Reactive metabolite covalent binding to protein targets has been correlated with downstream cytotoxicity for numerous chemicals. Target proteins have been identified with a select group of known cytotoxic chemicals, but the specific underlying mechanisms that lead to a toxic outcome are still unclear. While the correlation between reactive metabolite binding and toxicity provides presumptive evidence for their involvement in toxicity, simply establishing these relationships does little to enhance our understanding of the precise mechanisms of toxicity. The work described in this dissertation was conducted to test the hypothesis that differences between cytotoxic and non-cytotoxic compounds which form reactive metabolites are related to differences in: 1) the persistence of adducts on proteins, 2) the identity of adducted proteins, and/or 3) the amino acid targets modified by the reactive metabolite. Furthermore, these studies postulate that posttranslational modification of a critical protein at a critical site will result in decreased protein function. Comparisons of the cytotoxic polycyclic, aromatic hydrocarbon, naphthalene (NA) and the non-toxic glutathione-depleting compound, diethyl maleate (DEM) were used to address these hypotheses in well-defined sub compartments of the lung.
Previous studies have shown that administration of naphthalene to mice resulted in selective, cytotoxic injury to non-ciliated bronchiolar epithelial cells (Clara cells) which correlated with depletion of cellular glutathione levels and with the formation of covalent protein adducts. In contrast to NA, administration of DEM did not result in Clara cell injury when administered at doses that resulted in GSH depletion to the same extent as toxic doses of NA. Thus, an understanding of the differences between NA—where reactive metabolite formation correlated with the incidence and severity of toxicity—and DEM—where GSH depletion occurred in the absence of detectable toxicity—provided an approach for testing the hypotheses above.
Intraperitoneal administration of DEM (1000 mg/kg) or NA (300 mg/kg) to mice resulted in depletion of GSH from the airway epithelium in vivo to ∼20% of control. Both compounds covalently bound to proteins at levels that were nearly identical in the airway epithelium following depletion of GSH, with stable adducts that persisted for at least eight hours. Chapter 2 of this dissertation describes studies showing that glutathione depletion and covalent binding are not coupled to toxicity with DEM and provide in vivo data in target sub compartments of the lung which supported use of this model to address the hypotheses listed above.
In chapter 3, appropriate in vitro conditions were established to examine potential differences in target proteins adducted by NA and DEM. Incubation of NA (350 μM) or DEM (350 μM) with dissected airways resulted in rapid GSH depletion to approximately 25% of control at 1 hour, similar to that observed following in vivo administration. However, the levels of reactive metabolite binding in dissected airway incubations with DEM were less than 30% of those measured with NA. Increasing the concentration of DEM yielded levels of bound adduct that were similar to those observed with lower concentrations of NA. The adducted protein targets of DEM included actin, myosin, protein disulfide isomerase, heat shock proteins, and more. There was significant overlap of proteins adducted by DEM and NA and the entire list of all low-abundance proteins which are posttranslationally modified is in Chapter 3. Only two proteins, BiP/Grp78 and 14-3-3 zeta/delta were uniquely adducted by NA, and the protein-folding chaperone functions of BiP along with informatics approaches presented by other research groups suggested that this may be a key cytotoxicity-related target protein. However, the substantial overlap between proteins adducted by DEM and NA suggested that this may not be a key factor in controlling downstream toxicity.
In chapter 4, the amino acid adduction sites for DEM with purified proteins were examined with protein disulfide isomerase (PDI), aldehyde dehydrogenase 2 (ALDH2), actin, tubulin, Hsp60, Hsp70, and Hsp90. Binding to Cys occurred at almost every residue examined, lysine binding ranged from 40-98%, and histidine binding from 20-100%, depending on the protein. Sixty percent of the prolines were adducted in PDI. Previous work determined that NA metabolites adduct cysteine, histidine, and lysine but the percentage of amino acids covered was less than observed with DEM. Alteration of protein function also was explored using actin, PDI, and ALDH2. NA metabolites, including the epoxide and both quinones, inhibited the polymerization of actin as well as the redox activities of PDI but had variable effects on ALDH2. In contrast, DEM had minimal effects on protein function at concentrations that were 3-4 times higher than NA.
|Commitee:||Puschner, Birgit, Van Winkle, Laura|
|School:||University of California, Davis|
|Department:||Pharmacology and Toxicology|
|School Location:||United States -- California|
|Source:||DAI-B 76/02(E), Dissertation Abstracts International|
|Keywords:||Amino acid targets, Diethyl maleate, Naphthalene, Protein adducts, Reactive metabolites, Respiratory track toxicity|
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