Time‐dependent Inhibition of Cytochrome P450 2A13 (CYP2A13): by Cinnamaldehyde as a Lung Cancer Preventive Agent

Abstract

Nicotine is the addictive substance found in various tobacco products CYP2A13, an enzyme localized in the lungs, metabolizes tobacco‐specific nitrosamine carcinogens that contribute to lung cancer. Therefore, pinpointing CYP2A13 inhibitors is an approach to lower tobacco‐based lung cancer risk. Cinnamaldehyde is a common flavoring agent in the fluids of electronic nicotine vaping devices. Cinnamaldehyde was found to be a potent inhibitor of CYP2A6, another enzyme that metabolizes nicotine. Because CYP2A13 and CYP2A6 exhibit overlap in substrate/inhibitor selectivity, the goal here was to evaluate the inhibition of CYP2A13 by cinnamaldehyde. A time‐dependent inhibition coumarin assay was performed to determine the kinetic parameters for cinnamaldehyde in recombinant CYP2A13. Primary incubations contained cinnamaldehyde (5 to 200 micromolar) of (or DMSO for control), CYP2A13 Supersomes, and potassium phosphate buffer. Incubations were initiated with NADPH. Secondary incubations contained coumarin, NADPH, and potassium phosphate buffer. At selected time points (0 to 18 minutes), an aliquot of the primary incubation mixture was transferred to the secondary incubation tubes, which were terminated with trichloroacetic acid after heating at 37 degree C for 5.5 minutes. A linearity study o was conducted to determine the appropriate termination time. CYP2A13 activity was measured by detection of hydroxycoumarin using high performance liquid chromatography (HPLC) and a fluorescence detector. Standard curves of hydroxycoumarin were used to quantify product formation from experimental samples.Hydroxycoumarin formation decreased with time and inhibitor concentrations. Maximal inhibition following a 18 minute incubation was 38.3 ± 1.6 and 4.0 ± 0.6 % at 20 and 200 micromolar cinnamaldehyde, respectively. The maximal rate of inhibition was 0.109 per minute. The results provide evidence that cinnamaldehyde is a time‐dependent inhibitor of CYP2A13. Furthermore, cinnamaldehyde appears to be a more potent inhibitor of CYP2A13 than CYP2A6, based on the maximal rate of inhibition (0.109 and 0.056 per minute, respectively). The results imply that cinnamaldehyde could interfere with the bioactivation of nitrosamine lung carcinogens.. Additional kinetic studies are needed to confirm the results of this study, and to evaluate the safety and toxicity profiles of cinnamaldehyde in more complex physiological models.Figure 1 Standard Curve (7‐OH‐Coumarin) Percent uM Area uM Stds 1 Stds 2 Average Change Stds 1 4 1.1E+07 4 1.1E+07 11028898 11028898 0.0 2 5684098 2 5684098 5684098 5684098 0.0 Slope 2,764,695.99 1 2773892 1 2773892 2784915 2779404 0.4 Y‐intercept 29,108.47 0.5 1410325 0.5 1410325 1425656 1417991 1.1 R2 0.9998 0.25 713352 0.25 713352 724841 719097 1.6 0.125 355993 0.125 355993 364369 360181 2.4 0.0625 181197 0.0625 181197 182438 181818 0.7 0.03125 92767 0.03125 92767 92341 92554 −0.5 Stds 2 4 1.1E+07 AUC E 20 200 E 20 200 2 5684098 0 966263 897472 321950 873055 768870 272624 1 2784915 3 810370 715528 193710 871857 651272 169610 0.5 1425656 6 862911 554828 144832 3746286 804730 133450 0.25 724841 9 884632 520572 119480 841174 461582 109707 0.125 364369 12 853112 496090 87451 834152 423436 81136 0.0625 182438 15 871382 390425 76171 799519 326200 85743 0.03125 92341 18 855074 279226 66930 761978 340103 60431 OH‐Cou Concentration E 20 200 E 20 200 0 0.339 0.314 0.106 0.305 0.268 0.088 3 0.283 0.248 0.060 0.305 0.225 0.051 6 0.302 0.190 0.042 1.345 0.281 0.038 9 0.309 0.178 0.033 0.294 0.156 0.029 12 0.298 0.169 0.021 0.291 0.143 0.019 15 0.305 0.131 0.017 0.279 0.107 0.020 18 0.299 0.090 0.014 0.265 0.112 0.011