Acetaminophen-induced S-nitrosylation and S-sulfenylation signalling in 3D cultured hepatocarcinoma cell spheroids

Acetaminophen (APAP) is possibly the most widely used medication globally and yet little is known of its molecular effects at therapeutic doses. Using a novel approach, we have analysed the redox proteome of the hepatocellular cell line HepG2/C3A treated with therapeutic doses of APAP and quantitated both individual protein abundance and their reversible S-nitrosylation (SNO) and S-sulfenylation (SOH) modifications by mass spectrometry. APAP treatment results in a late, transient increase in ATP production and a multiplicity of alterations in protein abundance and modifications. The majority of the differentially SNO or SOH modified proteins are found in the endoplasmic reticulum and cytosol, suggesting that the source of reactive species is there. The cellular response indicates: constraint of fatty acid metabolism; reduction in ribosome construction and protein synthesis (to conserve ATP); maintenance of glutathione levels (by increased synthetic capacity); and an increased NADPH production (via the pentose phosphate pathway). This response appears to be coordinated, directly or indirectly, by the canonical Wnt and Nrf2 signalling pathways. Combined with the known role of NAPQI, these studies suggest that the physiological and toxicological responses form a continuum: therapeutic doses of APAP produce reactive species and NAPQI in the cytoplasm but result in little permanent damage. The cell mounts a multifaceted response which minimises disruption and repairs are effected within a day or two. Higher doses of APAP lead to intensified reactive species production, which increasingly disturbs mitochondrial function and eventually leads to cell death.