In summary these results are consistent with our
In summary, these results are consistent with our hypothesis that chemical nitrosation of BMAA to N-BMAA results in the formation of an alkylating agent. Furthermore, we have shown that N-BMAA is more toxic than BMAA itself. Previously, methylazoxymethanol (MAM), which is also present in the cycad nut (Kisby et al., 2009), was shown to be a classical alkylating agent causing toxic DNA damage (e.g. O6-methylguanine) in target Benzoylhypaconitine receptor (Esclaire et al., 1999) resulting in changes in cell-signalling gene expression pathways associated with human neurodegenerative disease (Kisby et al., 2011). The chemical structure of N-BMAA predicts that it will cause the same spectrum of DNA damage as that induced by MAM and other alkylating agents (Beranek et al., 1980). Analogously, N-nitrosation of glycine results in both methylating and carboxymethylating agents that can generate the DNA adducts, O6-methylguanine and O6-carboxymethylguanine respectively (Harrison et al., 1999): both of these adducts have been detected in human DNA (Povey et al., 2000, Lewin et al., 2006). In addition to dietary BMAA sources, BMAA may also be generated i n situ within the gastro-intestinal lumen as a result of cyanobacteria (Ley et al., 2006, Hill et al., 2014) which when coupled with heme-catalysed N-nitrosation (Lunn et al., 2007) may give rise to the formation of N-BMAA. These findings suggest a novel pathway of human exposure to potentially neurotoxic alkylating agents.
Conclusions These results demonstrate that N-nitrosation of BMAA results in an alkylating agent that damages DNA in vitro, generating single strand breaks, and is toxic to neuroblastoma cells. Our results suggest a novel hypothesis in which in situ nitrosation of BMAA, and potentially other amines, likely catalysed by the microbiome leads to the formation of a variety of alkylating agents that may contribute to the aetiology of ALS and other neurodegenerative diseases.
Conflict of interest
Introduction DNA-alkylating agents were the first chemotherapeutic drugs developed for the treatment of cancer . Although the classic, highly reactive DNA-alkylating agents (e.g., melphalan, chlorambucil, and platinum-based agents) are potent chemotherapeutic agents, they have limited clinical benefits mainly because of their toxicity, which results in severe adverse side effects . The toxicity associated with DNA-alkylating agents highlights the need to develop anticancer agents with greater selectivity. To overcome the drawbacks of alkylating agents, which lack intrinsic DNA-binding affinity and thus are genotoxic and carcinogenic , DNA-directed alkylating agents with increased DNA-binding selectivity were designed to avoid “off-target” effects on cellular components and hence reduce adverse side effects . Accordingly, we and many other scientists have previously synthesized a series of DNA-directed alkylating agents by coupling various DNA-affinic carriers (a DNA-intercalating agent or a DNA minor groove binder) that directly target DNA with the N-mustard pharmacophore , , , , , . These DNA-directed alkylating agents were shown to have increased chemical stability and improved anticancer activity. Based on structural-activity relationship studies, selecting the appropriate DNA-affinic carrier and the spacer, which links to the alkylating warhead, is critical for improving the antitumor activity of DNA-directed alkylating agents ,  In 1991, Denny and coworkers first synthesized N-mustard-quinoline conjugates that exerted potent cytotoxicity by forming DNA cross-links and bulky monoadducts . Additionally, N-mustard-quinoline derivatives were found to overcome the poor solubility and low DNA-binding selectivity of some DNA intercalators, such as acridine mustard conjugates . To improve the antitumor activity and increase the chemical stability of DNA-affinic N-mustards, we previously designed and synthesized a series of conjugates by coupling the N-mustard residue to quinoline moieties via a urea or hydrazinecarboxamide linker . We found that these N-mustard-quinoline conjugates were chemically stable in serum and possessed strong growth inhibitory activity against human lymphoblastic leukemia and a panel of solid tumor cell lines . These compounds also displayed limited systemic toxicity in animals. Among them, we noted that compound 18c, redesignated SL-1 (Figure 1A), was the most potent suppressor of the growth of breast carcinoma MX-1 xenografts, even achieving complete tumor remission . These results indicate that SL-1 is a potential lead and warrants further investigation.