Chemical reactivity measurements: Potential for characterization of respiratory chemical allergens

Allergic diseases of the skin and respiratory tract resulting from exposure to low molecular weight chemicals remain important issues for consumer product development and occupational/environmental health. Widespread opportunities for exposure to chemical allergens require that there are available effective methods for hazard identification and risk assessment. In the search for new tools for hazard identification/characterization there has been interest in developing alternative methods that will reduce, refine or replace the need for animals. One approach that shows promise is based on the measurement of the peptide reactivity of chemicals; the potential to form stable associations with protein/peptide being a key requirement for the induction of sensitization. Recent investigations using these systems have focused primarily on skin sensitizing chemicals. However, there is interest in the possibility of exploiting these same experimental approaches to distinguish between different forms of chemical allergens - as individual materials are primarily associated with one or the other form of sensitization in humans. These investigations may also provide insight into why chemical sensitizers can differ in the form of allergic disease they will preferentially induce. These opportunities are surveyed here against a background of the immunobiology of allergic sensitization and current state-of-the-art approaches to measurement of peptide/protein reactivity.     

Reactivity of chemical respiratory allergens in the Peroxidase Peptide Reactivity Assay

Sensitizing chemicals are commonly associated primarily with either skin or respiratory sensitization. In the Direct Peptide Reactivity Assay (DPRA), when compared with skin sensitizers, respiratory allergens have been demonstrated to selectively react with lysine rather than cysteine. The Peroxidase Peptide Reactivity Assay (PPRA) has been developed as a refinement to the DPRA. The PPRA incorporates dose–response analyses, mass spectroscopy for peptide detection and a horseradish peroxidase–hydrogen peroxide enzymatic system, increasing the potential to identify pro-haptens. In the investigations reported here, the PPRA was evaluated to determine whether it provides advantages for the identification of respiratory allergens. Twenty respiratory sensitizers, including five predicted to be pre-/pro-haptens were evaluated. The PPRA performed similarly to the DPRA with respect to identifying inherently reactive respiratory sensitizers. However, three respiratory sensitizers predicted to be pre-/pro-haptens (chlorhexidine, ethylenediamine and piperazine) were non-reactive and the general selectivity of the respiratory allergens for lysine was lost in the PPRA. Overall, the data indicate that the PPRA does not provide an advantage over the DPRA for discriminating allergens as either contact or respiratory sensitizers. Nevertheless, the PPRA provides a number of refinements to the DPRA that allow for an enhanced characterization of reactivity for both classes of chemical allergens.     

Peptide reactivity assay using spectrophotometric method for high-throughput screening of skin sensitization potential of chemical haptens

Haptens must react with cellular proteins to be recognized by antigen presenting cells. Therefore, monitoring reactivity of chemicals with peptide/protein has been considered an in vitro skin sensitization testing method. The reactivity of peptides with chemicals (peptide reactivity) has usually been monitored by chromatographic methods like HPLC or LC/MS, which are robust tools for monitoring common chemical reactions but are rather expensive and time consuming. Here, we examined the possibility of using spectrophotometric methods to monitor peptide reactivity. Two synthetic peptides, Ac-RWAACAA and Ac-RWAAKAA, were reacted with 48 chemicals (34 sensitizers and 14 non-sensitizers). Peptide reactivity was measured by monitoring unreacted peptides with UV–Vis spectrophotometer using 5,5′-dithiobis-2-nitrobenzoic acid as a detection reagent for the free thiol group of cysteine-containing peptide or fluorometer using fluorescamine? as a detection reagent for the free amine group of lysine-containing peptide. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine-containing peptide or 20% depletion of lysine-containing peptide. The sensitivity, specificity, and accuracy of this method were 82.4%, 85.7%, and 83.3%, respectively. These results demonstrate that spectrophotometric methods can be easy, fast, and high-throughput screening tools for the prediction of the skin sensitization potential of chemical haptens.     

Development of a peptide reactivity assay for screening contact allergens.

Allergic contact dermatitis resulting from skin sensitization is a common occupational and environmental health problem. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurate identification of skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency; information that is pivotal in successful management of human health risks. However, even with the significant animal welfare benefits provided by the LLNA, there is still interest in the development of nonanimal test methods for skin sensitization testing. One characteristic of a chemical allergen is its ability to react with proteins prior to the induction of skin sensitization. The majority of chemical allergens is electrophilic and as such reacts with nucleophilic amino acids like cysteine or lysine. In order to determine if reactivity correlates with sensitization potential, 38 chemicals representing allergens of different potencies (weak to extreme) and nonsensitizers were evaluated for their ability to react with glutathione or three synthetic peptides containing either cysteine, lysine, or histidine. Following a 15-min reaction time for glutathione or a 24 h reaction period for the three synthetic peptides, the samples were analyzed by HPLC. UV detection was used to monitor the depletion of glutathione or the peptide following reaction. The results demonstrate that a significant correlation (Spearman correlation) exists between allergen potency and the depletion of glutathione (p = 0.001), lysine (p = 0.025), and cysteine (p = 0.020), but not histidine. The peptide with the highest sensitivity was cysteine (80.8%) whereas histidine was the least sensitive (11.5%). The data presented show that measuring peptide reactivity has utility for screening chemicals for their skin sensitization potency and thus potential for reducing our reliance on animal test methods.     

Cytokine profiling of chemical allergens in mice: impact of mitogen on selectivity of response

A major constraint in the development of methods for the identification of chemical respiratory allergens is the continuing uncertainty regarding the mechanisms of this disease and in particular the role of IgE antibody. There is, however, increasing evidence that respiratory sensitization is favoured by the induction of a selective type 2 cytokine response. The current investigations focus on the potential application of cytokine profiling to the identification of chemical respiratory sensitizers. The objective is to determine the optimal configuration of the method for discrimination between chemical contact and respiratory sensitizers. The reference contact sensitizer 2,4‐dinitrochlorobenzene (DNCB) and reference respiratory sensitizer trimellitic anhydride (TMA), which have been shown to induce type 1 and type 2 cytokine profiles, respectively, were utilized. Variables investigated included cell concentration, time in culture, dosing regimens (a 13 day and a truncated 8 day protocol) and the impact of restimulation in vitro with T cell mitogens. Cell culture conditions were critical for the selectivity of the response, with the addition of mitogen resulting in a less discriminatory pattern of cytokine expression, particularly for the type 1 cytokine interferon γ (IFN‐γ). Furthermore, a 13 day exposure period was required for vigorous expression of IFN‐γ by DNCB‐activated cells, whereas type 2 cytokine expression by TMA‐stimulated cells was recorded after 8 days. These data demonstrate that the most optimal method for cytokine profiling is a chronic (13 day) exposure regime followed by culture of lymph node cells at 10⁷ cells ml^?1 for120 h in the absence of mitogen. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantification of chemical peptide reactivity for screening contact allergens: a classification tree model approach.

In the interest of reducing animal use, in vitro alternatives for skin sensitization testing are under development. One unifying characteristic of chemical allergens is the requirement that they react with proteins for the effective induction of skin sensitization. The majority of chemical allergens are electrophilic and react with nucleophilic amino acids. To determine whether and to what extent reactivity correlates with skin sensitization potential, 82 chemicals comprising allergens of different potencies and nonallergenic chemicals were evaluated for their ability to react with reduced glutathione (GSH) or with two synthetic peptides containing either a single cysteine or lysine. Following a 15-min reaction time with GSH, or a 24-h reaction time with the two synthetic peptides, the samples were analyzed by high-performance liquid chromatography. UV detection was used to monitor the depletion of GSH or the peptides. The peptide reactivity data were compared with existing local lymph node assay data using recursive partitioning methodology to build a classification tree that allowed a ranking of reactivity as minimal, low, moderate, and high. Generally, nonallergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as nonsensitizers and low, moderate, and high reactivity as sensitizers, it was determined that a model based on cysteine and lysine gave a prediction accuracy of 89%. The results of these investigations reveal that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods.     

Categorisation of protein respiratory allergens: The case of Subtilisin

Characterisation of the relative sensitizing potency of protein and chemical allergens remains challenging, particularly for materials causing allergic sensitization of the respiratory tract. There nevertheless remains an appetite, for priority setting and risk management, to develop paradigms that distinguish between individual respiratory allergens according to perceptions of the hazards and risks posed to human health. One manifestation thereof is recent listing of certain respiratory allergens as Substances of Very High Concern (SVHC) under the provisions of REACH (Registration, Evaluation, Authorisation and restriction of Chemicals). Although priority setting is a laudable ambition, it is important the process is predicated on evidence-based criteria that are transparent, understood and owned. The danger is that in the absence of rigorous criteria unwanted precedents can be created, and confidence in the process is compromised. A default categorisation of sensitisers as SVHC requiring assessment under the authorisation process is not desirable. We therefore consider here the value and limitations of selective assignment of certain respiratory allergens as being SVHC. The difficulties of sustaining such designations in a sound and equitable way is discussed in the context of the challenges that exist with respect to assessment of potency, and information available regarding the effectiveness of exposure-based risk management.     

Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.     

A murine model for assessing the respiratory hypersensitivity potential of chemical allergens

Using equimolar quantities of 2 chemical allergens, toluene diisocyanate (TDI), noted for its ability to cause respiratory hypersensitivity, and dinitrochlorobenzene (DNCB), noted for its dermal sensitizing activity, the mouse was evaluated as a possible model to indicate respiratory hypersensitivity. A previously published procedure (Garssen et al. (1989) Immunology 68, 51–58) was followed whereby chemicals were applied epicutaneously to the shaved flank of BALB/c mice. Eight days later, animals were challenged by intranasal application of the chemical. The lungs were evaluated at 48 h. Both TDI and DNCB elicited mild mononuclear inflammatory cuffing around pulmonary vasculature. No reaction was noted around pulmonary airways. Sera, drawn 48 h following the intranasal challenge with chemical allergen, were evaluated for total IgE, hapten-specific IgE and IgG, and for IL-2, IL-4, IL-5, IL-6, and interferon gamma. Animals exposed to TDI demonstrated decreased total IgE and the presence of TDI-specific IgG. Cytokine levels were unchanged in both groups. These results indicate that in this mouse model, total serum IgE and the production of hapten-specific IgG antibodies distinguished a respiratory from a contact sensitizing chemical. Further comparison of the serologic response of mice to these two classes of chemicals is required to determine if the murine model can be used to predict dermal versus respiratory sensitizing activity of chemical allergens.     

Utility and limitations of a peptide reactivity assay to predict fragrance allergens in vitro.

A key step in the skin sensitization process is the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. A published peptide depletion assay was used to relate the in vitro reactivity of fragrance molecules to LLNA data. Using the classical assay, 22 of 28 tested moderate to strong sensitizers were positive. The prediction of weak sensitizers proved to be more difficult with only 50% of weak sensitizers giving a positive response, but for some compounds this could also be due to false-positive results from the LLNA. LC-MS analysis yielded the expected mass of the peptide adducts in several cases, whereas in other cases putative oxidation reactions led to adducts of unexpected molecular weight. Several moderately sensitizing aldehydes were correctly predicted by the depletion assay, but no adducts were found and the depletion appears to be due to an oxidation of the parent peptide catalyzed by the test compound. Finally, alternative test peptides derived from a physiological reactive protein with enhanced sensitivity for weak Michael acceptors were found, further increasing the sensitivity of the assay.     

Mechanistic assessment of peptide reactivity assay to predict skin allergens with Kathon® CG isothiazolinones

Assessment of skin sensitization hazard of chemicals currently depends on in vivo methods. Considering the forthcoming European Union ban on in vivo testing of cosmetic/toiletry ingredients, the search for alternative non-animal approaches is an urgent challenge for investigators today. For the skin sensitization end-point the concept of protein/peptide haptenation, that could reflect the chemical modification of skin proteins, crucial to form immunogenic structures, has been used to develop in vitro assays to predict the sensitization potential of new chemicals. Using glutathione and nucleophile-containing synthetic peptides we confirmed previously the possibility to screen for skin sensitization potential by measuring peptide depletion following incubation with a set of allergens and non-allergens. In this paper, additionally to our model development work, we performed mechanistic based studies to confirm the peptide reactivity concept under the specific conditions used for haptens in the screening assay as they were somewhat different from the ones expected to happen in vivo. Following the reactivity toward the peptides of ¹³C labelled MI and MCI, models of true haptens, we showed that the initial step leading to the biological end-point was similar regardless the conditions used even if final adducts could be different. This confirmed the validity of the peptide reactivity concept as well as the choice made to look at peptide depletion rather than at adduct formation.     

A direct INT-dehydrogenase assay (DIDHA) for assessing chemical toxicity

A direct INT-dehydrogenase assay (DIDHA) was developed to measure chemical toxicity as well as toxicant interactions. This test consists of measuring quickly and directly the dehydrogenase activity (i.e., INT reduction) with the aid of a double beam spectrophotometer, thus avoiding formazan extraction and centrifugation steps. The assay was used to determine the toxicity of heavy metals, phenol, α-naphtol, and herbicides (Barban, propanil, swep, BIPC, and CIPC). It was also useful in demonstrating the protective effect of clays and humic acids with regard to herbicide toxicity as well as antagonistic and synergistic interactions among toxicants.     

The chemical and biochemical reactivity of dichlorvos

The chemical structure, reactivity and metabolic fate of the insecticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate) are discussed in relation to the possible genotoxicity of this and other methyl phosphate triesters. Recent attempts to demonstrate the methylation of DNA following exposure of bacteria and animals to dichlorvos are reviewed. On the basis of comparative data relating mutagenesis to methylation reactions, it seems entirely appropriate to conclude that the mutagenicity of dichlorvos to bacteria is due solely to methylation of the bacterial DNA under the conditions of these tests. However, the methylation of mammalian DNA could not be demonstrated under realistic exposure conditions (when the alkylating mutagen methyl methanesulphonate afforded clearly measurable methylation). The failure to detect methylation by dichlorvos in vivo is attributed to the operation of highly efficient enzyme-catalysed biotransformations which rely largely on the phosphorylating reactivity of dichlorvos. The biotransformation pathways, characterised mostly in the rat, appear to be common also to pig, mouse, hamster, and man.     

Assessing the potency of respiratory allergens: Uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.     

Assessing the potency of respiratory allergens: uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.     

Methods for the identification of chemical respiratory allergens in rodents: comparisons of cytokine profiling with induced changes in serum IgE

No validated or widely recognized test methods are currently available for the prospective identification of chemicals with the potential to cause respiratory allergy. The cellular and molecular mechanisms that result in the induction of chemical sensitization of the respiratory tract are unclear, although there is evidence for the selective development of T helper 2 (Th2)-type responses and, in some cases, the production of IgE antibody. We have therefore examined the utility of cytokine profiling using BALB/c mice, together with the measurement of induced increases in the total serum concentration of IgE in the Brown Norway (BN) rat, as markers for the prospective identification of chemical respiratory allergens. Responses provoked by the reference respiratory allergen trimellitic anhydride (TMA) have been compared with those stimulated by the respiratory sensitizing diisocyanates toluene diisocyanate (TDI) and hexamethylene diisocyanate (HDI) and by the acid anhydride hexahydrophthalic anhydride (HHPA). Topical exposure of BN rats to TMA, TDI and HHPA each provoked marked immune activation (increases in lymph node cellularity and proliferation). However, only treatment with TMA stimulated vigorous increases in the total serum concentration of IgE. In contrast, exposure to HHPA, TDI or HDI failed to provoke significant changes in serum IgE concentration or induced only transient and relatively weak increases in serum IgE levels. In parallel experiments using BALB/c strain mice, however, topical application of all four chemical respiratory allergens provoked a marked Th2-type cytokine secretion profile in draining lymph node cells. These data suggest that the measurement of induced changes in serum IgE is not sufficiently sensitive for the robust identification of chemical respiratory allergens. Furthermore, irrespective of the reasons for variations in TMA-induced IgE production among BN rats, doubts remain regarding the utility of these animals for the characterization of immune responses to chemical allergens. Cytokine profiling using the BALB/c strain mouse apparently provides a more robust method for the hazard assessment of chemical respiratory allergens.     

Induced changes in total serum IgE concentration in the Brown Norway rat: potential for identification of chemical respiratory allergens.

A variety of chemicals can cause sensitization of the respiratory tract and occupational asthma that may be associated with IgE antibody production. Topical exposure to chemical respiratory allergens such as trimellitic anhydride (TMA) has been shown previously to induce increases in the total serum concentration of IgE in BALB/c strain mice. Contact allergens such as 2,4-dinitrochlorobenzene (DNCB), which apparently lack respiratory sensitizing potential, fail to provoke similar changes. However, it became apparent with time that there was some inter-animal variation in constitutive and inducible IgE levels. We have now examined the influence of topical exposure to TMA and DNCB on serum IgE levels in the Brown Norway (BN) rat. Such animals can be bled serially and thus it is possible to perform longitudinal analyses of changes in serum IgE concentration. The kinetics of IgE responses therefore can be followed on an individual animal basis, allowing discrimination between transient and sustained increases in serum IgE concentration. Rats (n = 5) were exposed on shaved flanks to 50% TMA, to 1% DNCB (concentrations that elicit comparable immune activation with respect to draining lymph node cellularity and proliferation) or to vehicle alone. Total IgE was measured by enzyme-linked immunosorbent assay in serum samples taken prior to and 14-42 days following initial exposure. Those animals having high pre-existing IgE levels (>1.0 microg ml(-1)) were excluded from subsequent analyses. The levels of serum IgE in the majority of rats exposed to DNCB or vehicle alone remained relatively stable throughout the duration of all the experiments conducted, although some animals displayed transient increases in serum IgE. Only TMA treatment was associated with a significant and sustained increase in the level of serum IgE in the majority of experiments. The elevated concentrations of IgE induced by topical exposure to TMA are persistent, the results reported here demonstrating that induced changes in IgE are maximal or near maximal at approximately 35 days, with a significant increase in IgE demonstrable for at least 42 days following the initiation of exposure. Interestingly, although TMA and DNCB at the test concentrations used were found to be of comparable overall immunogenicity with regard to lymph node activation and the induction of lymph node cell proliferation, there were apparent differences in humoral immune responses. Thus, not only did exposure to TMA stimulate increases in total serum IgE concentration and the production of specific IgE antibody, but also a more vigorous IgG antibody response was provoked by TMA compared with DNCB. These data suggest that the measurement of induced changes in serum IgE concentration in the BN strain of rat is able to differentiate between different classes of chemical allergen. Given the inter-animal variation in IgE production, it would be prudent to incorporate a concurrent assessment of responses induced by treatment with TMA as a positive control against which to assess the activity of other test materials.     

Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses

The cellular and molecular mechanisms that result in the induction of chemical respiratory sensitization are unclear, although there is evidence for the development of T helper (Th) 2 type responses and, in some cases, the production of IgE. We have compared cytokine secretion patterns stimulated by topical exposure of BALB/c strain mice or Brown Norway (BN) strain rats to the reference respiratory allergen trimellitic anhydride (TMA), or to the reference contact allergen 2,4-dinitrochlorobenzene (DNCB). Under conditions where TMA and DNCB provoke similar levels of immune activation [increases in lymph node cell (LNC) cellularity and proliferation] divergent cytokine expression patterns are elicited. TMA-activated LNC isolated from BALB/c mice or BN rats elaborated high levels of the Th2-type cytokines interleukin (IL)-10 and IL-13, but relatively little of the Th1-type cytokines IL-12 or interferon γ. For LNC derived from both species there was a requirement for restimulation in vitro with the mitogen concanavalin A for IL-4 production. Generally, DNCB-stimulated LNC displayed the converse type 1 cytokine phenotype. The cytokine secretion profiles of LNC isolated from BN rats were considerably more variable than those observed for LNC from BALB/c mice. Statistically significant differences (P<0.01) between DNCB- and TMA-activated LNC were recorded for all cytokines in BALB/c strain mice. For the BN rat, differences reached statistical significance (P<0.01) only for the expression of IL-4 and IL-13. These data demonstrate that the intrinsic ability of DNCB and TMA to promote preferential Th1- and Th2-type responses, respectively, is species-independent and provide further evidence that chemical respiratory allergens are associated with polarized Th2-type responses. For the prospective assessment of chemical respiratory allergens as a function of induced cytokine secretion profiles, however, these data suggest that the use of the BALB/c strain mouse will provide the more robust method.