Evaluation of the role of mitochondria in drug-induced toxicity

Mitochondria are organelles where the cellular energy is produced and oxidative respiration as well as many other vital physiological processes take place. Therefore any structural or functional disorders in mitochondria may cause drastic reasults in the organism. In this thesis, it has been aimed to investigate the effects of various drugs on mitochondria whose toxic mechanisms are unclear. Mechanisms of clozapine-induced cardiotoxicity and paracetamol-induced nephrotoxicity have not been enlightened yet, but is thought to be related with the biotransformation of the drugs. Many adverse drug reactions are known to be mediated by the reactive metabolites of the drug molecules. Therefore the role of mitochondrial and microsomal bioactivation is investigated in the toxicity of the two drugs. To achieve this aim, microsomes that are obtained from liver and kidney tissues of the patients were incubated with paracetamol. Inter-individual differences between the amounts of reactive metabolites and inter-organ variations in enzyme activities that catalyze the bioacitvation of paracetamol were evaluated. Possibility of one of the hypotheses of the present study, "drugs can cause toxicity via producing local reactive metabolites in mitochondria" was tested for paracetamol and clozapine. For this aim, pure mitochondrial fractions were obtained by using various techniques and their purity was proven by Western blot analysis. Clozapine was incubated with mice liver and heart mitochondrial fractions and formation of reactive metabolite as well as several stable metabolites were shown. Similarly, reactive metabolite formation was shown for paracetamol following incubation with liver mitochondria. Mitochondrial Permeability Transition Pore (MPTP) formation was measured and utilized as an indicator of the structural and functional integrity of mitochondria. Our data indicated that mitochondria maintain their integrity for 3 months when they are kept in -86oC as the whole tissue or as isolated mitochondria. Next to clozapine and paracetamol, the effects of olanzapine, nifedipin, diclofenac, valproic acid, metformin, dapsone, tiaprofenic acid and sulphafenazole on mitochondria were investigated in the presence or absence of calcium as MPTP inducers. Diclofenac and valproic acid have been found to increase the formation of calcium-induced MPTP, but none of the drugs was found to induce MPTP formation alone. DNA methylation as an important determinant of the gene activity, has been further evaluated in mitochondrial DNA in the presence of the above drugs. All tested drugs were shown to cause statistically significant hypomethylation, and clozapine was shown to cause this effect via its reactive metabolite. The effect of the drugs on oxygen consumption rates, the most important metabolic process taking place in mitochondria, was further investigated. Clozapine, olanzapine, nifedipin, valproic acid, dapsone ve tiaprofenic acid were shown to decrease the oxygen consumption rates, where sulphafenazol interestingly caused an increase. Finally human liver and heart antioxidant and phase II conjugation enzyme activities that are important in terms of mitochondrial drug biotransformation, were evaluated in human tissues obtained via autopsy, and GSTT, GSTM, CAT and Se-GPx activities were shown in mitochondria from both organs in humans. By investigating the structural and/or functional effects of the drugs on mitochondria, data were obtained not only for toxic, but also for possible therapeutic targets in this thesis. As well as novel opinions have been put forward for the mechanisms of clozapine-associated cardiac toxicity and paracetamol-associated kidney toxicity. Developed experimental approaches may be used for screening the mitochondrial toxicity of other drugs, drug candidates or chemicals.