Quantification of selected aroma compounds in e-cigarette products and toxicity evaluation in HUVEC/Tert2 cells.

During recent years, the number of consumers using so-called e-cigarettes, which are electrical devices to aerosolize a liquid consisting of propylene glycol, glycerol, optional nicotine and flavoring chemicals, has been increasing. Aromas vary from common flavors such as mint to more unusual flavors such as buttermilk or pepperoni pizza. Consumers today can buy e-concentrates that consist of propylene glycol and aroma to blend their own desired flavor at home. Little is known about the composition and concentration of various aroma molecules in the different e-liquids and e-concentrates. In addition, the process of EU-wide regulation is still ongoing. The aim of this research study was to identify and quantify possible undesirable aroma compounds in e-liquids and e-concentrates. Flavoring chemicals such as estragole, benzaldehyde and cinnamaldehyde were quantified. The measurements were carried out on a GC-MS system. The results show the presence of highly concentrated flavoring compounds and limonene oxide in lemon-flavored e-concentrates. In the final step, samples and single-aroma standards were tested for their toxicity to HUVEC/Tert2 cells, where some single-flavoring chemicals such as cinnamic aldehyde revealed significant toxic effects.

Refill liquids for electronic cigarettes display peculiar toxicity on human endothelial cells.

The electronic cigarettes mimic combustible cigarettes through a heating technology that vaporizes a refill liquid consisting of solvents, flavors, and nicotine. E-cigarettes are sometimes still used as a support for smoking cessation, even if in 2019 an acute lung injury outbreak occurred in the USA, affecting mainly adolescents and young adults, and was correlated to eCigs. Therefore, due to the lack of a definite knowledge about the mechanism(s) of refill liquid toxicity and considering that previous investigations gave controversial results, the aim of the present study was the cytotoxicity assessment of different refill liquids on human endothelial cells, evaluated by means of two different in vitro approaches, i.e. the resazurin and the LDH release assays. Our results clearly demonstrated that different refill liquids (6 samples) display different levels of cytotoxicity in our cellular model, although their cytotoxicity was always lower than that observed for the condensate obtained from traditional cigarettes (3 samples). These results suggest that accurate evaluations should be provided for refill liquids, in particular to correlate their toxicity to their chemical composition, with the final aim of obtaining useful information for the agencies involved in the regulation of their components.

The Ala95-to-Gly substitution in Aerococcus viridans l-lactate oxidase revisited - structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors.

Aerococcus viridansl-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O2 into pyruvate and H2O2. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O2 with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O2 compared to wild-type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20 °C (pH 6.5), we determined that the A95G variant (fully reduced by L-lactate) was approximately three-fold more reactive towards DCIP (1.0 ± 0.1 × 10(6) M(-1) ·s(-1) ) than O2, whereas avLOX wild-type under the same conditions was 14-fold more reactive towards O2(1.8 ± 0.1 × 10(6) m(-1) ·s(-1)) than DCIP. Substituted 1,4-benzoquinones were up to five-fold better electron acceptors for reaction with L-lactate-reduced A95G variant than wild-type. A 1.65-Å crystal structure of oxidized A95G variant bound with pyruvate was determined and revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the accomodation of a new active-site water molecule within hydrogen-bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position.