NMR STUDIES OF CELLULAR METABOLISM

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Two lines of investigation of the metabolism of fluorinated compounds and the toxicological behavior of phenolic compounds for which adducts are formed as a result of the formation of quinone methide intermediates have recently converged in a study of the toxicity of 4-trifluoromethyl phenol (4-TFMP). This compound was initially used as a control in studies of cresol metabolism, since it is generally assumed that trifluoromethyl groups are metabolically inert. However, 4-TFMP was found to exhibit cytotoxicity similar to cresol in studies of percision-cut rat liver slices. Analysis of the glutathione adduct indicates formation of a difluoro-quinone methide intermediate as a result of spontaneous (non-enzymatic) defluorination. This behavior is somewhat analogous to proposals for the reaction of trifluridine with thymidylate synthetase. The development of an artificial liver represents a long standing goal of toxicology as well as medicine. Attempts to utilize various bioreactor models have had only moderate success, with systems being relatively short lived and showing poor expression of drug metabolizing enzymes such as cytochrome P450. However, recent advances in the development of stem cell technology have brought this goal considerably closer to reality. We have recently been working with Prof. Lola Reid and Dr. Jeffrey Macdonald at the University of North Carolina to utilize NMR as a non-invasive tool for assessing various metabolic and flow parameters in several hepatic bioreactor model systems. Dr. Reid?s group has recently developed an antibody screening method that will be used to negatively sort human hepatic progenitors by fluorescent activated cell sorting, allowing enrichment of hepatic progenitor populations. We also recently have been able to demonstrate the feasibility of NMR monitoring of fluorinated xenobiotic metabolism, as well as a number of other metabolic parameters in a commercially available bioreactor. Preliminary NMR studies utilizing a miniature bioreactor that mimics a human liver lobule have also been performed. A short project involving the metabolism of propionate by E. coli was recently completed. We were able to observe NMR resonances arising from peptide components of the cell wall. This approach may prove useful for evaluating cell wall turnover and for monitoring the effects of antibiotics which interfere with this process.