Tobacco Constituents Research Articles

Simultaneous analysis of urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, N\u2032-nitrosonornicotine, and cotinine by liquid chromatography-tandem mass-spectrometry

Biomarkers of exposure to harmful tobacco constituents are key tools for identifying individuals at risk and developing interventions and tobacco control measures. However, tobacco biomarker studies are scarce in many parts of the world with high prevalence of tobacco use. Our goal was to establish a robust method for simultaneous analysis of urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N′-nitrosonornicotine (NNN), and cotinine at the Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) in Mumbai, India. These biomarkers are validated measures of exposure to the carcinogenic tobacco nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and NNN and the addictive alkaloid nicotine, respectively. The established method is characterized by excellent accuracy, linearity, and precision, and was successfully applied to the analysis of 15 smokeless tobacco (SLT) users and 15 non-users of tobacco recruited in Mumbai. This is the first report of establishment of such procedure in a laboratory in India, which offers the first in-country capacity for research on tobacco carcinogenesis in Indian SLT users.

Evaluation of organic and inorganic fertilizers on chemical and biochemical quality constituents of burley tobacco: Influence of fertilizers on chemical and biochemical quality of burley tobacco

A field experiment was conducted during 2011-13 at Burley Tobacco Research Centre, Kalavacharla, East Godavari district, Andhra Pradesh to study the effect of organic and inorganic manures viz., four formulations of organic manures (neem cake, pongamia cake, filter press cake, poultry manure, FYM and vermicompost in different ratios) and two levels of inorganic nitrogen fertilizers (120 and 160 kg N /ha) on chemical and biochemical quality constituents of burley tobacco. Cured leaf samples from bottom, middle and top leaf positions in different treatments were analysed for chemical and biochemical quality constituents. Total chlorophylls, carotenoids and nitrate nitrogen varied from 0.116 to 0.388, 0.161 to 0.531, 3.87 to 8.06 mg/g, respectively. Chlorophylls and carotenoids were higher in manure treatments compared to inorganic treatments. Polyphenols viz., chromogenic acid and rutin ranged from 0.58 to 3.89 mg/g and 0.94 to 2.85 mg/g respectively. Polyphenols were less with organic manures compared to inorganic manures. Proline content varied from 0.506 to 3.7 mg/g. Organic manures showed higher proline compared to inorganic Nitrogen. Chemical quality parameters viz., nicotine, reducing sugars and chlorides ranged from 0.4 to 1.73%, 0.49 to 1.93% and 0.39 to 2.84% respectively. Reducing sugars content is high with organic manure application compared to inorganic nutrients. Activity of soil enzymes is high with organic compared to inorganic manures. Manure treatments not only increased the content of carotenoids, proline, reducing sugars, chlorophyll, and free fatty acids which play an important role in the quality of tobacco but also decreased the levels of polyphenols and nitrate nitrogen, which are negatively correlated with the quality of tobacco. Among the different organic manures, manure A (neem cake, pongamia cake, filter press cake, poultry manure, FYM and vermicompost in the ratio of 2:2:2:2:1:1) showed superiority followed by Manure C (neem cake, pongamia cake, filter press cake, poultry manure, FYM and vermicompost in the ratio of 1.5:1.5:5:2:1:2) in their influence on chemical quality parameters of Burley tobacco.

Identification and Confirmation of the miR-30 Family as a Potential Central Player in Tobacco-Related Head and Neck Squamous Cell Carcinoma.

Constituents of tobacco that can cause DNA adduct formation and oxidative stress are implicated in the development of head and neck squamous cell carcinoma (HNSCC). However, there are few studies on the mechanism(s) that underlie tobacco-associated HNSCC. Here, we used a model in which tumors were induced in rats using 4-nitroquinoline 1-oxide (4NQO), which mimicked tobacco-related HNSCC, and analyzed the expression profiles of microRNAs and mRNAs. Our results indicated that 57 miRNAs and 474 mRNA/EST transcripts exhibited differential expression profiles between tumor and normal tongue tissues. In tumor tissue, the expression levels of rno-miR-30 family members (rno-miR-30a, rno-miR-30a-3p, rno-miR-30b-5p, rno-miR-30c, rno-miR-30d, rno-miR-30e and rno-miR-30e-3p) were only 8% to 37% of those in the control group. The GO terms enrichment analysis of the differentially expressed miRNAs indicated that oxidation reduction was the most enriched process. Low expression of miR-30 family members in human HNSCC cell lines and tissues was validated by qPCR. The results revealed that the expression of miR-30b-5p and miR-30e-5p was significantly decreased in the TCGA HNSCC dataset and validation datasets, and this decrease in expression further distinguishes HNSCC associated with tobacco use from other subtypes of HNSCC. CCK8, colony formation, transwell migration and HNSCC xenograft tumor assays indicated that miR-30b-5p or miR-30e-5p inhibited proliferation, migration and invasion in vitro, and miR-30b-5p suppressed tumor growth in vivo. Moreover, we uncovered that KRAS might be the potential target gene of miR-30e-5p or miR-30b-5p. Thus, our data clearly showed that decreased expression of miR-30e-5p or miR-30b-5p may play a crucial role in cancer development, especially that of tobacco-induced HNSCC, and may be a novel candidate biomarker and target for this HNSCC subtype.

Determination of Free Solanesol Levels in Cigarette Filters by Liquid Chromatography-Mass Spectrometry.

Solanesol, a naturally occurring constituent of tobacco, has been utilized as a good marker for environmental tobacco smoke particulate and as a noninvasive predictor of mainstream cigarette smoke tar and nicotine intake under naturalistic smoking conditions. A fast and accurate method for measuring free solanesol to assess tobacco smoke exposure is highly desirable. We have developed and validated a new environmentally friendly, high-throughput method for measuring solanesol content in discarded cigarette filter butts. The solanesol deposited in the used filters can be correlated with mainstream smoke deliveries of nicotine and total particle matter to estimate constituent delivery to smokers. A portion of filter material is removed from cigarette butts after machine smoking, spiked with internal standard solution, extracted and quantitatively analyzed using reverse-phase liquid chromatography coupled to a triple-quadrupole mass spectrometer. The new method incorporates a 48-well plate format for automated sample preparation that reduces sample preparation time and solvent use and increases sample throughput 10-fold compared to our previous method. Accuracy and precision were evaluated by spiking known amounts of solanesol on both clean and smoked cigarette butts. Recoveries exceeded 93% at both low and high spiking levels. Linear solanesol calibration curves ranged from 1.9 to 367 µg/butt with a 0.05 µg/butt limit of detection.

Effect of Cigarette Constituent Messages With Engagement Text on Intention to Quit Smoking Among Adults Who Smoke Cigarettes

The US Food and Drug Administration (FDA) is required to communicate the risks of tobacco constituents to the public. Few studies have addressed how FDA media campaigns can effectively communicate about cigarette smoke constituents. To examine whether messages about cigarette smoke constituents are effective in reducing smoking intentions and behaviors among adults who smoke. This randomized clinical trial enrolled participants who were aged between 18 and 65 years, were English speakers, were living in the United States, and who smoked at least 100 cigarettes during their lifetime and now smoked every day or some days. Participants received daily messages via email for 15 days. Participants were randomized to 1 of 2 message conditions or a control group and reported their previous-day smoking behaviors daily. Follow-up surveys were conducted on days 16 and 32. Data were collected from June 2017 to April 2018 and analyzed from April to September 2018. The 3 groups were (1) constituent plus engagement messages (eg, "Cigarette smoke contains arsenic. This causes heart damage.") that included the FDA as the source and engagement text (eg, "Within 3 months of quitting, your heart and lungs work better. Ready to be tobacco free? You can quit. For free nicotine replacement, call 1-800-QUIT-NOW"); (2) constituent-only messages that did not list the FDA as the source or include engagement text; and (3) a control condition with messages about littering cigarette butts. The primary outcome was the change in quit intentions (range, 1-4, with higher scores indicating stronger intentions) from pretest to day 16. Secondary outcome measures included daily smoking behaviors and quit attempts. A total of 789 participants (mean [SD] age, 43.4 [12.9] years; 483 [61.2%] women; 578 [73.3%] White; 717 [90.9%] non-Hispanic) were included in the study. The mean (SD) quit intention score was 2.5 (0.9) at pretest. Mean (SE) change in quit intention score from pretest to day 16 was 0.19 (0.07) points higher in the constituent plus engagement condition than in the control condition (P = .005) and 0.23 (0.07) points higher in the constituent-only condition compared with the control condition (P = .001). Participant reports of cigarettes smoked, forgone, and butted out were similar across study conditions at baseline and did not differ significantly at days 16 and 32 across study conditions. Viewing more messages was associated with an estimated decrease of 0.15 (SE, 0.01) cigarettes smoked per day per message viewed overall across conditions. To our knowledge, this is the first longitudinal test of cigarette constituent campaign messages in a national sample of adults who currently smoke. Messages about cigarette smoke constituents, with or without engagement text and source information, increased participants' intentions to quit, lending support to FDA efforts to educate consumers about such constituents. ClinicalTrials.gov Identifier: NCT03339206.

The Evolving E-cigarette: Comparative Chemical Analyses of E-cigarette Vapor and Cigarette Smoke.

Background: E-cigarette designs, materials, and ingredients are continually evolving, with cotton wicks and diverse coil materials emerging as the popular components of atomisers. Another recent development is the use of nicotine salts in e-liquids to replicate the form of nicotine found in cigarette smoke, which may help cigarette smokers to transition to e-cigarettes. However, scientific understanding of the impact of such innovations on e-cigarette aerosol chemistry is limited.Methods: To address these knowledge gaps, we have conducted a comparative study analyzing relevant toxicant emissions from five e-cigarettes varying in wick, atomiser coil, and benzoic acid content and two tobacco cigarettes, quantifying 97 aerosol constituents and 84 smoke compounds, respectively. Our focus was the potential for benzoic acid in e-liquids and cotton wicks to form aerosol toxicants through thermal degradation reactions, and the potential for nickel–iron alloy coils to catalyze degradation of aerosol formers. In addition, we analyzed e-cigarette emissions for 19 flavor compounds, thermal decomposition products, and e-liquid contaminants that the FDA has recently proposed adding to the established list of Harmful and Potentially Harmful Constituents (HPHCs) in tobacco products.Results: Analyses for benzene and phenol showed no evidence of the thermal decomposition of benzoic acid in the e-cigarettes tested. Measurements of cotton decomposition products, such as carbonyls, hydrocarbons, aromatics, and PAHs, further indicated that cotton wicks can be used without thermal degradation in suitable e-cigarette designs. No evidence was found for enhanced thermal decomposition of propylene glycol or glycerol by the nickel–iron coil. Sixteen of the 19 FDA-proposed compounds were not detected in the e-cigarettes. Comparing toxicant emissions from e-cigarettes and tobacco cigarettes showed that levels of the nine WHO TobReg priority cigarette smoke toxicants were more than 99% lower in the aerosols from each of five e-cigarettes as compared with the commercial and reference cigarettes.Conclusions: Despite continuing evolution in design, components and ingredients, e-cigarettes continue to offer significantly lower toxicant exposure alternatives to cigarette smoking.

Analysis of Nicotine and Non-nicotine Tobacco Constituents in Aqueous Smoke/Aerosol Extracts by UHPLC and Ultraperformance Convergence Chromatography-Tandem Mass Spectrometry.

The non-nicotine constituents of tobacco may alter the reinforcing effects of nicotine, but the quantitative and qualitative profiles of these chemicals in tobacco products such as electronic cigarettes (e-cigarettes), cigars, and waterpipe tobacco are not well characterized. The objective of this work was to develop and validate analytical methods to utilize saline both as an extraction solvent for smoke condensates from cigarettes, little cigars, and waterpipe tobacco and aerosols from e-cigarettes and as a delivery vehicle of nicotine and non-nicotine constitents for nonclinical pharmacological studies. Ultrahigh-performance liquid chromatography was used to analyze nicotine and acetaldehyde, and a novel ultraperformance convergence chromatography-tandem mass spectrometry method was developed to analyze anabasine, anatabine, cotinine, myosmine, nornicotine, harmane, and norharmane. Linearity was confirmed for each standard curve with correlation coefficients (r) ≥ 0.99, and relative errors (RE) for the standards were ≤±10% over the calibration ranges. Method validation was performed by preparing triplicate samples in saline to mimic the composition and concentration of each analyte in the smoke or aerosol condensate and were used to determine method accuracy and precision. Relative standard deviation values were ≤15% and mean RE ≤15% for each analyte at each concentration level. Selectivity of the methods was demonstrated by the absence of peaks in blank vehicle or diluent samples. Storage stability was assessed over ∼45 days. Precision (%RSD ≤ 13) and recovery (percent of day 0 ≥ 80%) indicated that the saline formulations of all four products could be considered stable for up to ∼45 days at 4-8 °C. Therefore, the use of saline both as an extraction solvent and as a delivery vehicle adds versatility and improved performance in the study of the pharmacological effects of constituents from mainstream smoke and aerosols generated from cigarettes, little cigars, waterpipes, and e-cigarettes.

Blood lead levels and lung cancer mortality in the United States: an updated analysis using the National Health and Nutrition Examination Survey (NHANES) II and III

Background: Elevated blood lead levels (BLLs) have been associated with lung cancer in occupational studies. To assess whether this relationship extends to the general population (with lower BLLs) and with full adjustment for smoking (given that lead is a tobacco constituent), we conducted analyses within the National Health and Nutrition Examination Survey (NHANES) II (1976-1980) and III (1988-1994) cycles.Methods: We included 4,293 and 15,671 participants in NHANES II and III, respectively, aged ≥ 20 with BLL measurements and mortality follow-up through 2014. We fit multivariable Cox models adjusting for smoking and other potential confounders to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for BLL associated with mortality for lung cancer.Results: We observed a suggestive association between BLLs and lung cancer mortality in both NHANES II (189 deaths; HR 1.8, 95% CI 0.7, 4.7 for BLL ≥20.0 µg/dl vs <10.0 µg/dl, Ptrend =0.12) and NHANES III (363 deaths; HR 1.4, 95% CI 0.9, 2.0 for BLL ≥10.0 µg/dl vs <2.5 µg/dl, Ptrend =0.08). These associations were stronger among women (NHANES II: HR 2.7, 95% CI 0.7, 10.0, Ptrend =0.07; NHANES III: HR 2.7, 95% CI 1.2, 6.5, Ptrend =0.03). After stratifying on smoking status, the NHANES II association for women was present among both current smokers (HR 1.3, 95% CI 0.6, 2.7 for 90th vs 10th BLL percentile) and never/former smokers (HR 2.1, 95% CI 0.7, 6.2 for 90th vs 10th BLL percentile). In NHANES III, however, the association for women was observed only among current smokers (HR 1.8, 95% CI 1.2, 2.7).Conclusions: We observed a suggestive association between BLLs and lung cancer mortality among women. While residual confounding from tobacco use may explain the NHANES III finding, the NHANES II finding appears to be independent of smoking.

Nicotine uncovers endotoxic-like cardiovascular manifestations in female rats: Estrogen and nitric oxide dependency

Endotoxic manifestations are diminished in female populations due to immune boosting actions of sex steroids. Considering that tobacco constituents including nicotine inhibit estrogen synthesis, we tested the hypothesis that nicotine exposure unveils cardiovascular anomalies of endotoxemia in female rats. Studies were undertaken in conscious female rats treated with i.v. lipopolysaccharide (LPS, 10 mg/kg) in absence and presence of nicotine. In contrast to no effects for LPS when used alone, dose-related decreases in blood pressure (BP) and serum estrogen were noted in endotoxic rats treated consequently with nicotine (25, 50, or 100 μg/kg i.v.). Signs of cardiac autonomic dysfunction appeared in LPS/nicotine-treated rats such as (i) decreased time-domain indices of heart rate variability (HRV), e.g. standard deviation of R-R intervals (SDNN) and root mean square of successive differences in R-R interval durations (rMSSD), and (ii) reduced total power of the frequency spectrum and shifted cardiac sympathovagal balance toward sympathetic dominance. Nicotine reversed the LPS-evoked modest rises in serum TNFα and IL-1β while had no effect on associated arterial baroreflex dysfunction, inferring no roles for inflammation or baroreflexes in LPS-nicotine interaction. Estrogen or aminoguanidine (iNOS inhibitor), but not pentoxifylline (TNFα inhibitor), abolished LPS/nicotine hypotension. Together, nicotine acts probably via reducing estrogen availability to uncover nitric oxide-dependent hypotension and autonomic dysregulation in endotoxic female rats.

Smoke Chemistry, In Vitro Cytotoxicity, and Genotoxicity Demonstrates Enhanced Toxicity of Cigarillos Compared With Cigarettes

There has been limited toxicity testing of cigarillos, including comparison to cigarettes. This study compared the smoke chemistry and the cytotoxic and genotoxic potential of 10 conventional cigarettes and 10 cigarillos based on the greatest market share. Whole smoke and total particulate matter (TPM) were generated using the Canadian Intense and International Organization for Standardization puffing protocols. Tobacco-specific nitrosamines, carbonyls, and polycyclic aromatic hydrocarbons were measured using gas chromatography-mass spectrometry. TPM smoke extracts were used for the in vitro assays. Cytotoxicity was assessed in human bronchial epithelial continuously cultured cell line cells using the neutral red uptake assay. Genotoxic potential was assessed using the micronucleus (human lung adenocarcinoma continuously cultured cell line cells), Ames, and thymidine kinase assays. TPM from all cigarillos tested was more cytotoxic than cigarettes. Micronucleus formation was significantly greater for cigarillos compared with cigarettes at the highest dose of TPM, with or without rat liver S9 fraction. In the Ames test +S9, both tobacco products exhibited significant dose-dependent increases in mutation frequency, indicating metabolic activation is required for genotoxicity. In the thymidine kinase assay +S9, cigarillos showed a significantly enhanced mutation frequency although both tobacco products were positive. The levels of all measured polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, and carbonyls (except acrolein) were significantly greater in cigarillos than cigarettes. The Canadian Intense puffing protocol demonstrated increased smoke constituent levels compared with International Organization for Standardization. Even though the gas vapor phase was not tested, the results of this study showed that under the tested conditions the investigated cigarillos showed greater toxicity than comparator cigarettes. This study found that there is significantly greater toxicity in the tested U.S. marketed cigarillos than cigarettes for tobacco constituent levels, cytotoxicity, and genotoxicity. These findings are important for understanding the human health toxicity from the use of cigarillos relative to cigarettes and for building upon knowledge regarding harm from cigarillos to inform risk mitigation strategies.

Rapid quantification of constituents in tobacco by NIR fiber‐optic probe

AbstractThis study aims to develop, evaluate, and optimize the application of near‐infrared spectroscopy (NIR) autosampler device equipped with a fiber‐optic probe associated with chemometric methods for the fast quantification of routine chemical constituents such as total alkaloids, reducing sugars, nitrate, and ammonia in tobacco. For this purpose, an NIR autosampler device with capacity for 40 samples was used. The spectra were collected in the range of 950 to 2,500 nm, with three spins on the sample, a 3.0‐cm2 sample scanning area, and 2.0 ± 0.2 mm distance between the fiber‐optic probe and the sample. Four partial least‐squares (PLS) models were developed, and different preprocessing methods were investigated. The predicted results were compared with those obtained from the reference method (continuous‐flow analysis), and root mean square error of prediction values of 0.31%, 1.27%, 0.47%, and 0.026% were obtained for total alkaloids, reducing sugars, nitrate, and ammonia, respectively. The proposed method performed well for the analysis of total alkaloids and reducing sugars with an appropriate goodness‐of‐fit and fair precision. In conclusion, considering the performance of the regression models and the associated environmental and economic advantages, the application of NIR spectrometer autosampler device equipped with a fiber‐optic probe associated with a PLS and synergy interval PLS algorithms cannot replace the reference method, but it is a promising tool for tobacco monitoring.

Looking Inside the World of Granulosa Cells: The Noxious Effects of Cigarette Smoke.

The detrimental implications of tobacco smoke on systemic health have been widely established during the past few decades. Nonetheless, increasing evidence has begun to shed more light on the serious impact that smoke exposure could also have on mammal reproductive health in terms of overall ovarian dysfunction and gestation. A variety of these complications seem to be causally related to specific chemical substances contained in cigarette smoke and their possible effects on ovarian tissues and cells, such as granulosa cells. Granulosa cells represent the functional unit of the ovary and are able to establish a bidirectional cross-talk relationship with the oocyte during folliculogenesis, which makes them vital for its correct growth and development. Based on these premises, the current review focuses on the presence of related smoke-induced damages in granulosa cells. Data have been grouped according to the studied tobacco constituents and the molecular pathways involved, in order to synthesize their impact on granulosa cells and fertility. Attention is further brought to the correlation between electronic cigarettes and female reproduction, although there have been no investigations so far regarding e-cigarette-related granulosa cell exposure. We summarize how tobacco constituents are able to cause alterations in the “life” of granulosa cells, ranging from luteal steroidogenesis and follicular loss to granulosa cell apoptosis and activation of the autophagic machinery. Further studies have been conducted to elucidate the relationship between lifestyle and fertility as to reduce the morbidity connected with infertility.

Perceptions of nicotine in current and former users of tobacco and tobacco harm reduction products from seven countries

Purpose Nicotine is widely known as a tobacco constituent and for its use as a tobacco cessation aid. The development of new devices for nicotine delivery in recent years has led to uncertainty among consumers regarding the health risks of nicotine relative to tobacco. The purpose of this study was to discover if current and former consumers of tobacco and tobacco harm reduction (THR) products could distinguish between “nicotine” and “cigarettes” and examined the preceding media dialogue to determine if conflicting messages by the media influence public perceptions. Design/methodology/approach A quantitative survey was administered online in Norway (NO), Japan (JP), the United Kingdom (UK) and the United States (US), while face-to-face computer-aided interviews were conducted with randomly selected samples in India (IN), Greece (GR) and South Africa (SA). Participants were between 18 and 69 years of age and either current users of tobacco and THR products or previous users who quit within the past five years. Questions assessed beliefs about harmfulness of nicotine. Nicotine and other products and substances were also independently rated for harmfulness on a scale of 1–10 and subsequently compared. In addition, the authors examined the media dialogue of top media outlets in four countries to assess the potential influence on people’s beliefs. Findings A total of 54,267 participants (NO: 1,700, JP: 2,227, UK: 2,250, USA: 2,309, IN: 41,633, GR: 1,801, SA: 2,359) were sampled with the percentage of women participants ranging from 14.8% (IN) to 53.8% (UK). Between 68.3% (men, IN) and 88.7% (men, USA) of current consumers believed nicotine is harmful. Current consumers who agreed with the statement that nicotine is the primary cause of tobacco-related cancer ranged from 43.7% (men, UK) to 78.0% (men, SA). In six countries nicotine was rated nearly as harmful as cigarettes and alcohol, while other substances such as sugar, salt or caffeine, were usually rated as less harmful. Research limitations/implications A large proportion of consumers across all surveyed countries view nicotine and cigarettes similarly. Clearer communication on the harmful properties of both by the media is needed to help consumers make informed decisions about products across the continuum of risk. Messaging to consumers, especially via the media, propagates misinformation about the relative harms of tobacco and nicotine through reporting that is often incomplete and biased toward more negative aspects. Originality/value This study specifically assessed public perceptions of nicotine as opposed to products containing nicotine, which is the focus of previous studies. Apart from showing that consumers often incorrectly perceive nicotine and cigarettes as similar in terms of harmfulness, the authors highlight the need for more accurate and complete reporting by the media to clarify widespread misunderstandings and mitigate public uncertainty.

Abstract A42: The search for new targets for mitigation of metal toxicity based on molecular pathways of action: A case study using cadmium and iron

Abstract Cadmium is a nonessential heavy metal, ubiquitous in nature with a long half-life in human beings. It is widely used in various industrial applications such as toy and jewelry making and is one of the key constituents of tobacco and cigarette smoke. Cadmium, both at occupational and environmental levels, has been implicated in the causation of cancerous and noncancerous health outcomes. In comparison, iron is an essential metal and is a constituent of hemoglobin and many vital enzymes and proteins and plays a pivotal role in human health. However, increased consumption of iron from fortified diets and iron supplements presents opportunities for exposure to supraphysiologic iron concentrations and toxicity. Additionally, hereditary iron disorders (thalassemia, hereditary hemochromatosis), other disease conditions such as cancer and kidney diseases, and certain environmental exposures (e.g., steel-welding fumes, asbestos, and air pollution) could also lead to iron toxicity with disturbed homeostasis mechanisms affecting intracellular and systemic regulation of iron. Chelation therapy has traditionally been used to mitigate the toxicity of metals. This talk will present the results of systematic reviews and analyses for both cadmium and iron toxicity and highlight the diverse molecular pathways by which these metals can elicit toxic effects. Using the examples of cadmium and iron, I will discuss some recent advances in our understanding of the molecular mechanisms of metal toxicity and how this information can be used to identify novel targets for toxicity mitigation. (The views expressed in this abstract are those of the author and do not necessarily reflect the views or policies of EPA.) Citation Format: Anuradha Mudipalli. The search for new targets for mitigation of metal toxicity based on molecular pathways of action: A case study using cadmium and iron [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A42.

Menthol blunts the interoceptive discriminative stimulus effects of nicotine in female but not male rats.

Menthol is a widely used tobacco constituent that has shown to enhance nicotine's reinforcing effects. To determine whether injected menthol also alters nicotine's stimulus effects, we used a drug discrimination task. A total of 57 adult Sprague-Dawley rats (28M, 29F) received 20 positive and 20 negative days (intermixed) of discrimination training. On positive days, rats received a group-specific menthol and nicotine injection (VEH + 0.1 NIC, 1 M + 0.1 NIC, 5 M + 0.1 NIC, VEH + 0.4 NIC, 1 M + 0.4 NIC, 5 M + 0.4 NIC; mg/kg) before eight 15-s cue light presentations (conditioned stimulus (CS)), each followed by 4-s sucrose access. On negative days, all rats were injected with vehicle and saline before eight non-reinforced CS presentations. Next, rats underwent generalization testing with 30 dose combinations of menthol and nicotine. The change in drug-mediated anticipatory goal tracking during the CS was calculated as a difference score (CS minus pre-CS responding). All groups readily acquired drug discrimination. However, difference scores for the 5M + 0.1 NIC group were lower for females. Additionally, females had lower scores for 0.05, 0.1, and 0.4 mg/kg nicotine generalization tests. The lowest nicotine dose discriminable from saline was 0.05 mg/kg for females but 0.025 mg/kg for males. Co-administration with 5 or 10 mg/kg menthol weakened discrimination performance between 0.1 and 0.4 mg/kg and between 0.1 and 0.05 mg/kg nicotine for 0.1 mg/kg nicotine training groups. Female rats that were trained with 0.1 mg/kg nicotine were more sensitive to menthol's modulatory effects on nicotine's stimulus effects. This highlights the importance of taking sex and training dose into account when evaluating the interoceptive stimulus effects of nicotine and menthol.

Differential packaging of inflammatory cytokines/ chemokines and oxidative stress modulators in U937 and U1 macrophages-derived extracellular vesicles upon exposure to tobacco constituents

Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophage-derived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

Electronic cigarette vapour moderately stimulates pro-inflammatory signalling pathways and interleukin-6 production by human monocyte-derived dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells that play a critical role in bridging innate and adaptive immunity. Numerous studies have shown that tobacco constituents present in conventional cigarettes affect the phenotype and function of DCs; however, no studies have examined the effects of vapour from E-cigarettes on human DCs. Here, the effects of E-cigarette vapour extract (ECVE) on the phenotype and function of DCs were investigated by creating an in vitro cell culture model using human monocyte-derived DCs (MoDCs). Immature DCs were generated from peripheral blood monocytes and mature DCs were then produced by treatment with LPS or Poly I:C for 24 h. For LPS-matured DCs, 3% ECVE treatment slightly suppressed HLA-DR and CD86 expression, whereas 1% ECVE treatment enhanced IL-6 production. The overall expression of 29 signalling molecules and other cytoplasmic proteins (mainly associated with DC activation) was significantly upregulated in immature DCs by 1% ECVE, and in LPS-treated DCs by 3% ECVE. In particular, the condition that induced IL-6 production also upregulated MAPK pathway activation. These findings indicate that E-cigarette vapour moderately affects human DCs, but the effects are less pronounced than those reported for tobacco smoke.

Development of a Nicotinic Acetylcholine Receptor nAChR α7 Binding Activity Prediction Model.

Despite the well-known adverse health effects associated with tobacco use, addiction to nicotine found in tobacco products causes difficulty in quitting among users. Nicotinic acetylcholine receptors (nAChRs) are the physiological targets of nicotine and facilitate addiction to tobacco products. The nAChR-α7 subtype plays an important role in addiction; therefore, predicting the binding activity of tobacco constituents to nAChR-α7 is an important component for assessing addictive potential of tobacco constituents. We developed an α7 binding activity prediction model based on a large training data set of 843 chemicals with human α7 binding activity data extracted from PubChem and ChEMBL. The model was tested using 1215 chemicals with rat α7 binding activity data from the same databases. Based on the competitive docking results, the docking scores were partitioned to the key residues that play important roles in the receptor-ligand binding. A decision forest was used to train the human α7 binding activity prediction model based on the partition of docking scores. Five-fold cross validations were conducted to estimate the performance of the decision forest models. The developed model was used to predict the potential human α7 binding activity for 5275 tobacco constituents. The human α7 binding activity data for 84 of the 5275 tobacco constituents were experimentally measured to confirm and empirically validate the prediction results. The prediction accuracy, sensitivity, and specificity were 64.3, 40.0, and 81.6%, respectively. The developed prediction model of human α7 may be a useful tool for high-throughput screening of potential addictive tobacco constituents.

Can the Public Be Educated About Constituents in Smokeless Tobacco? A Three-Wave Randomized Controlled Trial.

The U.S. Food and Drug Administration (FDA) is required by law to inform the public about levels of harmful and potentially harmful tobacco constituents in a format that is "understandable and not misleading to a lay person." Our study addresses a critical gap in research on communicating such information for smokeless tobacco (SLT) products. The design included random assignment to one of the experimental (online interactive) conditions differing in presentation format or a control condition (receiving no information). Experimental respondents viewed information on levels and health risks of 5 harmful constituents in up to 79 products. Outcome measures included knowledge of health risks of constituents, perception of constituent variability in SLT products, disease risk ratings, self-reported SLT use, and side-by-side product comparisons. The sample of 333 SLT users, 535 cigarette smokers, and 663 nontobacco users participated at baseline, time of intervention, and 6 weeks postintervention. Presentation formats showed few systematic differences so were combined in analyses. Experimental condition respondents increased their knowledge about constituent health effects and their perceptions of constituent variability in SLT products, from baseline to postintervention, and relative to the control condition. Changes in respondents' ratings of disease risk and their estimates of constituent exposure from specific products were observed, but not in self-reported SLT use. Interactive online graphic and numeric presentation formats can be efficient in increasing people's knowledge of health effects and perceived variation of constituents in SLT products. Further research on longer-term behavioral assessment, and usefulness of this approach for regulatory agencies, is needed. Research on communicating the information about harmful constituents in SLT products to lay persons is critically lacking. This study proposes novel formats for effective communication about the levels and the health effects of SLT constituents to multiple user groups. The lack of misperceptions among study participants that some tobacco products are safe suggests that such formats can potentially be used for public display of SLT constituent data by the FDA and regulatory agencies in other countries.

Investigating the interoceptive stimulus effects of injected menthol in rats.

Menthol is a commonly used tobacco constituent that also modulates nicotine reinforcement and metabolism. Little is known about the stimulus effects of menthol that mediate the behavior associated with reinforcement-learning. Our present research explored the interoceptive stimulus effects of intraperitoneally administered menthol in a drug discrimination task. For Experiment 1, Sprague-Dawley rats (N = 20) received IP menthol (0.0183 or 5 mg/kg) or vehicle. For positive sessions, rats were given menthol before receiving 8 15-s light presentations, each followed by 4-s access to liquid sucrose. For intermixed negative sessions, rats were given vehicle before receiving 8 light presentations without sucrose delivery. After 32 sessions, rats previously receiving 0.0183 mg/kg menthol were switched to 15 mg/kg menthol. After 16 sessions, the injection-to-placement-interval was switched from 5 min to 15 min for 16 additional sessions. Lastly, a subset of rats (n = 10) received nicotine discrimination training for 40 sessions, with 0.4 mg/kg nicotine pretreatment on positive days and saline on negative days. In Experiment 2, naïve rats (N = 7) received nicotine discrimination training. Later sessions assessed nicotine discrimination performance in combination with 5 mg/kg menthol or vehicle. Menthol-vehicle discrimination was not evident regardless of dose or injection-to-placement interval in Experiment 1. However, rats that underwent nicotine training developed robust drug discrimination. In Experiment 2, co-exposure with menthol or vehicle did not modulate nicotine discrimination performance. These data suggest that menthol does not acquire control of responding in a drug discrimination task. Additional research is needed to further explore the interoceptive stimulus effects of menthol and nicotine combined. (PsycINFO Database Record (c) 2020 APA, all rights reserved).