Correlating the structure and reactivity of a contact allergen, DNCB, and its analogs to sensitization potential

Abstract

We report a study that seeks to find a correlation between the overall sensitization potential quantified by the expression of IL-8 by stimulated monocytes and the chemical structure of a model contact allergen, 2,4-dinitrochlorobenzene (DNCB). We show that structure and reactivity of the chemical compounds play an important role in activation of the monocytes and subsequent inflammation in tissue. However, we observed a non-linear correlation between the rate of reaction and biological activity indicating a required balance of stability and reactivity.

Keywords: 1-chloro-2,4-dinitrochlorobenzene (DNCB); Contact hypersensitivity; Interleukin (IL) – 8; Skin sensitization; Structure Activity Relationship (SAR).

Figure 1.

The overview of this paper. The sensitization potential of DNCB and derivatives is measured employing a variety of techniques

Figure 2.

The leaving group halides (a) and non-halide leaving groups (b) were explored. THP-1 cells were incubated with 50 μM of labelled compounds for 10 h and the percentage of IL-8 expressing cells were measured via flow cytometry. * = significance from resting, ¥ = significance from DNCB. *P ≤ 0.05 ***P ≤ 0.001 ****P ≤ 0.001 ¥¥ P ≤ 0.01

Figure 3.

The quality of electron-withdrawing groups was explored. A series of electron withdrawing groups were substituted in place of nitro group (a) or additional electron-withdrawing groups were substituted in ortho position (b). THP-1 cells were incubated with 50 μM of labelled compounds for 10 h and the percentage of IL-8 expressing cells were measured via flow cytometry. * = significance from resting, ¥ = significance from DNCB. *P ≤ 0.05 **P ≤ 0.01 ****P ≤ 0.0001

Figure 4.

IL-8 secretion by THP-1 cells stimulated with the parent compound (1) and the ester derivatives (18-21). THP-1 cells were incubated with 50 μM of labelled compounds for 20 h and the IL-8 secreted to supernatant was quantified using sandwich ELISA * = significance from resting, ¥ = significance from DNCB. **P ≤ 0.01 ***P ≤ 0.001 ****P ≤ 0.0001

Figure 5.

Correlation IL-8 activity to the rate of reactivity to cysteine. IL-8 activity of compounds was normalized to DNCB (1).

Figure 6.

Diagram showing DNCB modification to cysteine side chains of cellular protein (a) and the reaction mechanism (b). Proteins modified by DNCB (1) and its derivatives were analyzed by western blotting (c, left). Untreated lane shows no band, confirming that anti-DNP antibody is specific to DNP-like-moieties. Compounds 4, 6, and 7 show band intensity similar to that of DNCB. Proteins were separated using the same material and methods and Coomassie-stained to confirm that the same amount of proteins were used in all lanes (c, right).

Figure 7.

Skin inflammation study on mouse dorsum. (a) Examples of unswollen (left) and swollen (right) mouse dorsum. Mouse was treated with 0.5 % compound solution on day 0 and 1. The mouse was challenged with the same 0.3 % compound solution on day 5, 6, 7 and 8. (b) Ear thickness measured on day 9. * = significance from resting, **P ≤ 0.01 ***P ≤ 0.001 ****P ≤ 0.0001