Figure 9. Carnosine abrogates acrolein toxicity in vitro by inhibition of protein carbonylation.

(A) Incubation of HUVECs with 130 nM of acrolein for 24 hours led to cell death, which was completely reversed when co-incubated with carnosine. Analogous results were observed for acrolein concentrations as low as 1,3 nM. (B) Quantitative MTT analysis (n = 6 for each condition) revealed complete cytotoxicity for acrolein concentrations of 1.3 nM to 130 μM. Carnosine co-incubation restored cell viability. (C) Representative OxyBlot results of lysed cells after 24 hours incubation with acrolein in presence or absence of carnosine. Signal intensity of bands representing carbonylated proteins was increased in acrolein stimulated cells. This increase was diminished in presence of carnosine. Samples without addition of derivation solution served as negative controls. (D) Identification of carnosine-acrolein Michael adduct in cell supernatants. 1) Single ion chromatograms of cell supernatants containing a fixed amount of carnosine (20 mM) and no acrolein (black line), 0.13 mM acrolein (orange line), 1.3 mM acrolein (green line) or 13 mM acrolein (blue line) reconstituted by setting the filter ion at m/z 283.14014. 2) Tandem mass spectrum of the ion at m/z 283.14014 detected in cell supernatants. 3) Tandem mass spectrum of a standard carnosine-acrolein Michael adduct. 4) Carnosine-acrolein adducts detected in cell supernatants. *P < 0.01, ****P < 0.0001. Car: carnosine. Deri: derivation solution.