Novel superoxide dismutase mimetics for protection against paraquat nephrotoxicity

Paraquat (PO), a broad-spectrum herbicide, is primarily eliminated from the body unchanged via the kidneys. Unfortunately, it is nephrotoxic; and the PO-induced nephrotoxicity involves severe renal damage caused by reactive oxygen species (ROS), specifically by increasing superoxide anion (0/-) generation in the kidney. While proven to be of benefit in animal models of organ injury involving O2.-, superoxide dismutase (SOD) and superoxide dismutase mimetics (SOOm) can suffer problems regarding their bioavailability and toxicity. Since ROS has been incriminated in the pathogenesis of several disease conditions, the search for ideal SOOm therefore continues unabated. Thus, the current study was undertaken with the overall objective of investigating and comparing the therapeutic potentials of Mn (II) and Cu (II) complexes of ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) and ethylenebis hydroxyphenylglycine (EHPG), novel SOOm, against PO-induced nephrotoxicity using in vitro and in vivo models. The reno-cytotoxic effects of PO on confluent NRK-52E renal cell line in the absence and presence of SOOm were assessed using biochemical assays of cellular viability and cell death. The mode of injury produced by PO in vitro was evaluated using Hoechst-Propidium iodide staining. The ability of PO to increase ROS generation, specifically O2.- and hydroxyl radicals (OW) was investigated using nitroblue tetrazolium reduction/hydroxyethidium fluorescence and deoxyribose assay respectively. ROS-mediated cell damage was assessed via determination of lipid peroxidation, inhibition of DNA and protein synthesis, DNA single strand breaks and poly- (AOP-ribose) polymerase (PARP) activation using the thiobarbituric acid reactive substance assa¥., cellular incorporation of [3H]-thymidine and [3H]-leucine, a DNA precipitation and [ H]-NAO incorporation assays respectively. In addition, the effects of PO on the renal activities and expression of SOD, catalase (CAT) and glutathione peroxidase (GSH-Px) in confluent NRK-52E renal cells in culture were also evaluated. The reno-cytotoxic effects of paraquat were mediated in part by oxidative stress injury secondary to increase generation of ROS; specifically 0/- and OW. This was accompanied by a reduction in the activity and expression of SOD, a reduction in the activity with no significant change in the expression of CAT and an increased in the activity of GSH-Px. Oxidative stress injury was further confirmed by increased lipid peroxidation, inhibition of DNA and protein synthesis, and increased DNA single strand breaks with a subsequent increased in PARP activation. The doses of paraquat examined predominantly produced necrotic cell death. EUK-134 (30-300 IJM), tempol (0.1-1.0 mM) and Mn (II) and Cu (II) complexes of EGTA (10100 IJM) and EHPG (10-100 IJM) were able to improve cellular viability and reduce PO-mediated cell death in vitro via dismutation and/or scavenging of O2.- and reduced OH· generation. Mn (II) complexes displayed greater efficacy than Cu (II) complexes and at equivalent concentrations, Mn (II)-EHPG provided greatest protection. Thus, the hypothesis that, these novel SOOm, particularly Mn (II) complexes of EHPG and EGTA, could provide similar beneficial effect in vivo was tested in rodent model of paraquat-induced nephrotoxicity. Administration of a single intraperitoneal dose of PO (10-100 mg/kg) to male wistar rats (weighing 200-250 g) produced acute renal failure (ARF) within 24-48 hours as evidenced by a significant increase in serum creatinine levels and fractional excretion of sodium (FENa) and a concomitant reduction in creatinine clearance. Unlike Mn (II)-EGTA (2 mg/kg), Mn (II)-EHPG (4 mg/kg) was able to significantly attenuate the PO-induced ARF. In contrast to SOD and/or conventional SOOm which can display pro-oxidant actions at higher concentrations these complexes were not toxic at the doses examined. Since the clinical toxicity profiles of EGTA and EHPG are already known, these novel SOOm particularly Mn (II)-EHPG could be beneficial in attenuating disease conditions involving ROS generation.