Computational modeling method to estimate secondhand exposure potential from exhalations during e-vapor product use under various real-world scenarios.

Potential secondhand exposure of exhaled constituents from e-vapor product (EVP) use is a public health concern. We present a computational modeling method to predict air levels of exhaled constituents from EVP use. We measured select constituent levels in exhaled breath from adult e-vapor product users, then used a validated computational model to predict constituent levels under three scenarios (car, office, and restaurant) to estimate likely secondhand exposure to non-users. The model was based on physical/thermodynamic interactions between air, vapor, and particulate phase of the aerosol. Input variables included space setting, ventilation rate, total aerosol amount exhaled, and aerosol composition. Exhaled breath samples were analyzed after the use of four different e-liquids in a cartridge-based EVP. Nicotine, propylene glycol, glycerin, menthol, formaldehyde, acetaldehyde, and acrolein levels were measured and reported based on a linear mixed model for analysis of covariance. The ranges of nicotine, propylene glycol, glycerin, and formaldehyde in exhaled breath were 89.44-195.70 µg, 1199.7-3354.5 µg, 5366.8-6484.7 µg, and 0.25-0.34 µg, respectively. Acetaldehyde and acrolein were below detectable limits; thus, no estimated exposure to non-EVP users is reported. The model predicted that nicotine and formaldehyde exposure to non-users was substantially lower during EVPs use compared to cigarettes. The model also predicted that exposure to propylene glycol, glycerin, nicotine and formaldehyde among non-users was below permissible exposure limits.

A comprehensive physiologically based pharmacokinetic (PBPK) model for nicotine in humans from using nicotine-containing products with different routes of exposure.

Physiologically based pharmacokinetic (PBPK) modeling can be a useful tool for characterizing nicotine pharmacokinetics (PK) from use of tobacco products. We expand a previously published PBPK model to simulate a nicotine PK profile, following single or multiple use of various tobacco products [cigarettes, smokeless tobacco, and electronic nicotine delivery systems, or a nicotine inhaler (NICOTROL)] The uptake route in the model was designed to allow for three uptake compartments: buccal cavity (BC), upper respiratory tract (URT) (conducting and transitional airways) and lower respiratory tract (alveolar region). Within each region, the model includes product-specific descriptions of the flux of nicotine into plasma, as well as the flux of nicotine from the BC and URT to the gastrointestinal tract. These descriptions are based on regional deposition and diffusion models of nicotine into plasma, which depends on the product type. Regional deposition flux combined with regional differences in physiological parameters (e.g., blood perfusion ratio and tissue thickness) play a key role in the product-specific PK profile of nicotine. The current model describes the slower flux of nicotine into plasma across the BC and URT, as well as the rapid flux known to occur in the alveolar region. Overall, the addition of the BC and respiratory tract compartments to the nicotine model provided simulation results that are comparable to the nicotine time-course plasma concentrations reported from clinical studies for the four product categories simulated.

Smokeless tobacco mortality risks: an analysis of two contemporary nationally representative longitudinal mortality studies.

BACKGROUND: Assessments supporting smokeless tobacco (SLT) disease risk are generally decades old. Newer epidemiological data may more accurately represent the health risks associated with contemporary US-based SLT products, many of which contain lower levels of hazardous and potentially hazardous chemicals compared to previously available SLT products. METHODS: Data from two longitudinal datasets (National Longitudinal Mortality Study-NLMS, and the National Health Interview Survey-NHIS) were analyzed to determine potential associations between SLT use and/or cigarette smoking and all-cause and disease-specific mortality. Mortality hazard ratios (HR) were estimated using a Cox proportional hazards regression model applied to various groups, including never users of any tobacco or SLT product, and current and former SLT users and/or cigarette smokers. RESULTS: The two datasets yielded consistent findings with similar patterns evident for the specific causes of death measured. All-cause mortality risk for exclusive SLT users was significantly lower than that observed for exclusive cigarette smokers and dual SLT/cigarette users. Similar trends were found for mortality from diseases of the heart, chronic lower respiratory diseases, and malignant neoplasms. Mortality risk for lung cancer in exclusive cigarette smokers was increased by about 12-fold over never-tobacco users but was rarely present in exclusive SLT users in either survey (NHIS, < 5 cases/1,563 observations; NLMS, 3 cases/1,863 observations). While the data in the surveys are limited, SLT use by former cigarette smokers was not associated with an increase in the lung cancer risk HR compared to that by former cigarette smokers who never used SLT. CONCLUSIONS: Emerging epidemiological data provides a new perspective on the health risks of SLT use compared to risks associated with cigarette smoking. HR estimates derived from two current US datasets, which include data on contemporary tobacco products, demonstrate a clear mortality risk differential between modern SLT products and cigarettes. Cigarette smokers had an increased overall mortality risk and risk for several disease-specific causes of death, while SLT users consistently had lower mortality risks.

A Computational Model for Assessing the Population Health Impact of Introducing a Modified Risk Claim on an Existing Smokeless Tobacco Product.

Computational models are valuable tools for predicting the population effects prior to Food and Drug Administration (FDA) authorization of a modified risk claim on a tobacco product. We have developed and validated a population model using best modeling practices. Our model consists of a Markov compartmental model based on cohorts starting at a defined age and followed up to a specific age accounting for 29 tobacco-use states based on a cohort members transition pathway. The Markov model is coupled with statistical mortality models and excess relative risk ratio estimates to determine survival probabilities from use of smokeless tobacco. Our model estimates the difference in premature deaths prevented by comparing Base Case ("world-as-is") and Modified Case (the most likely outcome given that a modified risk claim is authorized) scenarios. Nationally representative transition probabilities were used for the Base Case. Probabilities of key transitions for the Modified Case were estimated based on a behavioral intentions study in users and nonusers. Our model predicts an estimated 93,000 premature deaths would be avoided over a 60-year period upon authorization of a modified risk claim. Our sensitivity analyses using various reasonable ranges of input parameters do not indicate any scenario under which the net benefit could be offset entirely.

Development/verification of methods for measurement of exhaled breath and environmental e-vapor product aerosol.

Concerns have been raised about the potential health effects of potential bystander exposure to exhaled aerosols from e-vapor products (EVPs). An exhaled breath collection system (EBS) was developed and analytical methods were verified for collection and analysis of exhaled breath from users of EVPs. Analytical methods were adapted and verified for collection of environmental air samples during EVP use in an exposure chamber. Analysis of constituents in exhaled breath focused on nicotine, propylene glycol, and glycerin (because these are reported as the major constituents in EVPs) and selected carbonyl compounds (acetaldehyde, acrolein, and formaldehyde). Analysis of environmental samples included nicotine, propylene glycol, glycerin, 12 volatile organic compounds (VOCs), 15 carbonyl compounds and 4 metals. The EBS and analytical methods used were found to be suitable for collection and analysis of the target constituents in exhaled breath. Environmental sampling for background levels of VOCs and carbonyl compounds found only acetone, acetaldehyde, benzene, ethylbenzene, formaldehyde, isoprene, methyl ethyl ketone, hexaldehyde, propionaldehyde, and toluene above the limit of quantification in some samples. None of the targeted metals were detected. Background levels of VOCs and carbonyl compounds were consistent with levels previously reported for ambient air.

A Well-Mixed Computational Model for Estimating Room Air Levels of Selected Constituents from E-Vapor Product Use.

Concerns have been raised in the literature for the potential of secondhand exposure from e-vapor product (EVP) use. It would be difficult to experimentally determine the impact of various factors on secondhand exposure including, but not limited to, room characteristics (indoor space size, ventilation rate), device specifications (aerosol mass delivery, e-liquid composition), and use behavior (number of users and usage frequency). Therefore, a well-mixed computational model was developed to estimate the indoor levels of constituents from EVPs under a variety of conditions. The model is based on physical and thermodynamic interactions between aerosol, vapor, and air, similar to indoor air models referred to by the Environmental Protection Agency. The model results agree well with measured indoor air levels of nicotine from two sources: smoking machine-generated aerosol and aerosol exhaled from EVP use. Sensitivity analysis indicated that increasing air exchange rate reduces room air level of constituents, as more material is carried away. The effect of the amount of aerosol released into the space due to variability in exhalation was also evaluated. The model can estimate the room air level of constituents as a function of time, which may be used to assess the level of non-user exposure over time.

Insights from two industrial hygiene pilot e-cigarette passive vaping studies.

While several reports have been published using research methods of estimating exposure risk to e-cigarette vapors in nonusers, only two have directly measured indoor air concentrations from vaping using validated industrial hygiene sampling methodology. Our first study was designed to measure indoor air concentrations of nicotine, menthol, propylene glycol, glycerol, and total particulates during the use of multiple e-cigarettes in a well-characterized room over a period of time. Our second study was a repeat of the first study, and it also evaluated levels of formaldehyde. Measurements were collected using active sampling, near real-time and direct measurement techniques. Air sampling incorporated industrial hygiene sampling methodology using analytical methods established by the National Institute of Occupational Safety and Health and the Occupational Safety and Health Administration. Active samples were collected over a 12-hr period, for 4 days. Background measurements were taken in the same room the day before and the day after vaping. Panelists (n = 185 Study 1; n = 145 Study 2) used menthol and non-menthol MarkTen prototype e-cigarettes. Vaping sessions (six, 1-hr) included 3 prototypes, with total number of puffs ranging from 36-216 per session. Results of the active samples were below the limit of quantitation of the analytical methods. Near real-time data were below the lowest concentration on the established calibration curves. Data from this study indicate that the majority of chemical constituents sampled were below quantifiable levels. Formaldehyde was detected at consistent levels during all sampling periods. These two studies found that indoor vaping of MarkTen prototype e-cigarette does not produce chemical constituents at quantifiable levels or background levels using standard industrial hygiene collection techniques and analytical methods.

Effect of chronic renal insufficiency on hepatic and renal udp-glucuronyltransferases in rats.

Significant evidence exists regarding altered CYP450 enzymes in chronic renal insufficiency (CRI), although none exists for the phase II enzymes. The objective of this study was to investigate the effect of CRI on hepatic and renal UDP-glucuronyltransferase (UGT) enzymes. Three groups of rats were included: CRI induced by the 5/6th nephrectomy model, control, and control pair-fed (CPF) rats. UGT activities were determined in liver and kidney microsomes by the 3- and 17-glucuronidation of beta-estradiol (E2-3G and E2-17G), glucuronidation of 4-methylumbelliferone (4-MUG), and 3-glucuronidation of morphine (M3G). UGT isoforms responsible for these catalytic activities were screened using recombinant rat UGT1A1, UGT1A2, UGT1A3, UGT1A7, UGT2B2, UGT2B3, and UGT2B8. UGT protein levels were examined by Western blot analysis using polyclonal antibodies. There was no significant difference between CRI and CPF rats in hepatic and/or renal E2-3G (UGT1A1), E2-17G (UGT2B3), 4-MUG (UGT1A6), and M3G (UGT2B1) formation. Formation of E2-17G and 4-MUG in the liver and E2-3G and 4-MUG in the kidney was significantly reduced (p < 0.05) in CPF and CRI rats compared with control rats. The down-regulated glucuronidation activities were accompanied by corresponding reductions in protein content of specific UGT isoforms. These results suggest that CRI does not seem to influence the protein levels or catalytic activity of most of the major hepatic or renal UGT enzymes. The observed down-regulation of hepatic and renal UGTs in CRI and CPF rats could be caused by restricted food intake in these groups of rats.

4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1alpha and HIF-1beta subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. HIF-1 activates downstream target genes such as vascular endothelial growth factor A (VEGF-A), which plays an important role in tumor progression and angiogenesis. Estrogen exposure is considered to be the major risk factor for ovarian cancer. Estradiol (E2) is usually metabolized by CYP1A1/1A2 and CYP3A4 to the 2-hydroxy estradiol (2-OHE2) and 4-hydroxy estradiol (4-OHE2) in human liver. Many reports have suggested that the formation of 4-OHE2 is important for mammary carcinogenesis. However, the formation of 2-OHE2 may play an important role in exhibiting anticarcinogenic effects. In the present study, we have demonstrated that one of the catechol estrogen metabolites of E2, 4-OHE2, induces HIF-1alpha and VEGF-A expression at protein level in two human ovarian cancer cell lines, OVCAR-3 and A2780-CP70 cells, in dose- and time-dependent manners, whereas the other catechol estrogen metabolite of E2, 2-OHE2, does not alter HIF-1alpha and VEGF-A expression. To explore the mechanism of 4-OHE2-induced HIF-1alpha and VEGF-A expression, we studied whether phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase (MAPK) signaling pathways are involved in 4-OHE2-induced HIF-1alpha and VEGF-A expression. Our findings indicate that PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1alpha and VEGF-A expression, whereas MAPK inhibitor, PD98059, did not alter HIF-1alpha and VEGF-A expression induced by 4-OHE2. 4-OHE2, but not 2-OHE2, also induced Akt phosphorylation at Ser473 in dose- and time-dependent manners, and LY294002 and wortmannin inhibited Akt phosphorylation at Ser473 induced by 4-OHE2. Our results also indicated that the mTOR/FRAP inhibitor, rapamycin, inhibited 4-OHE2-induced HIF-1alpha and VEGF-A expression. These results suggest that the PI3K/Akt/FRAP signaling pathway is required for HIF-1alpha and VEGF-A expression induced by 4-OHE2, whereas the MAPK pathway is not required. The finding that induction of HIF-1alpha and VEGF-A expression occurs via the activation of the PI3K/Akt/FRAP signaling pathway could be an important mechanism of 4-OHE2-induced carcinogenesis.

Down-regulation of hepatic CYP3A in chronic renal insufficiency.

PURPOSE: The objective of this study was to investigate the mechanisms underlying the decrease in hepatic clearance of some drugs metabolized by CYP450 enzymes in chronic renal insufficiency (CRI). METHODS: CRI was induced in male Sprague-Dawley rats (n = 7) by the remnant kidney model (RKM); control animals (C) (n = 12) underwent sham surgery, of which n = 6 rats were pair-fed (CPF) with CRI rats and others (n = 6) had free access to food. Serum creatinine (Scr) and urea nitrogen (SUN) were monitored every 2 weeks. On day 36, livers were isolated, and microsomes were prepared. Catalytic activities were measured through O-demethylation (CYP2D) and N-demethylation of dextromethorphan (CYP3A) and O-deethylation of 7-ethoxyresorufin (CYP1A2). CYP450 protein and mRNA levels were also measured. RESULTS: Compared with CPF, Scr and SUN levels in CRI rats were increased twofold (p < 0.01) and 2.5-fold (p < 0.01), respectively. No effect on CYP1A2 and CYP2D activities, mRNA, or protein levels was observed between the groups. There was a reduction (41.8 +/- 20%, p < 0.01) in CYP3A activity, mRNA (p < 0.05), and protein levels (p < 0.05) in CRI rats compared to CPF. CONCLUSIONS: CRI induced by RKM does not have an effect on hepatic CYP1A2 and CYP2D enzymes but does reduce CYP3A activity, probably through down-regulation of CYP3A2.

2-Methoxyestradiol, a promising anticancer agent.

Estrogens occurring naturally in the body are metabolized to catecholestrogens (2- and 4-hydroxyestradiol) by the cytochrome P450 enzymes. 2-Hydroxy catecholestrogens are further metabolized by catechol-O-methyltransferase to 2-methoxyestradiol, which is known to be protective against tumor formation. 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells. It also possesses antiangiogenic activity through a direct apoptotic effect on endothelial cells. Other molecular mechanisms, including microtubule stabilization by inhibition of the colchicine-binding site, have been reported. The exact mechanism of action of 2-methoxyestradiol is still unclear, but it has been shown to be effective in preventing tumor growth in a variety of cell lines. 2-Methoxyestradiol also possesses cardioprotective activity by inhibiting vascular smooth muscle cell growth in arteries. It has a lower binding affinity for estrogen receptor alpha compared with that of estradiol, and its affinity for estrogen receptor beta is even lower than that of estrogen receptor alpha, thus it has minimal estrogenic activity. 2-Methoxyestradiol is distinct because of its inability to engage estrogen receptors as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of estrogen receptors alpha and beta. A phase I clinical trial of 2-methoxyestradiol 200, 400, 600, 800, and 1,000 mg/day in 15 patients with breast cancer showed significant reduction in bone pain and analgesic intake in some patients, with no significant adverse effects. Another phase I study of 2-methoxyestradiol 200-1,000 mg/day in combination with docetaxel 35 mg/m2/week for 4-6 weeks performed in 15 patients with advanced refractory metastatic breast cancer showed no serious drug-related adverse effects. A phase II randomized, double-blind trial of 2-methoxyestradiol 400 and 1,200 mg/day in 33 patients with hormone-refractory prostate cancer showed that it was well tolerated and showed prostate specific antigen stabilizations and declines. We have started a phase I clinical trial to explore dosages greater than 1,000 mg/day.

Expression and cyclic variability of CYP3A4 and CYP3A7 isoforms in human endometrium and cervix during the menstrual cycle.

CYP3A4, a cytochrome p450 (p450) isoform metabolizes estrogens, whereas CYP3A7, a fetal liver p450 isoform, is involved in estriol biosynthesis. The goal of this study was to evaluate expression of these enzymes in human uterine tissue during the proliferative and secretory phases of the menstrual cycle. Endometrium and cervix specimens were collected from women undergoing hysterectomy (n = 36). Total mRNA was extracted, quantified, and reverse-transcription polymerase chain reaction (RT-PCR) was carried out using consensus primers for CYP3A. The 453 base pairs PCR product was hybridized with specific internal oligonucleotide probes for CYP3A4 or CYP3A7 end labeled with (32)P gamma-ATP. The relative intensity of hybridization was determined by autoradiography. Expression of CYP3A7 in endometrium was significantly greater (approximately 10-fold) in the proliferative phase compared with the secretory phase (p < 0.05). CYP3A4 expression was comparable between the two phases. Expression of both enzymes was minimal in the cervix. Fluorescence in situ hybridization of paraffinized sections indicated localized expression of CYP3A enzymes in the glandular epithelium as well as the stroma. Comparison of relative fluorescence intensity indicated differential expression of CYP3A7 in various phases of the menstrual cycle. These results suggest that CYP3A expression in the endometrium of premenopausal women may vary depending on the menstrual cycle phase.

Benzo(a)pyrene exposure induces CYP1A1 activity and expression in human endometrial cells.

Estrogen is a major risk factor for endometrial cancer and it has been well-established that smokers have a significantly reduced risk of endometrial cancer. Localized levels of estrogen within the uterus may determine the estrogenic response. The objective of this research was to investigate effects of cigarette smoke related hydrocarbons (benzo(a)pyrene, BP) on uterine CYP1A1/2 and 1B1, enzymes involved in estrogen metabolism. Human endometrium epithelial cells (RL95-2) were incubated with various concentrations (0.05, 0.1, 0.5, 1, and 10mM) of BP for 48h. CYP1 catalytic activity, protein and mRNA levels were determined. Selective chemical and immuno-inhibitors were used to determine the contribution of individual CYP1 isoenzymes. Cells expressing CYP1A1, CYP1A2 and CYP1B1 were used for comparisons. CYP1A1/2 protein and mRNA levels were significantly elevated by BP. Low level of constitutive CYP1 activity was observed in RL95-2 cells, which was significantly induced by BP exposure (12-fold at 1mM). CYP1 activity in BP-induced cells was significantly inhibited by specific anti-CYP1A1 and high concentration of alpha-naphthoflavone (ANF, 100nM), but not by selective CYP1A2 (furafylline) and CYP1B1 (homoeriodictoyl) inhibitors and low concentration of ANF (5nM). These studies suggest that CYP1A2 and CYP1B1 are not induced by BP in the endometrial cells. It also appears that CYP1A1 is one of the major CYP450 enzymes induced by BP.

Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes.

Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.

Mutagenic outcome of the urinary carcinogen 4-aminobiphenyl is increased in acidic pH.

One of the carcinogens from cigarette smoke, 4-aminobiphenyl, is excreted in urine as 4-N-hydroxyl aminobiphenyl metabolite conjugates that undergo pH mediated activation to nitreneum ions leading to bladder tumorigenesis via DNA adduct formation. We hypothesize that changing the pH of smokers' urine might impact the mutagenic outcome. 4-NOHABP was synthesized (∼98% purity), structure confirmed by TLC and NMR; mutagenic activity, assessed by the Ames test, was measured after pre-incubation in buffer solutions or human urine at pH 4, 6, 7 and 8. An inverse correlation was observed between number of histidine revertant colonies and pH (r(2)=0.89), 4-NOHABP exhibiting significant mutagenicity at pH 4.0 (P<0.05).