Inhibition and stimulation of long-chain fatty acid oxidation by chloroacetaldehyde and methylene blue in rats.

The effects of chloroacetaldehyde (CAA) and methylene blue, both alone and together, on mitochondrial metabolism, hepatic glutathione content, and bile flow were investigated in rats. Oxidation of [1-14C]palmitic acid, [1-14C]octanoic acid, and [1,4-14C]succinic acid allowed for the differentiation between carnitine-dependent long-chain fatty acid metabolism, medium chain fatty acid oxidation, and citric acid cycle activity, respectively. CAA, a metabolite of the anticancer drug ifosfamide, which may be responsible for ifosfamide-induced encephalopathy, inhibited palmitic acid metabolism but not octanoic or succinic acid oxidation, depleted hepatic glutathione, and stimulated bile flow. Methylene blue, which is clinically used to either prevent or reverse ifosfamide-associated encephalopathy, markedly stimulated palmitic acid oxidation either in the presence or absence of CAA, but did not affect the oxidation of octanoic and succinic acid or hepatic glutathione. Taken together, this study demonstrates that CAA inhibits palmitic acid metabolism. Methylene blue stimulates long-chain fatty acid oxidation, most likely by facilitating the translocation of fatty acids into mitochondria, and compensates for the CAA effect in vivo.

Cellular and subcellular heterogeneity of glutathione metabolism and transport in rat kidney cells.

Selective permeabilization of plasma membranes with digitonin produced separation of cytosolic and mitochondrial compartments of proximal tubular (PT) and distal tubular (DT) cells from a rat kidney. Subcellular distributions of several intracellular glutathione (GSH)-dependent enzymes were similar in the two cell types but specific activities were significantly higher in PT cells, indicating that DT cells, particularly in their mitochondrial fraction, have a diminished capacity to detoxify reactive oxygen species. To enable isolation of suspensions of mitochondria, renal cells were treated with digitonin followed by the bacterial protease nagarse and were filtered through polycarbonate membranes. Activity distributions of enzymatic markers for subcellular fractions were quantitated and uptake of GSH was studied in suspensions of PT and DT cell mitochondria. While PT cell mitochondria catalyzed rapid uptake of GSH that was inhibited by malate, indicating involvement of dicarboxylate carriers, DT cell mitochondria exhibited limited capacity for GSH uptake that was not inhibited by substrates for the two dicarboxylate carriers. This report provides the first description of methodology for the preparation of mitochondria from renal cells derived from specific nephron cell types and shows that mitochondria from DT cells have a significantly lower capacity to use GSH for detoxification and regulation of redox status.

Thiodiglycolic acid is excreted by humans receiving ifosfamide and inhibits mitochondrial function in rats.

Thiodiglycolic acid has been identified as a major metabolite of the anticancer drug ifosfamide in humans. Patients treated with 12-16 g ifosfamide/m2.day excreted thiodiglycolic acid ranging from 0.10 +/- 0.02 mmol on the first day of therapy, to a maximum of 3.27 +/- 0.15 mmol on the fourth day of ifosfamide infusion. This amounted to 5.4 +/- 0.2% of the administered dose of ifosfamide appearing as thiodiglycolic acid in urine during a 5 days' continuous ifosfamide infusion. Thiodiglycolic acid (50mg/kg) administered to rats inhibited the carnitine-dependent oxidation of [1-14C]palmitic acid by 55%, but affected neither the oxidation of [1-14C]octanoic acid, which is carnitine-independent, nor the oxidation of [1, 4-14C]succinic acid, a marker of Kreb's cycle activity. Additionally, thiodiglycolic acid (30 microM) inhibited oxidation of palmitic acid but not palmitoyl-L-carnitine in isolated rat liver mitochondria, indicating that it either sequesters carnitine or inhibits carnitine palmitoyltransferase I. This study elucidates a specific mitochondrial dysfunction induced by thiodiglycolic acid which may contribute to the adverse effects associated with ifosfamide chemotherapy.

Stimulation of respiration by methylene blue in rat liver mitochondria.

The effect of methylene blue on isolated rat liver mitochondria in the presence and absence of chloroacetaldehyde was investigated. Fatty acid oxidation was inhibited by chloroacetaldehyde and subsequently stimulated by methylene blue. Assessment of tightly coupled mitochondria revealed decreasing respiratory control ratios induced by increasing concentrations of methylene blue and methylene blue provoked mitochondrial swelling. In uncoupled mitochondria, methylene blue promoted a concentration-dependent stimulation of respiration. These findings provide evidence that methylene blue, the redox dye currently used as an antidote for encephalopathy associated with alkylating chemotherapy, uncouples oxidative phosphorylation and acts as an electron transfer mediator to stimulate mitochondrial respiration.

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney.

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.