In vivo genotoxic effects of dietary heme iron on rat colon mucosa and ex vivo effects on colon cells monitored by an optimized alkaline comet assay

In vivo comet assay is increasingly used although this assay has serious limitations as standard conditions does not allow the use of frozen samples nor the processing -at the same time- of an important number of samples generated by in vivo studies. Therefore, the aim of our work was firstly to optimize samples preparation and alkaline comet assay protocol for frozen tissue and also to increase the number of samples processed in one experiment; secondly it was to use this optimized protocol to evaluate the genotoxic effects of dietary heme iron on colon mucosa and colon epithelial cells. To optimize samples preparation and include a step of samples freezing for further experiments, different experimental parameters were tested (i.e. buffer composition, temperature of freezing/defreezing… ). The lack of induced DNA strand breaks by the freezing process of colon rat samples was assessed by comparing the results obtained between frozen and fresh samples from the same rat. The extend of DNA damage was determined by alkaline comet assay and quantified by median % Tail DNA calculation on 100 cells from 2 experimental points. By modifying different experimental parameters, we determined the optimal conditions to prepare samples and perform alkaline comet assay on cryopreserved blood and colon mucosa without DNA strand breaks induction. In order to perform middle throughput comet assays, we replaced standard glass microscope slides by 20 wells Trevigen® comet slides. Quantification of DNA breaks by median % tail DNA was undertaken by using the Komet 6.0 software (Andor Technology). Once the protocol was suitable, we used it to study the effects of dietary heme iron. Epidemiological studies revealed that high red meat intake, rich in heme iron, is associated with an increased colorectal cancer risk. In rats, heme iron-induced colorectal carcinogenesis is associated with increased fecal lipoperoxidation. In our previous study (Bastide, Chenni et al. 2015), we showed that luminal lipid peroxidation induced by the hemoglobin diet is associated with a higher anaphase bridge index in the epithelium of intestine. The genotoxic effect of the hemoglobin diet in C57BL/6J mice was confirmed with immunolabelling of gammaH2AX, a Ser-139 phosphorylation of the histone variant H2A, a well-known marker of the occurrence of DNA double strand breaks and/or replication fork arrest. The aim of our present study was to check, with alkaline comet assay, if the heme-induced lipoperoxidation increase is associated with an increment of genotoxicity in the rat colon mucosa. For this purpose, four groups of rats received either control diet or hemin diet, added or not with calcium carbonate for 3 weeks. Calcium carbonate is used to increase the level of proof between heme-induced peroxidation and genotoxicity, since it annihilates the heme-induced increase of peroxidation via heme chelation. As expected, heme iron intake increased fecal lipoperoxidation and also genotoxicity. When calcium carbonate was added to the hemin diet, these parameters were normalized. These in vivo results were completed with ex vivo experiments to determine if aldehydes from heme-induced lipoperoxidation are responsible for an increase of genotoxicity. We used fecal water, the soluble part of colonic content, from control or hemin fed rats. Fecal waters were used before and after treatment with a polymer resin (4-Fmoc-hydrazinobenzoyl AM NovaGel™) for aldehyde trapping. With this trapping of aldehydes from fecal water, the weight of aldehydes in the genotoxic activity of fecal water can be determined. Fecal waters were used on murine colonic epithelial cells and genotoxicity was assayed. Similarly to the results obtained from in vivo study, fecal waters from hemin-fed rats induced genotoxicity. When fecal waters were depleted in aldehydes, the genotoxicity was normalized. Therefore, using the resin to specifically trap fecal aldehydes, we demonstrated that aldehydes alone are responsible -at least in great part- for fecal water genotoxicity. In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.