesis. These data suggest a previously unrecognised role for GSTs in mouse adipocytes.

Expression of the aflatoxin B1-8,9-epoxide-metabolizing murine glutathione S-transferase A3 subunit is regulated by the Nrf2 transcription factor through an antioxidant response element.

High expression of the aflatoxin B1 (AFB1)-8,9-epoxide-conjugating glutathione S-transferase A3 (mGSTA3) subunit in mouse liver confers intrinsic resistance to AFB1 hepatocarcinogenesis. It is not known how the gene encoding this protein is regulated. The murine mGSTA3 gene has been identified using bioinformatics. It localizes to mouse chromosome 1 (A3-4), spans approximately 24.6 kilobases (kb) of DNA, and comprises seven exons. High levels of mGSTA3 mRNA are present in organs associated with detoxification. Expression of mGSTA3 in Hepa1c1c7 mouse hepatoma cells was found to be inducible by sulforaphane, an organic isothiocyanate that can transcriptionally activate genes through the antioxidant response element (ARE). Sulforaphane also induced transcription of a luciferase reporter containing a 1.5 kb fragment of the mGSTA3 5'-upstream region. A putative ARE, with sequence 5'-TGACATTGC-3', was identified within this fragment, approximately 150 base pairs upstream of exon 1. Mutation of this sequence abrogated both basal and sulforaphane-inducible reporter activity. Overexpression of the basic-region leucine zipper Nrf2 transcription factor augmented activity of the mGSTA3-luciferase reporter through this ARE. Electrophoretic mobility shift assays demonstrated that Nrf2 binds the mGSTA3 ARE. Measurement of mGSTA3 mRNA levels in tissues isolated from both wild-type and nrf2-null mice revealed that loss of the Nrf2 transcription factor is associated with a reduction in basal expression of mGSTA3. Collectively, these data demonstrate a role for Nrf2 and the ARE in regulating transcription of mGSTA3.

Biochemical and genetic characterization of a murine class Kappa glutathione S-transferase.

The class Kappa family of glutathione S-transferases (GSTs) currently comprises a single rat subunit (rGSTK1), originally isolated from the matrix of liver mitochondria [Harris, Meyer, Coles and Ketterer (1991) Biochem. J. 278, 137-141; Pemble, Wardle and Taylor (1996) Biochem. J. 319, 749-754]. In the present study, an expressed sequence tag (EST) clone has been identified which encodes a mouse class Kappa GST (designated mGSTK1). The EST clone contains an open reading frame of 678 bp, encoding a protein composed of 226 amino acid residues with 86% sequence identity with the rGSTK1 polypeptide. The mGSTK1 and rGSTK1 proteins have been heterologously expressed in Escherichia coli and purified by affinity chromatography. Both mouse and rat transferases were found to exhibit GSH-conjugating and GSH-peroxidase activities towards model substrates. Analysis of expression levels in a range of mouse and rat tissues revealed that the mRNA encoding these enzymes is expressed predominantly in heart, kidney, liver and skeletal muscle. Although other soluble GST isoenzymes are believed to reside primarily within the cytosol, subcellular fractionation of mouse liver demonstrates that this novel murine class Kappa GST is associated with mitochondrial fractions. Through the use of bioinformatics, the genes encoding the mouse and rat class Kappa GSTs have been identified. Both genes comprise eight exons, the protein coding region of which spans approx. 4.3 kb and 4.1 kb of DNA for mGSTK1 and rGSTK1 respectively. This conservation in primary structure, catalytic properties, tissue-specific expression, subcellular localization and gene structure between mouse and rat class Kappa GSTs indicates that they perform similar physiological functions. Furthermore, the association of these enzymes with mitochondrial fractions is consistent with them performing a specific conserved biological role within this organelle.

Prostaglandin D2 synthase enzymes and PPARgamma are co-expressed in mouse 3T3-L1 adipocytes and human tissues.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a critical regulator of adipocyte differentiation. Whilst 15-deoxy-delta(12,14)-prostaglandin J2 (15-d-PGJ2) has been identified as a putative endogenous ligand for this transcription factor, it is unclear whether the enzymes necessary for 15-d-PGJ2 biosynthesis are co-expressed with PPARgamma. Prostaglandin D2 synthase (PGDS) enzymes represent the terminal enzymatic components responsible for 15-d-PGJ2 production. Both glutathione (GSH)-dependent and GSH-independent PGDS isoenzymes exist. We have, therefore, examined the expression of PGDS isoenzymes in mouse 3T3-L1 adipocytes, and various human tissues. The GSH-independent PGDS was found to be expressed in 3T3-L1 cells both before and after their differentiation into adipocytes. By contrast, we were unable to detect expression of the GSH-dependent PGDS at any stage during the adipose conversion of 3T3-L1 cells. Quantitative analysis of mRNA levels for PPARgamma and each PGDS isoenzyme revealed their co-expression in a number of human tissues and cell types, including adipose tissue, placenta, prostate, and macrophages. These data reveal the potential for de novo 15-d-PGJ2 synthesis in the context of PPARgamma expression, suggesting that this prostaglandin may contribute to PPARgamma signalling in vivo.