Decomposition and quality control considerations in biological work with fecapentaene preparations

Abstract

Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found to decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed to be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of an all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. The mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella typhimurium tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application at hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.

Noncovalent DNA Binding and the Mechanism of Oxidative DNA Damage by Fecapentaene-12

“…A number of reports have detailed the instability of the fecapentaenes (1,2,33,34). We confirm that 1 is, indeed, unstable and further demonstrate that decomposition yields products with vastly decreased ability to cause DNA strand breaks (Figure 2).…”

“…For example, following preincubation of 1 in ethanol-containing buffer for 5 h, essentially no DNAcleaving activity remains (Figure 2). Earlier work has shown that the mutagenic activity of the fecapentaenes diminishes upon preincubation (34). It is noteworthy that the decomposition of 1 in ethanol-containing buffer is more rapid than in the acetonitrile-containing buffers used in our typical DNA damage experiments.…”

The fecapentaenes, potent mutagens from human feces

“…The fecapentaenes are also unstable in ultraviolet light. Thus fecapentaene 12 decomposes almost completely on irradiation for as little as 30 s; the decomposition products have not been characterized (45).…”

“…In this study the decomposition of fecapentaene 12 was investigated under various conditions, and it was concluded that unchecked decomposition can profoundly affect biological test results. The authors commented that "we found fecapentaene 12 to be more unstable and difficult to handle than any other substance we have as yet attempted to test for carcinogenic activity" (45). A particular concern was that decomposition of fecapentaene 12 occurred under very variable conditions, and the authors recommend storage in amber vessels, avoidance of strong light, nucleophiles, and acidic conditions, the use of minimum quantities, and maximum protection from air, as standard methods for handling fecapentaene 12.…”

Fecapentaenes and risk for colorectal cancer

“…In the same study, no colonic tumors were found in F-344 rats after intrarectal infusion of FP-12, whereas an insignificantly increased incidence of fibrosarcomas and mammary carcinomas was found by Shamsuddin e/ a/., following intrarectal injection in male F-344 rats (130). It has repeatedly been suggested that in order to establish actual exposure levels, appropriate quality control measures for fecapentaene decomposition should be included in each carcinogenicity experiment (130,137).…”