Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells

Abstract

The anticancer 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 beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were 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 the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this 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 than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.

Figure 1

Effect of AOAA on Pt binding to protein in kidneys from mice treated with cisplatin. Mice were pretreated with saline (open circles) or AOAA (filled circles) followed by an injection of 15 mg/kg cisplatin. Twenty-four h after cisplatin treatment, kidney fractions were isolated and the protein in each fraction precipitated with acid. The amount of Pt bound to protein was measured with FAAS. There was significantly more Pt bound to protein in the mitochondrial fraction (M1) than in the cytosol. AOAA had no effect on the amount of Pt bound to proteins in the cytosol (cyto), but significantly reduced the binding to proteins in the M1 fraction. Treatment with AOAA did not significantly reduce the amount of Pt bound to protein in the M2 fraction.

Figure 2

Western blot of LLC-PK₁/mitAspAT and LLC-PK₁/C1 cell lysates. LLC-PK₁/C1 (Lane 1) or LLC-PK₁/mitAspAT (Lane 2) cell lysate were loaded in each lane (15 µg protein per lane). Rabbit anti-rat liver mitAspAT whole serum was used as the primary antibody. A prominent protein band with molecular mass of ~45 kDa (arrow) was detected in the LLC-PK₁/mitAspAT cell line, but not in the LLC-PK₁/C1 cell line. The molecular mass of this band is consistent with that of mitAspAT monomer. The positions of molecular mass markers are indicated.

Figure 3

Toxicity of cisplatin in confluent LLC-PK₁/mitAspAT and LLC-PK₁/C1 cells. Confluent monolayers of LLC-PK₁/mitAspAT cells (open diamonds) or LLC-PK₁/C1 cells (filled diamonds) were treated with cisplatin in DMEM for 3 h. The cisplatin was removed at the end of the 3-h exposure and replaced with fresh DMEM, containing 5% FBS and 400 µg/ml G418. The viability of the cells was measured at 72 h. A representative experiment is shown. Each point represents the mean of triplicate samples ± S.D.

Figure 4

Effect of AOAA on cisplatin toxicity in confluent LLC-PK₁/mitAspAT cells. Confluent monolayers of LLC-PK₁/mitAspAT were preincubated with AOAA for 30 min. The cells were then treated for 3 h with DMEM containing no cisplatin (filled diamonds) or 120 µM cisplatin (filled triangles) and the concentration of AOAA used in the preincubation. The cisplatin and AOAA were removed at the end of the 3-h exposure and replaced with fresh DMEM, containing 5% FBS and 400 µg/ml G418. The viability of the cells was measured at 72 h. The experiment was done three times. A representative experiment is shown. Each point represents the mean of triplicate samples ± S.D.

Figure 5

Toxicity of cisplatin in dividing LLC-PK₁/mitAspAT and LLC-PK₁/C1 cells. Dividing cells of LLC-PK₁/mitAspAT (open diamonds) or LLC-PK₁/C1 (solid diamonds) were treated with cisplatin in DMEM for 3 h. The cisplatin was removed at the end of the 3-h exposure and replaced with fresh DMEM, containing 5% FBS and 400 µg/ml G418. The viability of the cells was measured at 72 h. The experiment was done three times. A representative experiment is shown. Each point represents the mean of triplicate samples ± S.D.

Figure 6

Effect of cisplatin on the relative specific activities of several enzymes of energy metabolism in control LLC-PK₁ cells expressing empty vector (LLC-PK₁/C1 cells) and in LLC-PK₁ cells overexpressing mitAspAT (LLC-PK₁/mitAspAT cells). The cells were treated with 50 µM cisplatin or with 100 µM cisplatin for 3 h. The cisplatin-solution was removed and the cells were incubated in DMEM containing 5% FBS for an additional 24 h. At the end of the incubation the specific activities of selected enzymes in the cisplatin-treated cells were compared to those of control cells. The n and absolute values are as shown in Table 4. AspAT = mitAspAT + cytAspAT. The specific activities of the enzymes measured in the cisplatin-treated cells are reported as a percentage of the specific activity of the enzyme in the control cells for each individual cell preparation. Significance values were determined using one-way ANOVA. The data are shown as the mean ± SEM. The * symbol indicates p < 0.05. In addition, the two-tailed paired t-test indicates that the relative specific activity of KGDHC is significantly lower in the LLC-PK₁/mitAspAT cells treated with 100 µM cisplatin than in the LLC-PK₁/C1 treated with 100 µM cisplatin (p = 0.008).