Toward Better Characterization of a Free-Base Nicotine Fraction in e-Liquids and Aerosols.

Given that nicotine salts are a growing market, methods are needed to characterize nicotine forms in e-cigarette vaping products. By lowering the free-base nicotine fraction (αfb) in favor of protonated forms, the addition of organic acids to the e-liquid mix greatly modulates nicotine pharmacokinetics and improves vapers' craving. This research investigated (1) the performance of pH measurement, liquid-liquid extraction (LLE), and acid/nicotine molar ratio calculation methods for αfb estimation in 6 nicotine benzoate and nicotine salicylate e-liquids and (2) nicotine protonation in the aerosol post vaporization. Aerosols were generated with a JUUL device and another mod-pod on a vaping machine to assess device effects. E-liquid and aerosol samples were then analyzed after further analytical optimization of previous methods and careful consideration of biases. Globally, performances were comparable between methods. αfb accounted for less than 5% of nicotine content regardless of experimental conditions. αfb were consistent between e-liquids and aerosols irrespective of e-cigarette devices. Hence, e-liquids are adequate surrogates for aerosols, facilitating the establishment of regulations. pH measurement is one of the most used methods and enables the establishment of relative scales for e-liquid classification but lacks automation possibility. Until now, the extent of sample dilution remained arbitrary. The dilution factor was fixed at 10, as usually achieved, since no effect of dilution was noted. pH values ranged from 5.3 to 6.3 in accordance with the literature. By contrast, LLE relies on the specificity of organic solvent for free-base nicotine extraction, causing discrepancies in previous studies. Here, the results were similar to αfb values from pH determination. Yet, LLE presented the highest variability and was the most time-consuming protocol. Finally, αfb calculation from molar ratio was the most robust and versatile method. Estimations can be made in silico from reported composition data and/or after liquid chromatography routine analysis.

Metabolism and genotoxicity of polycyclic aromatic hydrocarbons in human skin explants: mixture effects and modulation by sunlight.

Cutaneous exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH) occurs frequently in the industrialized workplace. In the present study, we addressed this topic in a series of experiments using human skin explants and organic extracts of relevant industrial products. PAH mixtures were applied topically in volumes containing either 10 or 1 nmol B[a]P. We first observed that although mixtures were very efficient at inducing expression of CYP450 1A1, 1A2, and 1B1, formation of adducts of PAH metabolites to DNA, like those of benzo[a]pyrene diol epoxide (BPDE), was drastically reduced as the complexity of the surrounding matrix increased. Interestingly, observation of a nonlinear, dose-dependent response with the least complex mixture suggested the existence of a threshold for this inhibitory effect. We then investigated the impact of simulated sunlight (SSL) on the effects of PAH in skin. SSL was found to decrease the expression of CYP450 genes when applied either after or more efficiently before PAH treatment. Accordingly, the level of DNA-BPDE adducts was reduced in skin samples exposed to both PAH and SSL. The main conclusion of our work is that both increasing chemical complexity of the mixtures and co-exposure to UV radiation decreased the production of adducts between DNA and PAH metabolites. Such results must be taken into account in risk management.

Influence of exposure dose, complex mixture, and ultraviolet radiation on skin absorption and bioactivation of polycyclic aromatic hydrocarbons ex vivo.

Combined exposure to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) and ultraviolet radiation (UVR) is suspected to enhance PAH skin permeability and skin cancer risk depending on PAH bioactivation. The impact of PAH mixtures (exposure dose, composition, and complexity) and UVR was assessed for PAH cutaneous absorption and metabolism using realistic exposure conditions and human skin explants. PAH complex mixtures were extracted from the industrial products coal tar pitch (CTP-I) and petroleum coke (PC-I). The synthetic mixture (CTP-S) was identically reconstituted using PAH standards. The applied dose was adjusted to 1 (PC-I, CTP-I) or 10 nmol (CTP-I, CTP-S) of benzo[a]pyrene (B[a]P). Unmetabolized PAHs were recovered from the skin surface, skin and medium, and then quantified by HPLC-fluorescence detection. PAH metabolites were collected from the medium and analyzed by GC-MS/MS. B[a]P and PAH penetration was lower for the highest B[a]P dose, industrial mixtures, and CTP-I compared to PC-I. Skin irradiation increased PAH penetration only for CTP-I. PAH uptake was poorly influenced by the different experimental conditions. PAH metabolism markedly decreased in the application of mixtures, leading to unmetabolized PAH accumulation in human skin. PAH metabolism was similar between CTP-I and PC-I, but was lower for the highest dose and the industrial mixtures, suggesting a saturation of xenobiotic metabolizing enzymes, as confirmed in a time-course study. UVR strongly inhibited all PAH metabolism. Altogether, these results underline the necessity to consider the reality of human exposure (PAH complex mixtures and UVR) during in vitro experiments to properly estimate skin absorption and metabolism.

A realistic human skin model to study benzo[a]pyrene cutaneous absorption in order to determine the most relevant biomarker for carcinogenic exposure.

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous pollutants, among which benzo[a]pyrene (B[a]P) is the only compound classified carcinogenic to humans. Besides pulmonary uptake, skin is the major route of PAH absorption during occupational exposure. Health risk due to PAH exposure is commonly assessed among workers using biomonitoring. A realistic human ex vivo skin model was developed to explore B[a]P diffusion and metabolism to determine the most relevant biomarker following dermal exposure. Three realistic doses (0.88, 8.85 and 22.11 nmol/cm2) were topically applied for 8, 24, and 48 h. B[a]P and its metabolites were quantified by liquid chromatography coupled with fluorimetric detection. The impact of time, applied dose, and donor age were estimated using a linear mixed-effects model. B[a]P vastly penetrated the skin within 8 h. The major metabolites were 3-hydroxybenzo[a]pyrene (3-OHB[a]P) and 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-tetrol). This latter predominantly derives from the most carcinogenic metabolite of B[a]P, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), as well as benzo[a]pyrene-9,10-diol-7,8-epoxide (reverse-BPDE). Benzo[a]pyrene-trans-7,8-dihydrodiol (B[a]P-7,8-diol) was a minor metabolite, and benzo[a]pyrene-trans-4,5-dihydrodiol (B[a]P-4,5-diol) was never quantified. Unmetabolized B[a]P bioavailability was limited following dermal exposure since less than 3% of the applied dose could be measured in the culture medium. B[a]P was continuously absorbed and metabolized by human skin over 48 h. B[a]P-tetrol production became saturated as the applied dose increased, while no effect was measured on the other metabolic pathways. Age had a slight positive effect on B[a]P absorption and metabolism. This work supports the relevance of B[a]P-tetrol to assess occupational exposure and carcinogenic risk after cutaneous absorption of B[a]P.

Solar simulated light exposure alters metabolization and genotoxicity induced by benzo[a]pyrene in human skin.

Skin is a major barrier against external insults and is exposed to combinations of chemical and/or physical toxic agents. Co-exposure to the carcinogenic benzo[a]pyrene (B[a]P) and solar UV radiation is highly relevant in human health, especially in occupational safety. In vitro studies have suggested that UVB enhances B[a]P genotoxicity by activating the AhR pathway and overexpressing the cytochrome P450 enzymes responsible for the conversion of B[a]P into DNA damaging metabolites. Our present work involved more realistic conditions, namely ex vivo human skin explants and simulated sunlight (SSL) as a UV source. We found that topically applied B[a]P strongly induced expression of cutaneous cytochrome P450 genes and formation of DNA adducts. However, gene induction was significantly reduced when B[a]P was combined with SSL. Consequently, formation of BPDE-adducts was also reduced when B[a]P was associated with SSL. Similar results were obtained with primary cultures of human keratinocytes. These results indicate that UV significantly impairs B[a]P metabolism, and decreases rather than increases immediate toxicity. However, it cannot be ruled out that decreased metabolism leads to accumulation of B[a]P and delayed genotoxicity.