Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK₁ Cells

Abstract

The anti-cancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate β-lyase reaction. This reaction would generate pyruvate, ammonium and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In the present work, the cellular location and identity of the PLP-dependent cysteine S-conjugate β-lyase was investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice.The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP-enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins, but had no effect on the amount of Pt bound to proteins in cytAspAT, cytosolic aspartate aminotransferase; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DMEM, Dulbecco's Modified Eagle's Medium; GFAAS, graphite furnace atomic absorption spectrometry; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GDH, glutamate dehydrogenase; GGT, γ-glutamyl transpeptidase; GTK, glutamine transaminase K; HBSS, Hanks' balanced salt solution; KGDHC, α-ketoglutarate dehydrogenase complex; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; mitAspAT, mitochondrial aspartate aminotransferase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; pmitAspAT, precursor of mitochondrial aspartate aminotransferase; PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phosphate; SD, standard deviation; SEM, standard error of the mean; TFEC, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2014 August 14. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLPdependent cysteine S-conjugate β-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes β-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a β-lyase reaction with the cisplatin-cysteine S-conjugate. In the present study mitAspAT was stably overexpressed in LLC-PK 1 cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK 1 cells that overexpressed mitAspAT when compared to control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspATtransfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity to the PLP-dependent cysteine S-conjugate β-lyase. Treatment with 50-or 100 µM cisplatin for 3 h, followed by removal of cisplatin from the medium ...

Cisplatin Binding and Inactivation of Mitochondrial Glutamate Oxaloacetate Transaminase in Cisplatin-Induced Rat Nephrotoxicity

“…The present study indicates that cisplatin itself binds to mitochondrial GOT. A study by Zhang et al 39) confirms our results. They found that a pyridoxal 5-phosphate-dependent cysteine S-conjugated GOT reaction formed a platinum-cysteine S-conjugate of cisplatin.…”

Possible role of cysteine-S-conjugate β-lyase in species differences in cisplatin nephrotoxicity

“…Additionally, male and female laboratory cats aged 1-3 years (3.1 ± 0.3 kg) and male beagle dogs aged 2-3 years (12.5 ± 0.8 kg) were selected from among non-treated or control animals (n = 5-6) in other projects. Since the mitochondrial AST was reported to catalyze the bioactivation of cisplatin and to transfect into renal cells (Zhang et al, 2006), this enzyme was selected among the C-S lyases. The mitochondrial AST activity was measured by a previously reported method (Cooper and Meister 1985) with some modifications.…”

“…The Pt-cysteine S-conjugates are reabsorbed via renal cysteine heterodimetric amino acid transporters into the proximal tubular epithelia, where they are metabolized by C-S lyase to produce toxic reactive metabolites (Hanigan and Devarajan 2003;Townsend et al 2003;Townsend and Hanigan 2002;Zhang et al 2006;Zhang and Hanigan 2003).…”

Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

“…It is worth noting that GDH in brain supplies 2-oxoglutarate for its oxidation, which occurs through the 2-oxoglutarate dehydrogenase complex (OGDHC), forming a supramolecular structure with GDH which in turn binds aspartate aminotransferase [189,190]. Since OGDHC dysfunction is also associated with neurodegeneration [191,192], the importance of GDH in the development of neurodegenerative processes may be mediated by impaired 2-oxoglutarate supply to OGDHC.…”