Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach

Abstract

Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride ( 13 C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.

Covalent Binding of Reactive Anhydride of Cantharidin to Biological Amines

“…Cantharidin did not bind to glutathione. This preference is consistent with the reactivity of the reported anhydrides (Khong et al, 2021;Jeppsson et al, 2008).…”

“…Anhydride-containing compounds have been used for labeling or masking the lysine residue of proteins through their reactivity toward amines (Han et al, 2012;Spicer and Davis, 2014;. The reactivity of phthalic anhydride toward amines has been reported to be associated with respiratory sensitization (Khong et al, 2021). But to our knowledge, the binding of cantharidin anhydride to primary amines has not been reported.…”

“…Several well-known anhydride-containing compounds enable covalent binding with primary amino groups by the electrophilic anhydride moiety, such as succinic anhydride, maleic anhydride, citraconic anhydride, and phthalic anhydride (Kahsai et al, 2014;. Some anhydride-containing compounds, such as phthalic anhydride and hexahydrophthalic anhydride, cause allergies attributed to the highly reactive anhydride (Ahlfors et al, 2005;Khong et al, 2021). This study aimed to examine the chemical reactivity of cantharidin toward nucleophiles and characterize conjugates of cantharidin with biological amines in vitro and in mice.…”

Phthalic anhydride (PA): a valuable substrate in organic transformations

“…The adduct demonstrated high activities against Gram-negative and Gram-positive bacteria and high cytotoxic activity against MCF7 and HCT-116 (Scheme 21). 125 The pyrazole-containing substrate (50) was achieved via the one-pot coupling reaction of N,N-dimethylamino benzaldehyde (47), malononitrile (48), and phenyl hydrazine (49).…”

“… 48 Lepoittevin et al in 2021 investigated the reactivity of PA as a chemical respiratory sensitizer toward reconstructed human epidermis (RHE). 49 …”