Cytotoxicity in Human Kidney HK‐2 Cells of the Flavoring Aldehydes Cinnamaldehyde and Vanillin

Abstract

Vaping and e‐cigarette use has markedly increased in adults as well as middle and high school age children. Flavoring agents are not tightly regulated resulting in widely variable levels present in various commercial preparations. Cinnamaldehyde and vanillin are popular flavoring agents contained in e‐liquids. Unfortunately, the safety as well as the long term effects of these compounds is not well understood. While the lung is the primary site of contact for such flavoring agents, aldehydes absorbed following inhalation can distribute throughout the body to other tissue sites including the kidney. These flavoring compounds can then impact cell function in tissues such as the kidney. The purpose of this study was to test the hypothesis that cinnamaldehyde and vanillin may have cytotoxic effects in the kidney. In the present study, the effects of cinnamaldehyde or vanillin at final concentrations of 0‐100 uM or 0‐1000 uM, respectively, were evaluated using human renal proximal tubular epithelial cells (HK‐2) for 24‐ and 48‐hour time periods. Cells were obtained by ATCC and cultured according to vendor specifications. All experiments were a minimum of 4 independent experiments utilizing 4 different cell passages. Viability was determined by use of the MTT assay and conversion to formazan. Cell Countess was done to visualize percent live and dead cells. Western blots were performed to assess expression of oxidative stress as well as mitophagy‐related proteins LC3B‐II, PINK, and PARKIN. Vanillin was not cytotoxic between 0‐100 uM relative to vehicle control at 24 hours. Cinnamaldehyde was cytotoxic beginning at 25 uM when compared to vehicle control using the MTT assay. Expression of mitophagy‐related protein LC3B‐II increased significantly at higher concentrations of cinnamaldehyde (p<0.05). Oxidative stress was evaluated using Western blot for protein modifications. Cinnamaldehyde did not increase 4‐hydroxynonenal (4HNE) adducted proteins within a 24 hour period. Mitochondrial function was evaluated using a Seahorse analyzer following a 24 hour exposure. Mitochondrial oxygen consumption was diminished by cinnamaldehyde. These findings suggest that cinnamaldehyde was more cytotoxic to HK‐2 cells than vanillin. Cinnamaldehyde further altered mitochondrial function and increased LC3B‐II expression. Further studies are required to explore the specific mechanisms of cytotoxicity to the kidney by flavoring aldehydes.