Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol

Abstract

The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens.

Figure 1

(i) Reaction of BQ with NBT in 50% acetonitrile in a pH 7.4 phosphate buffer at 25 °C. Absorbance readings were performed at 412 nm. Using [NBT]₀ = 0.1 mM. BQ concentrations were varied from (a) 0.0 mM (b) 0.0125 mM (c) 0.025 mM (d) 0.05 mM (e) 0.1 mM (f) 0.2 mM (g) 0.4 mM (h) 0.8 mM. (ii) NBB was reacted with 0.1mM NBT in a phosphate buffered 50% acetonitrile. Initial NBB concentrations were (a) 0.0 mM (b) 0.1 mM (c) 0.25 mM (d) 0.5 mM (e) 1 mM (f) 2 mM (g) 4 mM. (iii) NBT at a concentration of 0.1 mM was reacted with {(a) 0 (b) 0.01 (c) 0.02 (d) 0.05} mM TDI in acetone.

Figure 1

(i) Reaction of BQ with NBT in 50% acetonitrile in a pH 7.4 phosphate buffer at 25 °C. Absorbance readings were performed at 412 nm. Using [NBT]₀ = 0.1 mM. BQ concentrations were varied from (a) 0.0 mM (b) 0.0125 mM (c) 0.025 mM (d) 0.05 mM (e) 0.1 mM (f) 0.2 mM (g) 0.4 mM (h) 0.8 mM. (ii) NBB was reacted with 0.1mM NBT in a phosphate buffered 50% acetonitrile. Initial NBB concentrations were (a) 0.0 mM (b) 0.1 mM (c) 0.25 mM (d) 0.5 mM (e) 1 mM (f) 2 mM (g) 4 mM. (iii) NBT at a concentration of 0.1 mM was reacted with {(a) 0 (b) 0.01 (c) 0.02 (d) 0.05} mM TDI in acetone.

Figure 2

Initial rate vs [E⁺] plots for (i) BQ, (ii) NBB and (iii) TDI. The rate constants are calculated from the slope of the [E⁺] vs initial rate for NBT depletion.

Figure 2

Initial rate vs [E⁺] plots for (i) BQ, (ii) NBB and (iii) TDI. The rate constants are calculated from the slope of the [E⁺] vs initial rate for NBT depletion.

Figure 3

Pseudo-first order plots for (i) BQ, (ii) NBB and (iii) TDI representing the Michael acceptor, S_N1/S_N2 and acylating domains respectively. Data obtained from full kinetics were used to plot the depletion of NBT with time.

Figure 3

Pseudo-first order plots for (i) BQ, (ii) NBB and (iii) TDI representing the Michael acceptor, S_N1/S_N2 and acylating domains respectively. Data obtained from full kinetics were used to plot the depletion of NBT with time.

Figure 4

Linear plots for k’ (−k_s[E⁺]₀) vs (i) [BQ], (ii) [NBB] and (iii) [TDI]. The k_s values obtained from the slopes of the linear plots were agreeable to the k_a values obtained using equation 3. The intercepts of the linear plots are the rate constants (k_i) for any competing reactions.

Figure 4

Linear plots for k’ (−k_s[E⁺]₀) vs (i) [BQ], (ii) [NBB] and (iii) [TDI]. The k_s values obtained from the slopes of the linear plots were agreeable to the k_a values obtained using equation 3. The intercepts of the linear plots are the rate constants (k_i) for any competing reactions.

Figure 5

(a) Logk_a vs pEC3 for Michael acceptors, acetylating agents and S_N1/S_N2 reactivity domains. Significant positive correlations between LLNA pEC3 values and NBT reactivity was observed within the domains. (b.) Correlation between NBT reactivity and potency in the LLNA was still observed across all reactivity domains (r² = 0.74). DNCB NBT reactivity was observed, but was outside the predictive bands at 95% CI.

Figure 5

(a) Logk_a vs pEC3 for Michael acceptors, acetylating agents and S_N1/S_N2 reactivity domains. Significant positive correlations between LLNA pEC3 values and NBT reactivity was observed within the domains. (b.) Correlation between NBT reactivity and potency in the LLNA was still observed across all reactivity domains (r² = 0.74). DNCB NBT reactivity was observed, but was outside the predictive bands at 95% CI.