Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the 
internalization of the epidermal growth factor (EGF) receptor and the 
EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 
(Eps15) in fibroblasts. In addition, it was suggested that EGF receptor 
internalization might be inhibited by H2O2 by inhibition of ubiquitination of 
proteins involved in endocytosis. Here, we show that H2O2 also inhibits the 
poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of 
the cells resulted in re-establishment of ubiquitination of both the EGF 
receptor and Eps15 and coincided with restoration of internalization of those 
receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor 
internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), 
indicating that intact SH groups might be required for receptor-mediated 
endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio 
of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 
resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these 
results suggest a relation between the inhibition of internalization 
ubiquitination and an increase in GSSG:GSH ratio, which strengthens the 
hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of 
ubiquitination of proteins involved in EGF receptor-mediated endocytosis.