The role of oxygen metabolism for the pathological phenotype of Fanconi anemia

Abstract

The molecular defect of the hereditary disease Fanconi anemia (FA) remains unknown. The two theoretical possibilities are (1) an impaired DNA crosslink-repair system or (2) a disturbed oxygen metabolism either by overproduction of reactive oxygen intermediates (ROI) or by diminished detoxification of ROI. In order to gain further insight into the molecular mechanism of this disease, we have determined the repair capacity of FA cells challenged by crosslinking agents and have analyzed diverse biological systems that are involved in oxygen metabolism. We have tested normal and FA cells for oxygen consumption and for the activity of the antioxidant phospholipid-hydroperoxide-glutathione-peroxidase (PHGPx). FA cells show a reduced oxygen consumption and an increased PHGPx activity. Since spontaneous and induced chromosomal instability is a main cellular feature of FA, we have analyzed the redox state of cells and the effect of cytochrome P-450 (Cyt P-450) inhibitors and inducers on chromosomal breaks and micronuclei production. Our results indicate that Cyt P-450 enzymes, especially Cyt P-450 1A2, play a crucial role in radical metabolism in FA cells. Furthermore, we have determined NF-kappa B activity in untransformed cells and in SV40-transformed cells by gel shift experiments. NF-kappa B is a multiunit transcription factor that is known to be induced by ROI and that activates the expression of various genes involved in cellular responses to stress. NF-kappa B is constitutively induced in SV40-transformed FA cells probably as a consequence of an increased ROI level. Our results suggest that enzymatic defects in oxygen metabolism mediate the FA phenotype via impaired reactivity with ROI. Cyt P-450 1A2 appears to be a good candidate for the defective enzyme, even though no differences have been measured in the activity of this enzyme in FA and control fibroblasts in pilot experiments.

Cyclophosphamide metabolism in children with Fanconi’s anaemia

“…[12][13][14] CY is a prodrug which requires metabolic transformation to generate active alkylating species. The initial activation is thought to be mediated by a hepatic cytochrome P450 reaction.…”

“…4,[7][8][9] The increased sensitivity of FA patients to CY has been ascribed either to deficiencies in DNA repair 10,11 or to disturbed oxygen metabolism with overproduction of reactive oxygen intermediates. [12][13][14] CY is a prodrug which requires metabolic transformation to generate active alkylating species. The initial activation is thought to be mediated by a hepatic cytochrome P450 reaction.…”

Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase

“…21,22 A potential endogenous source of superoxide, the NADPH cytochrome P-450 reductase (RED) system, has also been implicated in FA. Not only were chromosomal breaks in FA cells reduced by cytochrome P-450 inhibition, but evidence of a direct physical interaction between FANCC and RED was reported, 23,24 leading to the hypothesis that FANCC might protect cells from ROS via regulation of RED activity.…”

“…21,22 A potential endogenous source of superoxide, the NADPH cytochrome P-450 reductase (RED) system, has also been implicated in FA. Not only were chromosomal breaks in FA cells reduced by cytochrome P-450 inhibition, but evidence of a direct physical interaction between FANCC and RED was reported, 23,24 leading to the hypothesis that FANCC might protect cells from ROS via regulation of RED activity.Mice with a targeted disruption of the gene encoding the cytosolic Cu/Zn SOD (Sod1) exhibit normal growth and development; however, they show a distinctive motor axonopathy 25,26 and impaired gametogenesis. 27 The limited spontaneous pathology of Sod1 Ϫ/Ϫ mice suggested that although this enzyme might function to modulate superoxide-mediated effects in some tissues under basal conditions, that it was of critical importance during exposures to specific pro-oxidant stimuli.…”

Overexpressed thioredoxin compensates Fanconi anemia related chromosomal instability

“…Antioxidants exert a stabilizing in¯uence on FA DNA and FA cells (Dallapiccola et al, 1985;Ruppitsch et al, 1997). Interestingly, the amount of antioxidative enzymes like catalase, superoxide dismutase, glutathione peroxidase, and phospholipid hydroperoxide glutathione peroxidase are normal in FA ®broblasts (Gille et al, 1987;Ruppitsch et al, 1997). Nevertheless, the intracellular content of reactive oxygen intermediates is elevated (Korkina et al, 1992).…”