Carcinogenicity and haemoglobin synthesis induction by cytidine analogues

Abstract

Summary We investigated 5-azacytidine and five of its analogues for: (1) carcinogenicity, in the male Fischer rat; (2) toxicities using changes in rat weights in vivo and a cytotoxicity assay in vitro; and (3) haemoglobin gene expression, using minor haemoglobin synthesis in sheep, mice and rats. 5-Azacytidine was found to be a complete carcinogen. It increased the incidence of testicular tumours as well as non-testicular tumours in rats treated for 12 months. 5-Azacytidine also had hepatic tumour promoting properties and was able to induce transplacental carcinogenesis when administered to pregnant rats on day 21 of timed pregnancies. None of the other 5 analogues that were tested appeared to be carcinogenic in small experiments. All the analogues which are known to have hypomethylating activity were found to be cytotoxic in vitro; the most potent being 5-azacytidine. As judged by decreased rat weight compared to untreated controls, the fluorinated cytidine analogues and 5'-deoxyazacytidine were more toxic than 5-azacytidine. Altered haemoglobin synthesis was seen in rats and DBA/2J mice, but not in sheep. In mice, where the clearest haemoglobin changes were noted, an increase in minor haemoglobin synthesis was found using both high and low doses of 5-azacytidine, and with 5,6-dihydro-5-azacytidine and 5-aza-2'-deoxycytidine. These last two analogues appear to be relatively non-toxic, noncarcinogenic in these experiments, and retain haemoglobin activating properties with a potency similar to that of 5-azacytidine.Enzymatic DNA methylation occurs as a postreplicative process, producing, in mammals, only the minor base 5-methylcytosine. The methylation of specific DNA cytosine residues is inversely correlated with transcription of most genes, although there are exceptions. Inhibitors of DNA methylation will often activate previously silent genes and this is the strongest single line of evidence implicating methylation in gene control. There have been several recent reviews of mammalian DNA methylation (Holliday, 1979;

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“…Multicycle repeated-dose toxicity studies in rats and rabbits indicated that the primary toxicity was myelosuppression, which was reversible on cessation of treatment [28][29][30][31][32]. Gastrointestinal toxicity was also observed.…”

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“…22 An early study reported that this agent was not carcinogenic in the rat model. 7 More interestingly, recent studies have shown that treatment of mice with a genetic disposition for colon or lung cancer with decitabine results in a marked reduction in tumor formation. 23,24 It is believed that this reduction in tumorogenesis may reflect demethylation of tumor suppressor genes.…”

“…In spite of these promising preliminary results, this agent was never tested in large-scale clinical trials due to concerns about its potential carcinogenic effects, since a previous study conducted in laboratory rats showed that 5-azacytidine increased the incidence of tumors. 7 Interestingly, controversy over the mechanism of induction of HbF by 5-azacytidine led to the studies which showed that hydroxyurea, another cytostatic agent, can also result in a marked induction of HbF in Baboons. 8 …”

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“…For instance, given the effect of global DNA hypomethylation on genomic stability, therapyinduced hypomethylation might promote tumor formation on the long run, although this hypothesis still needs verification (Eden et al, 2003;Yang et al, 2003). Epigenetic therapy might also cause activation of imprinted or silenced genes and has indeed been shown to be mutagenic and possibly even carcinogenic (Carr et al, 1988;Jackson-Grusby et al, 1997). However, these concerns should not be exaggerated, as DNMT inhibitors only act on dividing cells, while leaving other cells unaffected.…”