Work-related asthma has become the most frequently diagnosed occupational respiratory illness, accounting for 10–25% of adult asthma with occupations in healthcare having the highest risk (Kogevinas et al., 2007
). Of the identified 250 substances suspected to cause occupational asthma, ~90 are low molecular weight (LMW) organic chemicals (Jarvis et al., 2005
). The mechanisms by which LMW chemicals cause asthma due to sensitization are believed to be different from that of high molecular weight substances and remain poorly defined (Wild and Lopez, 2003
). Glutaraldehyde (100.13 MW) and OPA (134.132 MW) are dialdehydes capable of cross-linking proteins, thus functioning as effective biocides and tissue fixatives. Covalent bonding to primary amines and other protein moieties can result in the formation of hapten-carrier complexes with host proteins and may induce immunological responses. Defining the mechanism by which these chemicals induce sensitization is a critical step toward early diagnosis and prevention of work-related asthma.
While research on OPA is limited, there is extensive literature available on the adverse health effects associated with glutaraldehyde exposure (Gannon et al., 1995
; Rideout et al., 2005
; Waters et al., 2003
). Our laboratory has previously investigated the allergic sensitization caused by exposure to glutaraldehyde. It was identified as a moderate contact sensitizer in LLNA with an EC3 value of 0.089% (Azadi et al., 2004
). Although precise comparisons cannot be made because the experiments were not conducted simultaneously, the OPA studies attempted to parallel those previously described for glutaraldehyde; vehicle, mouse strain, and reagents were kept consistent between the studies. summarizes the results from the two studies. The EC3 value for glutaraldehyde was extrapolated because the lowest concentration tested yielded an SI value greater than 3. For comparison purposes, two EC3 values are presented for OPA, one calculated based on the equation described by Basketter et al. (1999)
and the other calculated based on the equation used to determine the EC3 value for glutaraldehyde as cited by Azadi et al. (2004)
. The EC3 values calculated for OPA, using both equations (0.079 and 0.051%), are similar to that described for glutaraldehyde (0.089%). In addition, the SI values for the 0.1 and 0.75% exposure groups were 3.5 and 12.7 for glutaraldehyde and 7.5 and 47.6 for OPA, respectively.
Comparison of Glutaraldehyde and OPA Sensitization Potentials
OPA and glutaraldehyde were both shown to induce irritation; however, in vitro
studies showed that lower concentrations of OPA (0.4125%) had increased toxicity compared to glutaraldehyde (1.1%) when both chemicals were tested simultaneously. Skin irritation and the associated inflammatory response may be an important component to the development of a robust acquired immune response and the process of sensitization. In this light, data from both in vitro
and in vivo
studies suggest that OPA is an irritant as evidenced by the direct cytotoxicity to primary skin cultures and the marked increase in ear swelling observed following topical exposure in mice. Concomitant with irritation was significant elevations in total and OPA-specific IgE serum antibodies and IgE+B220+ cell population in the DLNs of OPA-exposed mice. Manetz and Meade (1999)
have shown that select chemicals capable of inducing IgE-mediated allergic responses have similar peak increases in the percent IgE+B220+ and B220+ populations and tend to become significantly elevated at equivalent concentrations. A similar trend was observed after treatment with 0.75% OPA (IgE+B220+ population increased to 23.42 ± 3.23% and B220+ population increased to 31.80 ± 1.2% of total lymphocytes). Additionally, the OPA concentration that induced significant increases in the IgE+B220+ cell population (0.1%) was lower than the concentration significantly elevating total serum IgE (0.75%). The elevation in IgE+B220+ cells relates to local binding of soluble IgE to the CD23 receptor on B cells in the DLNs, which may occur before IgE elevations in the serum. These data along with the observed elevations in IL-4 expression further support the involvement of IgE in the allergic response. Similar elevations in IL-4 and IgE+B220+ cells in DLN were observed following exposure of mice to glutaraldehyde although higher concentrations were required relative to OPA (Azadi et al., 2004
). IL-4 is crucial for IgE expression because it is required for increased expression of CD23 on the B cells, B-cell proliferation, isotype switching, and IgE synthesis, and its expression often supports polarization to a Th2 hypersensitivity response. Further supporting the Th2 polarization, a significant elevation in OPA-specific IgG1
, a Th2-driven isotype, was observed for the 0.75% treatment group compared to vehicle control (Snapper et al., 1988a
). With the exception of a single mouse, this elevation was approximately five- to eightfold greater than that observed for OPA-specific IgG2a
antibody levels. Elevations in IgG2a
typically represent an inflammatory response and suggest polarization to a Th1 response with class switching most often caused by elevations in IFN-γ and tumor necrosis factor-α (Snapper et al., 1988b
). However, no increase in IFN-γ mRNA was observed when the DLNs of these mice were analyzed, further supporting a Th2 response. These results suggest that OPA acts as a Th2 sensitizer and may have implications for respiratory allergy. It is also possible that OPA may exacerbate existing allergy by establishing a Th2-supporting immunological milieu. Hasegawa et al. (2009)
showed significant increases in OVA-specific serum IgE, IL-4 mRNA, and IL-5 mRNA in animals exposed to OVA and OPA compared to OVA only. Consistent with our results, IL-4 mRNA was also enhanced when the animals were exposed to OPA without OVA. A positive result in the LLNA (EC3 = 0.051% extrapolated from current LLNA) along with significant elevations in IgE and IL-4 expression at concentrations below or similar to that designated as the working solution (0.55%) strongly suggests that OPA is a sensitizing chemical and that this chemical would be expected to cause significant activation of the immune system following skin exposure.
These are the first studies to describe immunotoxicity induced by dermal exposure to OPA. The LLNA is a test method that was developed and validated for the identification of contact sensitizers, and while LMW chemical respiratory allergens, such as toluene diisocyanate and trimellitic anhydride (TMA), induce positive responses in the LLNA, not all LLNA-positive chemicals are associated with respiratory allergy or asthma. Although it is often thought that the most common route of exposure to respiratory allergens is inhalation, published animal and human data have shown that dermal exposure may result in respiratory tract sensitization (Fukuyama et al., 2009
; Herrick et al., 2002
; Petsonk et al., 2000
). Studies have shown that topical application is effective in sensitizing rats to TMA, resulting in airway reactivity after inhalational challenge (Zhang et al., 2004
). In addition, other literature has also shown that dermal exposure of mice to natural rubber latex can induce latex-specific IgE and airway hyperreactivity upon respiratory challenge (Howell et al.
, 2002). Therefore, skin exposure needs to be addressed in the risk assessment for OPA.
The identification of OPA as an irritant and sensitizing chemical along with significant increases in IgE+B220+ cells in the DLNs, IL-4 mRNA and protein expression, total and specific IgE, OPA-specific IgG1, and published case reports raises concern that OPA may function as an IgE-mediated sensitizer. Comparison of these data to that obtained for glutaraldehyde demonstrates that the sensitizing potential for OPA is comparable to that of glutaraldehyde, suggesting that it may not be a safe alternative. Similar to glutaraldehyde, in an effort to reduce and prevent occupational exposure and disease, regulations for the use of this chemical may need to be established.