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 induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic 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 conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 microM BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP 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 exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with 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 prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.
Pfeiffer
E
E
Department of Chemistry, University of Karlsruhe, Germany.
Metzler
M
M
D000818:Animals; D016227:Benzoquinones / Q000737:chemistry / Q000633:toxicity*; D001713:Biphenyl Compounds / Q000737:chemistry / Q000633:toxicity*; D002417:Cattle; D005978:Glutathione / Q000737:chemistry; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008868:Microtubule Proteins / Q000187:drug effects*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011809:Quinones / Q000737:chemistry / Q000633:toxicity*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry
C101556:4-biphenoquinone; D016227:Benzoquinones; D001713:Biphenyl Compounds; D008868:Microtubule Proteins; D011809:Quinones; D013438:Sulfhydryl Compounds; C004532:quinone; D005978:Glutathione