Human exposure to acrolein results from its presence in car exhaust and frying oil vapors, due to lipid peroxidation and polyamine catabolism, and it has recently been identified to be an endogenous product of glycerol metabolism by human gut microbes. Despite the recent designation of acrolein as a probable human carcinogen, there remains limited data concerning molecular initiating and key cellular events associated with biomolecular interactions of endogenously produced acrolein, especially with respect to exposure in the gastrointestinal tract. Acrolein is a highly reactive and volatile molecule, that is not stable in buffer or cell culture media making it difficult to characterize its biological effects in vitro. To circumvent the volatile nature of acrolein and emulate the persistent production of acrolein by gut bacteria for toxicological evaluation, we established a cell culture-compatible coupled-enzyme system for the in-situ production and testing the effects of acrolein. The continuous acrolein production is executed by combining a polyamine catabolism enzyme, polyamine substrate, and catalase to scavenge hydrogen peroxide formed as a side product. We performed cell-culture compatibility studies with the coupled enzyme system and observed reductions in cell viability after exposure of colon epithelial cells to acrolein generated by the coupled enzyme system. To validate this system for controlled continuous in vitro acrolein exposure, we first characterized the formation of the most abundant Acrolein-DNA adduct acrolein-deoxyguanosine (Acr-dG) following acute exposure of colon epithelial cells to acrolein. There was a dose-dependent increase in adduct levels after 6 and 24 hours. Next, we evaluated changes in levels of gene transcripts of HMOX1 and SRXN1, which are activated by the Nrf2 pathway under conditions of oxidative stress and, also, observed a dose-dependent induction after 6 and 24 hours. We used DNA adduct formation as an internal molecular dosimeter for acrolein produced by the enzyme system, consistent with exogenous acute exposures of 50-75 µM acrolein. Therefore, the system can produce acrolein at levels that cause DNA damage when compared to acute exposure and provides a novel adaptable in vitro method to understand biological consequences of endogenous acrolein formation.

Biosynthesis of acrolein with a coupled enzyme system to emulate continuous acrolein production by gut microbiota

ACS Fall 2021

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