Prior exposure to immunosuppressive organophosphorus or organochlorine compounds aggravates the T_H1- and T_H2-type allergy caused by topical sensitization to 2,4-dinitrochlorobenzene and trimellitic anhydride

Figures & data

Table 1.  Chemical doses used.

Oral pre-treatment at 4 weeks
Chemical	Classification	Oral LD50 values (mg/kg)	Doses in this study (mg/kg)
Parathion	Organophosphorus compound	5^a	0, 0.15, 1.5
Methoxychlor	Organochlorine compound	2900^a	0, 30, 300
Dermal sensitization at 8 weeks
Chemical	Classification	EC3 values for LLNA (%)	Doses in this study
			LLNA (%)	Detailed analysis (%)
Dinitrochlorobenzene	TH1-type allergen	0.06^b	0, 0.03, 0.1, 0.3	0.1
Trimellitic anhydride	TH2-type allergen	0.20^b	0, 0.1, 0.3, 1	0.3

^aRegistry of Toxic Effects of Chemical Substances NIOSH CD-ROM (2003). ^bFukuyama et al. (2008).

EC₃ value is an estimation of the amount of test solution required to induce in the LLNA a stimulation index of 3; LD₅₀, dose at which 50% of animals died; LLNA: local lymph node assay.

Figure 1.  Annexin-V (A: Jurkat T-cells; D: EL4 cells) and caspase-3 (B: Jurkat T-cells; E: EL4 cells) expression, and amount of DNA fragmentation (C: Jurkat T-cells; F: EL4 cells) in cells treated with parathion and methoxychlor. Annexin-V and caspase-3 expression values are expressed as mean (%) ± SD (n = 4 per group). Statistical significance indicated by asterisks: *p < 0.05 and **p < 0.01 compared with the control value (Dunnett’s multiple comparison test); ^Φp < .05 and ^ΦΦp < 0.01 compared with the high-dose treatment group (Dunnett’s multiple comparison test). For DNA fragmentation, molecular weight markers are shown on the left in C and F.

Figure 2.  Incorporation of [³H]-TdR in auricular lymph node in (A) TMA pre-treated with parathion, (B) DNCB pre-treated with parathion, (C) TMA pre-treated with methoxychlor, and (D) DNCB pre-treated with methoxychlor, in local lymph node assay. Incorporation of [³H]-TdR values are expressed as mean (dpm) ± SD (n = 5 per group). Statistical significance indicated by asterisks: *p < 0.05, **p < 0.01 compared with the vehicle control group (Dunnett’s multiple comparison test). DNCB, 2,4-dinitrochlorobenzene; TMA, trimellitic anhydride. [³H]-TdR, [³H]-methyl thymidine

Figure 3.  Helper (CD4⁺CD8⁻) (A: parathion; B: methoxychlor) and cytotoxic (CD4⁻CD8⁺) (C: Parathion; D: Methoxychlor) T-lymphocyte counts in auricular lymph nodes of mice treated with test solution. Helper- and cytotoxic T-lymphocyte counts are expressed as mean (× 10⁵ cells) ± SD (n = 6 per group). Statistical significance indicated by asterisks: *p < 0.05, **p < 0.01 compared with the vehicle control group (Dunnett’s multiple comparison test). AOO, acetone + olive oil, DNCB, 2,4-dinitrochlorobenzene; TMA, trimellitic anhydride.

Figure 4.  T_H1 cytokine production (IFNγ (A: parathion; D: methoxychlor), TNFα (B: Parathion; E: Methoxychlor), IL-17 (C: parathion; F: methoxychlor)) in auricular lymph nodes of mice treated with test solution. T_H1 cytokine production values are expressed as mean (pg/ mL) ± SD (n = 6 per group). Statistical significance is indicated by asterisks: *p < 0.05, **p < 0.01 compared with the vehicle control group (Dunnett’s multiple comparison test). AOO, acetone + olive oil; IFNγ, interferon-γ; IL, interleukin; TMA, trimellitic anhydride; DNCB, 2,4-dinitrochlorobenzene; TNFα, tumor necrosis factor-α.

Figure 5.  T_H2 cytokine production (IL-4 (A: parathion; D: methoxychlor), IL-5 (B: parathion; E: methoxychlor), IL-13 (C: parathion; F: methoxychlor)) in auricular lymph nodes of mice treated with test solution. T_H2 cytokine production values are expressed as mean (pg/mL) ± SD (n = 6 per group). Statistical significance is indicated by asterisks: *p < 0.05, **p < 0.01 compared with the vehicle control group (Dunnett’s multiple comparison test). AOO, acetone + olive oil; DNCB, 2,4-dinitrochlorobenzene; IL, interleukin; TMA, trimellitic anhydride.

Figure 6.  Expression of genes (T-bet (A: parathion; D: methoxychlor), GATA-3 (B: parathion; E: methoxychlor), ROR-γt (C: parathion; F: methoxychlor)) in auricular lymph nodes of mice treated with test solution. Gene expression values are represented the increase in expression compared to the value for the expression of GAPDH, and are expressed as mean ± SD (n = 6 per group). Statistical significance indicated by asterisks: *p < 0.05, **p < 0.01 compared with the vehicle control group (Dunnett’s multiple comparison test). PCR bands in photographs of the gels were visualized with a fluoroimager (Ettan DIGE) with 32, 35, and 35 amplification cycles for T-bet, ROR-γt and GATA-3, respectively. AOO, acetone + olive oil; DNCB, 2,4-dinitrochlorobenzene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GATA 3, GATA binding protein 3; PCR, polymerase chain reaction; ROR-γt, retinoic acid-related orphan receptor-γt; T-bet, T-box expressed in T-cells; TMA, trimellitic anhydride.