INTERACTION OF P-BENZOQUINONE AND P-BIPHENOQUINONE WITH MICROTUBULE PROTEINS IN-VITRO

p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to in duce aneuploidy in mammalian cells. Because a possible mechanism for t he aneuploidogenic effect of benzene may be the disruption of the mito tic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free condition s. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentratio n-dependent manner. This interaction is accompanied by a spectral chan ge of the quinones and loss of free sulfhydryl groups of MTP. With 40 mu M BQ or BPQ, 50% inhibition of MT assembly was observed and associa ted with the loss of 1.3 thiol groups per tubulin dimer. Further analy sis showed that native MTP form monoadducts, but no diadducts nor disu lfide bonds with both BQ and BPQ. The formation of covalent quinone/MT P monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually ex clusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently w ith thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not pron e to oxidative disulfide bond formation. This proposition was supporte d by the lack of native MTP to form disulfide bridges upon treatment w ith hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the be nzene metabolites BQ and BPQ to critical thiol groups of tubulin inhib its MT formation under cell-free conditions and may also interfere wit h the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy in duction reported for benzene.