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RE: Multiple factors conferring high radioresistance in insect Sf9 cells. (Mutagenesis, 24, 259-269, 2009)

2010; Oxford University Press; Volume: 25; Issue: 4 Linguagem: Inglês

10.1093/mutage/geq022

1464-3804

Sudhir Chandna,

Insect behavior and control techniques

In their recently published study in Mutagenesis, Cheng et al. (1) reported potential cellular factors responsible for radioresistance of a lepidopteran insect cell line, Sf9, established from the ovarian tissue of Fall armyworm, Spodoptera frugiperda. Their study using X-ray dose range 5—20 Gy demonstrates that multiple factors including reduced induction of DNA damage, apoptosis and reactive oxygen species (ROS) contribute to radioresistance of Sf9 cells, besides indicating a radioprotective role of cellular glutathione. As a radiobiologist working extensively on radioresistance of lepidopteran cells, I find that some of the major aspects that the authors report in this paper have been published earlier, including the role of efficient DNA repair (2) as well as the reduced induction of DNA damage and apoptosis (3). Further, certain results and inferences reported by Cheng et al. need to be addressed quite carefully due to limitations in experimental designs and/or a low radiation dose range chosen for these rather radioresistant cells. Therefore, certain major points brought out in the study by Cheng et al. are critically discussed here in order to provide an improved perspective of the understanding gained so far in this important field of insect cell radiobiology. The authors used X-ray doses as low as 5—20 Gy, whereas lepidopteran cell lines are known to withstand much higher doses ranging 200–400 Gy (3,4). While observing almost undetectable growth inhibition by 72 h post-irradiation at all these doses (also known from previous reports), a rather unacceptable lethal dose (50% survival) or LD50 dose of 49.7 Gy is derived from the growth curves, which is misleading and presents a falsely reduced level of radioresistance for these cells. As a result, the LD50 of 49.7 Gy derived for Sf9 cells is about three times the LD50 value shown for human gastric carcinoma SC-M1 cell line (18.4 Gy) (1), whereas earlier studies have shown 10–100 times higher radioresistance for lepidopteran cells compared to mammalian/human cells using cellular end points including growth inhibition (2,3). It should also be noted that negligible growth inhibition at 72 h postirradiation could well be a transient ‘cytostatic’ effect rather than a ‘cytotoxic’ effect. Using elaborate cell cycle progression analysis, we have actually shown that c-radiation doses up to 50 Gy induce transient growth inhibition in Sf9 cells, which gets released during 48—96 h post-irradiation (3). The authors further show that Sf9 cells do not undergo phosphatidylserine externalization (annexin-V labelling) or apoptotic DNA fragmentation following irradiation at up to 20 Gy, which is evidently a sublethal dose. Quite surprisingly, they also mention that radioresistance of insect cells has never been correlated with the induction of apoptosis, whereas these cells have already been shown to be excessively resistant against ionizing radiation-induced apoptosis at much higher doses of 100–200 Gy (3). It is however not yet known what mechanisms make these cells resist radiation-induced apoptosis. It has been shown that Sf9 cells carry a mitochondriamediated apoptotic pathway homologous to human cells (5,6), which is also induced at high doses of ionizing radiation (S. Chandna, S. Suman, A. Pandey, unpublished results). A significant reduction in radiation-induced DNA strand breaks has been shown in this study at 5–20 Gy using the comet assay, an effect already shown at higher doses by us using the same technique (3). Cheng et al. mention that the lower DNA damage is attributable to more efficient DNA repair and may act as a key factor in increased survival of irradiated insect cells. However, it is important to remember that the lower DNA damage induced (in comparison with human cells) is exclusive from the more efficient DNA repair reported previously in these cells (2). Efficient repair would primarily reduce DNA damage manifestations in the form of cytogenetic damage, mutations and cell death. Incidentally, we have shown that Sf9 cells with a genome size roughly half that of the human genome have a more compact chromatin organization with shorter loops between matrix attachment regions, although the nuclear volume (genome target size) is larger than that of human nuclei (3). An important effect shown by the authors is the relatively lower level of ROS generation in Sf9 cells at up to 20 Gy. Our laboratory has also demonstrated significantly less ROS induction in Sf9 cells as compared to human cells at high c-radiation doses up to 200 Gy (7), and recently, we reported that lepidopteran cells may have a stronger antioxidant defence system (8,9). All these findings thus suggest that lepidopteran cells are more tolerant to oxidative stress, and the free radicals formed during irradiation. Cheng et al. also show that radiation-induced Sf9 cytotoxicity is enhanced following pretreatment with the glutathione inhibitor buthionine sulfoximine, thus implying the important role of glutathione. It would be further important to investigate whether lepidopteran cells enjoy a relatively higher level of radioprotection by intracellular glutathione pool and whether this effect can be observed at high doses. In my opinion, an ideal design should include assessing buthionine sulphoximine (BSO)-induced increase in cytotoxicity, ROS generation and DNA damage at the iso-inhibitory BSO concentrations in insect and human cell lines. The study by Cheng et al. also mentions that the holokinetic nature of lepidopteran chromosomes could lead to radioresistance, a hypothesis that was contradicted by the less radioresistant hemipteran insect cells also carrying holokinetic chromosomes. As discussed earlier by Koval (2), this aspect could still contribute partially. Importantly, the first indirect