Inactivation of electrophilic metabolites by glutathione S-transferases and limitation of the system due to subcellular localization

Benzo(a)pyrene was activated to metabolites mutagenic for Salmonella typhimurium TA 98 by liver microsomes from control and phenobarbital treated mice. Under these conditions benzo(a)pyrene 4,5-oxide accounts for most of the mutagenicity. We have therefore investigated (1) the conjugation of benzo(a)pyrene 4,5-oxide with glutathione and (2) the effect of glutathione on the mutagenicity of benzo(a)pyrene.The spontaneous conjugation occurred only very slowly. The rate of this reaction was slightly augmented by microsomes and very greatly augmented by the cytosol fraction of liver homogenate. With respect to the mutagenicity of benzo(a)pyrene, glutathione had only a weak effect when benzo(a)pyrene was activated by microsomes in the absence of the cytosol fraction. In its presence, however, glutathione was able to strongly reduce the mutagenicity. But this reduction depended on the spatial relationship between microsomes and bacteria. The strongest inactivation was found when bacteria and microsomes were in separate agar layers. In contrast, no inactivation was observed when all the microsomes were in direct contact with the bacteria. When the test was performed according to the Ames procedure the topographical situation was intermediate: some microsomes were adsorbed onto the bacteria and some were free. Accordingly, the effect of glutathione was intermediate. When the premutagen trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene was activated in the presence of the cytosol fraction, glutathione again reduced the mutagenicity, when microsomes and bacteria were separated from each other, but did not reduce the mutagenicity, when all the microsomes were bound to the bacteria.Obviously in the situation where a direct diffusion within the lipophilic environment from the site of formation to the target bacteria was physically possible the mutagenic metabolites diffused preferentially directly to the bacteria and not through the hydrophilic environment of the medium. Therefore they could not be inactivated by components of the cytosol fraction. This could be of significance also for the situation in the eucaryotic cell, since the endoplasmic reticulum is in direct contact with other cell structures such as the nuclear envelope. Thus, hydrophobic metabolites generated in the endoplasmic reticulum could reach such sites by lateral diffusion within the membranes. The observation that benzo(a)pyrene 4,5-oxide was a very good substrate for the cytosol localized glutathione S-transferase, but that it was not inactivated by this system when bacteria and microsomes were in direct contact, indicates that a severe limitation for the inactivation of benzo(a)pyrene metabolites by this enzyme is imposed by its localization in the cytosol.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977