Nicotinamide adenine dinucleotide phosphate (NADPH), 2,6-dichloroindophenol and dicumarol were purchased from Sigma-Aldrich Ltd. (St. Louis, MO, USA), croton oil, hexadecyltrimethylammonium bromide (HTAB) and allyl nitrile (3-butenenitrile, CAS No. 109-75-1, purity > 98%) from Tokyo Kasei Organic Chemicals (Tokyo, Japan), rabbit anti-myeloperoxidase polyclonal antibody (Ab-1) from Thermo Fischer Scientific (Fremont, CA, USA), Vectastain ABC kit (peroxidase rabbit IgG) from Vector Laboratories (Burlingame, CA, USA), and sodium pentobarbital from Dainihon Seiyaku Co. (Tokyo, Japan). All other chemicals were purchased from Nacalai Tesque (Kyoto, Japan).

All animal experiments were performed according to the Guidelines of the Committee on Animal Experimentation of Kanazawa University. Male ddY mice weighing 26 - 30 g obtained from Japan SLC Co. (Shizuoka, Japan) were used. Animals were maintained at 22˚C ± 2˚C under a 12:12 h light/dark cycle and allowed free access to tap water and laboratory food (CRF-1; Charles River Japan Inc., Yokohama, Japan).

Groups of 3 - 4 animals were administered allyl nitrile at subtoxic levels (100, 200, or 400 μmol/kg) or vehicledistilled water (control; 4 mL/kg) daily for five days by gastric intubation, based on our previous findings [13]. For antioxidant and phase 2 enzyme analyses, the animals were sacrificed on the sixth day. For induction of dermatitis, the animals were given croton oil on the sixth day as described in Section 2.4.

Mice were anesthetized with 100 mg/kg sodium pentobarbital and perfused transcardially with 1.15% KCl. An ear plug was immediately taken and stored at −80˚C until analysis. Cytosolic supernatant for enzyme analyses was prepared as previously described [12]. Protein concentrations were measured according to the method of Bradford [21] using bovine serum albumin as the standard. QR activity was measured according to the spectrophotometric method of Ernster [22], and GST activity according to the spectrophotometric method of Habig et al. [23] using 1-chloro-2,4-dinitrobenzene as the substrate. Catalase activity was measured using the method of Abei [24] using H₂O₂ as the substrate, and glutathione peroxidase activity was measured according to the spectrophotometric method of Paglia and Valentine [25] using H₂O₂ as the substrate and NADPH as the source of reducing equivalents.

Skin acute inflammation was induced on the right ear by application of 40 μg of croton oil dissolved in 15 μL of acetone, while 15 μL of acetone was applied on the left ear. At selected times following the croton oil application, animals were sacrificed and a 63.3 mm² tissue punch was taken from both the right and left ears to evaluate oedema and the leukocyte infiltrate. Quantitative assessment of oedema was performed by measuring the difference in weight between the portions taken from the right and left ears. The effect of repeated exposure to allyl nitrile on oedema development was quantified up to 48 h following croton oil application.

Quantitative assessment of the cellular infiltrate was performed by measuring myeloperoxidase activity, an index of the presence of neutrophilic granulocytes [20]. Myeloperoxidase was extracted by HTAB according to Bradley et al. [26]. A portion of ear (63.6 mm²) was suspended in 1 mL of 0.1 M phosphate buffer pH 6.0, containing 0.1% HTAB, and then homogenized by Polytron (Kinematica GmbH, Switzerland) for 1 min setting at 9. The homogenates were centrifuged at 15,000 g at 4˚C for 20 min, and the resulting supernatant was used for the enzymatic assay. Myeloperoxidase activity was measured according to the method of Maehly and Chance [27] with guaiacol as the substrate, and was expressed as μmol of guaiacol oxidized/min/1mL of supernatant.

Ear biopsies, fixed in 4% paraformaldehyde followed by in 30% sucrose, were sectioned by cryostat (16 μm, performed as previously described [10]. Tissue sections were incubated with primary antibody (rabbit anti-myeloperoxidase, 1:800) for four days at 4˚C. Sections were then incubated in Tris-HCl buffered saline (pH 7.4) containing 0.04% diaminobenzidine, 0.08% nickel ammonium sulfate, and 0.003% H₂O₂ for 10 min for visualizetion.

Statistical analyses were performed by analysis of variance followed by the Dunnett’s test for multiple comparisons. The level of significance was set at P < 0.05.

Exposure to five days repeated allyl nitrile at 100, 200, and 400 μmol/kg/day levels resulted in an increase in catalase activities in the ear (P < 0.05) (Figure 1(a)). Allyl nitrile at 100 and 200 μmol/kg/day had no effect on QR activity, while QR activity was elevated at 400 μmol/ kg/day (P < 0.05) (Figure 1(b)). The activities of GST and glutathione peroxidase in the ear were too low to measure at any dose of allyl nitrile (0 - 400 μmol/kg/day).

Control animals at 0 μmol/kg/day showed oedema after croton oil application, peaking at 6 h post-dosing, and then decreasing 24 and 48 h post-dosing (Figure 2). There was a significant reduction in oedema at 24 h post-dosing in the 100 μmol/kg/day allyl nitrile group (P < 0.05), and at 6 and 24 h post-dosing in the 400 μmol/ kg/day group (P < 0.05). There was no effect of 200 μmol/kg/day allyl nitrile on oedema throughout the observation period.

The recruitment of leukocytes in the inflamed ear tissue was measured as myeloperoxidase activity. In control animals exposed to 0 μmol/kg/day allyl nitrile, myeloperoxidase activity appeared at 6 h post-dosing with croton oil, reached a peak at 24 h, and decreased at 48 h (Figure 3). There was a significant increase in myeloperoxidase activity at 24 h post-dosing in the 100 μmol/ kg/day allyl nitrile group (P < 0.05), and at 6 h postdosing in the 400 μmol/kg/day (P < 0.05), while there was no effect in the 200 μmol/kg/day group.

Immunostaining with antimyeloperoxidase revealed evidence of tissue oedema and leukocyte infiltration after

croton oil application. The ear from animals pre-treated with 0 μmol/kg allyl nitrile for five days showed the oe0 dematous response and leukocyte infiltration at 6 h post-dosing with croton oil (Figure 4(A)), while there was no response at 6 h after acetone only application (Figure 4(E)). At 24 post-dosing the oedema had recovered while leukocyte infiltration was still evident (Figure 4(B)). The ear from the 400 μmol/kg/day group also displayed the oedematous response and leukocyte infiltration at 6 h post-dosing with croton oil (Figure 4(C)), while at 24 h the oedema had recovered and there was evidence of myeloperoxidase-positive cells (Figure 4(D)).