## 4 Discussion
Obviously, our conclusions need confirmatory, in vivo experiments because the cytotoxicity of DKP could have disturbed the micronucleeli scoring in our studies, while our QSAR model predicting DKP carcinogenicity/genotoxicity was not sufficiently reliable to provide unquestionable conclusions. In addition to this, no other DKP derivatives of ACE-Is had been subjected to in vitro micronucleeli assay before, thus no comparative analysis was possible. Despite these limitations, we believe that the evidence from our investigations is sufficient to set follow-up in vivo experiments, without further confirmatory in vitro protocols [38]. Therefore, we strongly suggest that the aneugenicity threshold of DKP and its impact on living organisms should to be evaluated under in vivo conditions by marketing authorization holders in order to validate the acceptable limits of this impurity in formulated drugs. Finally, due to the presence of a diketopiperazine structural alert in the RAM degradation product, potentially leading to the formation of mutagenic nitroso compounds, we performed mutagenicity evaluation for pure DKP and its nitrosation product using the Ames test. To that end, the NAP test was first carried out using SGJ, which served as an in vitro model of DKP nitrosation in the stomach. Then, pure DKP and the product of its nitrosation (N-DKP) were subjected to mutagenicity evaluation. Both compounds were tested only at high concentrations, since mutagens have no threshold mechanism and their exposure is hazardous at any level. High concentrations were therefore applied to increase the sensitivity of our assay. The obtained results (Table 5) showed that pure DKP was not mutagenic, which was consistent with our QSAR predictions. In other words, pure DKP is not reactive against DNA. On the other hand, for N-DKP, a mutagenic effect was observed in the _Salmonella typhimurium_ T100 system, with S9 fraction at 4.5 mg/mL (FIB = 3.09; B = 1.00, Table 6). This indicates that DKP nitrosation products cause mutations by base substitutions upon activation by liver cytochromes. The possible products of DKP-nitrite interaction could involve the N-nitrosoramipril formed by the DKP ring opening or N-nitrosodiketopiperazine derivative, formed secondary to the loss of carboxyphenylalanine moiety. Our result corresponds with the reported mechanism of mutagenic action of N-nitrosamines. In fact, their emerging electrophilic metabolites, produced by cytochrome catalytic activity, easily alkylate nuclear DNA and thus, contribute to cancer initiation [39]. This justifies the fact that the mutagenic effect of N-DKP occurred in the experiment with metabolic activation. This also means that there is no safe level of N-DKP and its presence in human blood at any concentration could induce cancer. Given that the only source of N-DKP in humans is DKP formed secondary to RAM degradation, the content of this impurity in drug formulation must be minimized. Based on our results, it can be therefore concluded that the carcinogenic effect of DKP in humans is probable and it may be manifested via two independent mechanisms: direct aneugenic activity at excessive doses as a secondary factor, and indirect mutagenic action secondary to in vivo transformation to nitroso-metabolites. Here, it must be also noted that independent aneugenicity of pure DKP at standard blood levels is rather unlikely. The nitroso-metabolites of DKP, in turn, are mutagenic irrespective of blood level, and they could be the reason for increased cancer incidence among RAM users independently. The relevance of these in vitro findings must be verified in in vivo settings. Until this is done, one strategy to minimize the risk of human exposure to mutagenic DKP-nitrite metabolites might be employing additional stabilization methods by manufacturers so that DKP is not formed. Furthermore, antioxidants such as ascorbic acid or \(\alpha\)-tocopherol added to final RAM formulation could serve as a mitigation strategy to eliminate electrophilic nitroso-radicals endogenously formed from DKP.