ABSTRACT
Saccharomyces cerevisiae possesses both a cytoplasmic and a mitochondrial fumarate reductase, encoded by FRDS1 and OSM1, respectively. While previous studies have shown that mutants lacking FRDS1 and OSM1 cannot grow under anaerobiosis (Arikawa et al., 1998), the physiological role of fumarate reductase (FR) remains poorly understood. Here, we report that an osm1 frds1 mutant is unable to grow anaerobically, even with glutamate as a sole nitrogen source, when succinate can be produced by the TCA oxidative branch. We also show that the growth of the mutant is not restored by adding acetoin, an alternative sink for NADH oxidation, but it is at least partly restored by the addition of oxygen or menadione, which can oxidize FADH(2) in addition to NADH. These data indicate that the growth inhibition of the mutant is due to an inability to reoxidize FAD, rather than an indirect effect on NADH or an inability to produce succinate per se. During anaerobic growth, FRDS1 expression was two to eight times higher than that of OSM1, and fumarate reductase activity was higher in the osm1 mutant than in the frds1 mutant. FRDS1 expression was induced by anaerobiosis, and this induction was abolished in a rox1 mutant. We conclude that the formation of succinate is strictly required for the reoxidation of FADH(2) during anaerobiosis, and that it is regulated through the control of FRDS1 expression when oxygen is limiting. Based on these data, we discuss the potential role of fumarate reductase in the regeneration of the FAD-prosthetic group of essential flavoproteins.
Subject(s)
Saccharomyces cerevisiae/enzymology , Succinate Dehydrogenase/physiology , Acetoin/pharmacology , Anaerobiosis , Blotting, Northern , Flavin-Adenine Dinucleotide/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Isoenzymes/genetics , Isoenzymes/metabolism , Mutation/physiology , Oxidation-Reduction , RNA, Fungal/chemistry , RNA, Fungal/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Succinate Dehydrogenase/genetics , Succinate Dehydrogenase/metabolism , Succinic Acid/metabolism , Vitamin K 3/pharmacologyABSTRACT
Ochratoxins are a class of naturally occurring compounds produced by several fungi. The most toxic is ochratoxin A (OTA), and occurrence of some human nephropathies and tumors correlate with enhanced OTA exposure. In this Account, the following areas are examined: molecular details of the binding of OTA to human serum albumin (HSA), the influences of binding to HSA on the trans-port of OTA across epithelial cell membranes by organic anion transport proteins, the oxidative activation of OTA, and the formation of OTA adducts with biological molecules. These studies are beginning to provide a detailed chemical model for the trans-port, accumulation, and genotoxic and carcinogenic effects of OTA.
Subject(s)
Ochratoxins/metabolism , Ochratoxins/toxicity , Albumins/metabolism , Autoradiography , Biological Transport , Humans , Oxidation-Reduction , Protein BindingABSTRACT
Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is a potent renal carcinogen in male rats and is suspected of being the etiological agent of Balkan endemic nephropathy (BEN) and its associated urinary tract cancers. Conflicting results have been obtained regarding the genotoxicity of OTA and its ability to react directly with DNA upon oxidative bioactivation to yield covalent DNA adducts. To characterize DNA adduction by OTA, the present study utilizes the photooxidative properties of the toxin to generate authentic C8 OTA-3'-monophosphate-deoxyguanosine (3'-dGMP) adducts for use as cochromatographic standards for (32)P-postlabeling detection of OTA-mediated DNA adduction in the kidney of rat and pig. Our results show evidence for the photooxidation of OTA to yield carbon (C)- and oxygen (O)-bonded C8-3'-dGMP adducts (C-C8 and O-C8) that have been isolated and characterized by LC/MS with in-line UV and electrospray negative ionization (ES(-)) detection. A comparison to previously published work on related C8-dG adducts supports C8 attachment by OTA. The C-C8 OTA-3'-dGMP adduct standard is shown by (32)P-postlabeling to comigrate with the major lesion detected in the kidney of rat following chronic exposure to OTA and with one of four adducts detected in the kidney of pig following subacute exposure to the toxin. The O-C8 OTA-3'-dGMP adduct standard is also shown to coelute with a lesion detected in rat kidney. These findings suggest a role for the OTA phenoxyl radical in OTA-mediated DNA adduction in vivo, provide a rationale for the tumorigenesis of OTA, and strengthen the OTA hypothesis in the etiology of BEN and the associated urinary tract tumors.
