Mitomycin C–Induced DNA Damage in Fanconi Anemia: Cross-Linking or Redox-Mediated Effects?

To the Editor:

The article by Carreau et al1 reports on the in vivo effects of mitomycin C (MMC) in mice carrying the Fanconi anemia (FA) group C mutation (Fac−/−). Among the mechanistic scenarios underlying FA pathogenesis, the authors refer to a phenotypic feature of FA cells related to oxygen hypersensitivity. Unfortunately, the use of citations on this subject appears to be quite inappropriate. First, the authors attributed a “secondary” role for oxygen sensitivity in FA cells2 which, however, may have been made oxygen-resistant after the immortalization procedure. In fact, the loss of O₂ sensitivity in transformed cells has been recognized as a general phenomenon, not confined to FA cell lines.3 A general statement was then made1 about the published results of studies which “have demonstrated overproduction of reactive oxygen species (ROS) and increased susceptibility to oxygen, as well as an increase in ROS-induced DNA lesions, particularly 8-hydroxy-2′-deoxyguanosine (8OHdG).” Unfortunately, the three references reported4-6 (cited as 37-39 in the report) neither dealt with FA nor with ROS-induced DNA damage. The above statement about excess ROS production and 8OHdG formation in FA was true, but rather should refer to the reports by Takeuchi and Morimoto7 and Degan et al.8 It is worthwhile to consider the subject of oxidative stress in FA based on both in vitro and ex vivo evidence, as reviewed by us recently.9 

A role for oxidative stress in FA has been documented for two decades, with reports providing evidence for an improvement of either chromosomal instability or cell growth after exposure of either primary lymphocyte cultures or fibroblasts from FA patients to: (1) catalase or superoxide dismutase, (2) low-molecular-weight antioxidants, or (3) decreased oxygen levels.10-14 A G₂ cell cycle delay, observed in FA cells, was counteracted by culturing cells in 5% O₂,15 and a major role was suggested for free iron in inducing G₂ arrest in FA cells.16 The report by Takeuchi and Morimoto7 provided evidence for excess oxidative DNA damage (8OHdG) in FAA cells challenged with H₂O₂ that was related, at least in part, to catalase deficiency. A recent report by Ruppitsch et al17provided elegant evidence for the loss of both MMC and diepoxybutane (DEB) sensitivity of FAA cells transfected with cDNA causing overexpression of thioredoxin, a nonenzymatic antioxidant protein.18 Hence, both exogenous and endogenous antioxidants can decrease the phenotypic defect of FA cells, both including O₂ and MMC sensitivity. In turn, the action mechanisms of MMC can either be ascribed to DNA cross-linking or to redox cycling, as reported in early studies of MMC.19,20That MMC sensitivity in FA cells may be attributed to redox mechanisms rather than to DNA cross-linking has been shown by four independent reports11,13,21,22 focused on as many different endpoints (chromosomal instability, cytotoxicity, apoptosis, and mutagenesis). Together, the results of these studies showed that: (1) MMC-induced toxicity was confined to normoxic conditions which, unlike hypoxia, were associated to enhanced redox-cycling mechanisms, not to DNA cross-linking,21,22 and (2) MMC toxicity was both removed by antioxidant enzymes and by low-molecular-weight antioxidants.11,13 

The observation of redox abnormalities in FA is not confined to in vitro conditions. A series of ex vivo studies provided evidence for abnormal O₂ metabolism in FA patients and in their parents. Freshly drawn white blood cells from both FA homozygotes and heterozygotes produced excess ROS as detected by luminol-dependent chemiluminescence (LDCL),23,24 and displayed excess 8OHdG levels that were significantly correlated with LDCL as well as with chromosomal instability.8 Thus, both ex vivo and in vitro evidence pointed to a direct link between ROS formation, oxidative DNA damage, and chromosomal breakages in FA.

Based on the available evidence, one might suggest that the authors1 could carry out a new series of experiments by exposing Fac−/− mice to different oxygen levels, with or without MMC administration. As additional endpoints worth being evaluated in Fac−/− mice, one might suggest to include the evaluation of oxidative DNA damage as well as of ROS-detoxyfying activities. This study could provide a formidable insight both into the FAC defect and the in vivo action mechanisms of MMC.

In conclusion, the current view attributing the FA-associated defect(s) to the phenotypic sensitivity to MMC and DEB related to cross-linking mechanisms may be viewed as a fading dogma relying on the definition of FA as a DNA repair disorder. While no conclusive evidence has thus far related FA gene products to any function in DNA repair, a thriving body of evidence has associated MMC (and DEB) sensitivity to an impairment of redox balance in FA cells, both in vitro and in vivo. This evidence should no longer be disregarded in the forthcoming studies of FA.