Androgenic steroids in Over-the-Counter dietary Supplements: Analysis for association with adverse health effects.

From 2012 to 2013, approximately 16 New York residents reported vague, nonspecific adverse health effects which included fatigue, loss of scalp hair, and muscle aches. One patient was hospitalized for liver damage. An epidemiological investigation identified a common factor among these patients; the consumption of B-50 vitamin and multimineral supplements from the same supplier. To investigate whether these nutritional supplements might have been responsible for the adverse health effects observed, comprehensive chemical analyses of marketed lots of the supplements were performed. To determine presence of organic components and contaminants, organic extracts of samples were prepared and analyzed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR). These analyses revealed the presence of significant levels of methasterone (17ß-hydroxy-2α,17α-dimethyl-5α-androstane-3-one), an androgenic steroid and schedule III-controlled substance; dimethazine, an azine-linked dimer of methasterone; and methylstenbolone (2,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one), a related androgenic steroid. Methasterone and extracts of certain supplement capsules were identified as highly androgenic in luciferase assays by using an androgen receptor promoter construct. This androgenicity persisted for several days after cell exposure to the compounds. The presence of these components in implicated lots were associated with adverse health effects and the hospitalization of one patient and the presentation of symptoms of severe virilization in a child. These findings underscore the need for more rigorous oversight of the nutritional supplement industry.

Elevated levels of per- and polyfluoroalkyl substances (PFAS) in freshwater benthic macroinvertebrates from the Hudson River Watershed.

Per- and polyfluoroalkyl substances (PFAS) are contaminants of global concern due to their persistence and associated negative health effects. Considerable attention has been given to monitoring PFAS in the aquatic environment, however, few investigations have done so using freshwater benthic macroinvertebrates (BMIs). As these bottom-dwelling animals are known to bioconcentrate exogenous pollutants to a high degree, studying their PFAS levels may provide a more integrated view of PFAS contamination in the aquatic environment. In this study, BMIs, sediment, and surface water were collected from two streams in the Hudson River Watershed (one historically-impacted by PFAS) and analyzed for 44 PFAS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Orbitrap high-resolution mass spectrometry (HRMS) was used to confirm the identities of quantitated analytes. Across all matrices, 17 analytes were detected with PFOA dominating in surface water and PFOS in sediment/BMIs. PFOS bioaccumulation factors (BAFs) were approximately one order of magnitude higher than those of PFOA and ranged from 857 to 5151 L kg-1 across different BMI taxa. While PFAS concentrations in surface water and sediment were not excessively high, elevated levels were still measured in most BMI taxa. This observation suggests that the extent of PFAS contamination in a local system may be severely underestimated if only surface water and sediment are used for monitoring. Moreover, these findings have relevance for human exposure assessment considering BMIs are the primary food source of many fish.

Chemotyping of ∆8-THC-Containing e-Liquids Analyzed during the 2019-2020 New York State EVALI Investigation.

The investigation of the 2019-2020 E-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) outbreak in New York State provided a unique opportunity to examine the formulations and chemical components found in clandestine cannabis-containing e-liquids. In this EVALI investigation, it was determined that an unusually high proportion (16%) of the cannabis e-liquids analyzed contained significant levels of ∆8-tetrahydrocannabinol (∆8-THC). Although not thought to be the causative agent in the outbreak, the manufacturing origin of vaping e-liquids containing large concentrations of ∆8-THC was of great interest, since high ∆8-THC concentrations are not observed in the extracts of common cannabis strains. A principal component analysis of multiple cannabinoid concentrations revealed clusters of similar or identical ∆8-THC-containing products. This technique may be useful in identifying common manufacturing sources in this and future investigations. Several possible manufacturing methods to enrich ∆8-THC appear in literature and are discussed based on their likelihood as sources of this cannabinoid in these samples from the EVALI investigation. The presence of high levels of ∆8-THC in numerous illicit vaping products may implicate cannabidiol, which is readily available at low cost, as its synthetic precursor.

Investigation of Vaping Fluids Recovered From New York State E-Cigarette or Vaping Product Use-Associated Lung Injury Patients.

E-cigarette or vaping product use-associated lung injury (EVALI) is a serious pulmonary condition that is associated with the extended use of certain vaping products. EVALI was first characterized in the summer of 2019 and has since been reported in all 50 U.S. states. From August 2019 through June 2021, the New York State Department of Health has reported more than 197 confirmed cases emanating from all regions of the state. The Wadsworth Center at the New York State Department of Heath received vaping cartridges recovered from EVALI patients for chemical analysis of their contents. Untargeted analytical methods using gas chromatography-mass spectrometry and liquid chromatography-high-resolution mass spectrometry as well as targeted analyses for a variety of analytes including cannabinoids, pesticides, vitamin E acetate (VEA) and mycotoxins were used to characterize the composition of the vaping fluids and several commercial vaping fluid additives. From the analyses of the 284 e-cigarette devices recovered from patients, 82 were found to be nicotine-containing pods, and 202 devices containing cannabis oil, apparently from unauthorized or black-market dealers. The fluids from the cannabis-oil cartridges tended to have lower levels of THCs (Δ9-tetrahydrocannabinol + Δ8-tetrahydrocannabinol) and total cannabinoids compared with those of commercially produced formulations and contained significant levels of diluents including VEA, medium-chain triglycerides, polyethylene glycol, and castor oil. VEA was the diluent most frequently detected, which was present in 132 (65.3%) of the vaping fluids that contained cannabis oil. When present, VEA ranged from 2.0 to 67.8% of the total mass of the oil with a mean content of 37.0%. In some cases, two or three diluents were detected in the same sample. The ratio of VEA to THCs varied widely, from 0.07 to 5.34. VEA and specifically the high ratios of VEA to THCs in black-market vaping fluids may be causative in EVALI. The safety of additional components and additives that are present in vaping fluids are likewise of concern.

Noninvasive detection of human dehydroepiandrosterone, progesterone and testosterone using LC-MS/MS revealed effects of birth control pills/devices and body weight on ovulatory prediction.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to measure steroids in human saliva. We studied the performance of a conventional LC-MS/MS for measuring dehydroepiandrosterone (DHEA), testosterone and progesterone in human saliva. These three steroids were co-extracted by liquid-liquid extraction and derivatized. Derivatives were resolved on a C18 column and quantified using an LC-MS/MS (AB Sciex API 2000) instrument. The assay's limits of quantification were 0.03 ng/mL for all three steroids. Inter-assay coefficients of variation were 16.6-18.8% (DHEA), 12.0-15.8% (testosterone), and 12.7-19.3% (progesterone). Assay linearity analysis showed R2 of 0.9926, 0.9750 and 0.9949 for DHEA, testosterone and progesterone, respectively. No carry-over between samplings was observed. An ion-enhancement effect of 11.6% for DHEA determination and ion-suppression effects of 13.9% and 20.7% for analysis of progesterone and testosterone, respectively, were determined. No interferences by 9 steroid analogs were detected. Spiked recoveries were 85.5% (DHEA), 86.5% (testosterone), and 92.6% (progesterone). Comparison with laboratory developed test (LDT)-LC-MS/MS methods by other New York State Department of Health certified laboratories revealed R2 = 0.9425 (DHEA, LC-MS/MS = 1.0267 LDT + 21.989), R2 = 0.9849 (testosterone, LC-MS/MS = 0.9447 LDT + 9.8037), and R2 = 0.9736 (progesterone, LC-MS/MS = 1.1196 LDT + 0.0985). Reference intervals for the 3 steroids in saliva for young males and females were estimated. Results of intra-individual salivary progesterone analysis indicated that caution should be exercised when using progesterone concentrations in predicting ovulation for females who are under treatment with birth control pills/devices or has body a weight of > 90 kg.

Legacy and Emerging Per- and Polyfluoroalkyl Substances: Analytical Techniques, Environmental Fate, and Health Effects.

Due to their unique chemical properties, per- and polyfluoroalkyl substances (PFAS) have been used extensively as industrial surfactants and processing aids. While several types of PFAS have been voluntarily phased out by their manufacturers, these chemicals continue to be of ecological and public health concern due to their persistence in the environment and their presence in living organisms. Moreover, while the compounds referred to as "legacy" PFAS remain in the environment, alternative compounds have emerged as replacements for their legacy predecessors and are now detected in numerous matrices. In this review, we discuss the historical uses of PFAS, recent advances in analytical techniques for analysis of these compounds, and the fate of PFAS in the environment. In addition, we evaluate current biomonitoring studies of human exposure to legacy and emerging PFAS and examine the associations of PFAS exposure with human health impacts, including cancer- and non-cancer-related outcomes. Special focus is given to short-chain perfluoroalkyl acids (PFAAs) and ether-substituted, polyfluoroalkyl alternatives including hexafluoropropylene oxide dimer acid (HFPO-DA; tradename GenX), 4,8-dioxa-3H-perfluorononanoic acid (DONA), and 6:2 chlorinated polyfluoroethersulfonic acid (6:2 Cl-PFESA; tradename F-53B).

Enhanced Sensitivity for the Analysis of Perfluoroethercarboxylic Acids Using LC-ESI-MS/MS: Effects of Probe Position, Mobile Phase Additive, and Capillary Voltage.

Perfluoroethercarboxylic acids (PFECAs) have recently emerged as replacements for toxic per- and polyfluorinated alkyl substances (PFAS) including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). Compared with other PFAS, many PFECAs including hexafluoropropylene oxide dimer acid (HFPO-DA, trade name GenX) exhibit poor sensitivity during analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and are therefore often difficult to quantify. This study examined changes in ESI probe position, mobile phase additive, and capillary voltage with the goal of enhancing PFECA sensitivity. In addition, the relative contributions of existing mechanistic theories for PFAS ionization during ESI are discussed. Results indicated that the LC-ESI-MS/MS sensitivity for 9 PFECAs can be improved significantly by altering the ESI probe position. At the optimal probe position, lowering the capillary voltage from 2.0 to 0.5 kV universally enhanced the LC-ESI-MS/MS sensitivity for PFAS analysis. For most analytes, the use of ammonium bicarbonate rather than ammonium acetate as a mobile phase additive also enhanced the analytical response. These effects have not been previously reported and suggest that many laboratories may be conducting analyses of PFECAs under suboptimal conditions. Using the strategies outlined in this study, PFECAs can be more easily incorporated into comprehensive methods for PFAS analysis. Here, we describe analytical parameters that enhance the sensitivity for some PFECAs by up to 36-fold while maintaining high sensitivity for legacy PFAS. This work not only highlights solutions to mitigate inadequate PFECA sensitivity but also provides insight into the mechanisms underlying PFAS ionization efficiency during LC-ESI-MS/MS.

Analysis of Cannabinoid-Containing Fluids in Illicit Vaping Cartridges Recovered from Pulmonary Injury Patients: Identification of Vitamin E Acetate as a Major Diluent.

Beginning in June of 2019, there was a marked increase in reported cases of serious pulmonary injury associated with vaping. The condition, referred to as e-cigarette or vaping product use-associated lung injury (EVALI), does not appear to involve an infectious agent; rather, a chemical adulterant or contaminant in vaping fluids is suspected. In August of 2019, the Wadsworth Center began receiving vaporizer cartridges recovered from patients with EVALI for analysis. Having no a priori information of what might be in the cartridges, we employed untargeted analyses using gas chromatography-mass spectrometry and high-resolution mass spectrometry to identify components of concern. Additionally, we employed targeted analyses used for New York medical marijuana products. Here, we report on the analyses of 38 samples from the first 10 New York cases of EVALI for which we obtained cartridges. The illicit fluids had relatively low cannabinoid content, sometimes with unusual Δ9-/Δ8-tetrahydrocannabinol ratios, sometimes containing pesticides and many containing diluents. A notable diluent was α-tocopheryl acetate (vitamin E acetate; VEA), which was found in 64% of the cannabinoid-containing fluids. To investigate potential sources of the VEA, we analyzed six commercial cannabis-oil diluents/thickeners. Three were found to be >95% VEA, two were found to be primarily squalane, and one was primarily α-bisabolol. The cause(s) of EVALI is unknown. VEA and squalane are components of some personal care products; however, there is growing concern that vaping large amounts of these compounds is not safe.

Kratom Adulterated with Phenylethylamine and Associated Intracerebral Hemorrhage: Linking Toxicologists and Public Health Officials to Identify Dangerous Adulterants.

INTRODUCTION: Kratom is derived from the plant Mitragyna speciosa which is indigenous to Southeast Asia. Active compounds, mitragynine and 7-hydroxymitragynine, cause mild stimulant and opioid agonist effects. Although reported to have potential benefits in the treatment of opioid use disorder, efficacy remains uncertain while adverse health effects have been reported. A compounding concern is the presence of adulterants given that this is an unregulated product. CASE DETAILS: A 54-year-old fitness instructor who used an online purchased kratom product regularly for one year developed stimulatory effects and suffered a large hemorrhagic stroke with a close temporal relationship to ingestion of a different kratom product from the one he regularly used. A collaborative investigation by medical toxicologists, a regional poison center, the state public health laboratory, and public health officials determined that his new kratom product was adulterated with phenylethylamine (PEA). DISCUSSION: We report a case of PEA adulterated kratom purchased and used with resultant adverse effects. PEA is structurally similar to amphetamine and is known to produce sympathomimetic effects. It is possible the stimulatory effect of PEA resulted in a marked and transient increase in blood pressure resulting in hemorrhagic stroke. CONCLUSION: Medical toxicologists should form working relationships with laboratories and public health officials to aid in early identification of adulterated products that carry risk to the general population.

Potency Analysis of Medical Marijuana Products from New York State.

Introduction: In the United States, medical marijuana programs have been established in 29 states and the District of Columbia. In 2014, New York State (NYS) approved medical marijuana legislation, and its program became fully operational in January of 2016. Products manufactured under the auspices of the program may be used by certified patients in NYS for the treatment of 1 of 12 qualifying medical conditions. The NYS statute requires rigorous testing of each product lot manufactured in the state for its cannabinoid profile, bacterial and fungal contamination, mycotoxins, heavy metals, plant-growth regulators, and pesticides. Here, we report on the analysis of product cannabinoid profiles. Methods: A method employing a simple extraction/dilution technique and reversed-phase high-performance liquid chromatography with photodiode array detection (HPLC-PDA) was developed for the analysis of 10 cannabinoids: cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol (CBD), tetrahydrocannabivarin, cannabinol, Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene, cannabidivarin, and Δ9-tetrahydrocannabinolic acid-A. The method employed internal standard quantitation and incorporated a surrogate to monitor extraction efficiency and analytical recovery. Results: The HPLC-PDA method was validated using sample matrices composed of medium-chain triglycerides, hemp oil, sesame oil, and an ethanol-propylene glycol tincture. Limits of detection, limits of quantitation, accuracy, precision, and inter- and intraday reproducibility were found to be highly satisfactory. The validated method has been used to analyze over 3500 samples from over 700 lots of medical marijuana products manufactured in NYS from January 2016 through April 2018. Quality-control data showed quantitative spike recoveries and, for the analysis of samples from the same lot, the coefficients of variation for the principal analytes, Δ9-THC and CBD, averaged <3%. Using the HPLC-PDA method, the NYS medical marijuana products were analyzed to verify the potencies on the product labels and to determine the stability of the products. Conclusions: An HPLC-PDA-based method was developed, validated, and employed to analyze 10 cannabinoids in a variety of medical marijuana products. The method has proven to be accurate, precise, stable, and very robust. Its use is an integral part of the NYS Medical Marijuana program for validation of the content and consistency of medical marijuana products.

Introduction to the virtual special issue monitoring ecological responses to air quality and atmospheric deposition in the Athabasca Oil Sands region the wood Buffalo environmental Association's Forest health monitoring program.

The expansion of oil sands resource development in the Athabasca Oil Sands Region in the early 1990's led to concerns regarding the potential ecological and health effects of increased emissions and deposition of acidic substances. Conditions attached to a 1994 approval for an oil sands facility expansion led to the creation of the Wood Buffalo Environmental Association, and its Terrestrial Environmental Effects Monitoring committee. This multi-stakeholder body was tasked with development and operation of an environmental (forest health) monitoring program for the detection of ecological responses to atmospheric emissions and deposition. Initially focused on acid deposition monitoring, jack pine forest, growing on sandy soils with limited acid buffering capacity, was selected as the receptor system. An initial set of 10 monitoring locations was established using the Canadian Acid Rain Network Early Warning System methodology (since increased to 27, with three lost to development). Ecological monitoring is on a 6-year cycle, with concurrent measures of soil, needle and lichen chemistry, and tree and understory condition, together with ongoing measurements of air quality and atmospheric deposition. Because jack pine forest edges facing the emissions sources were expected to be more exposed to acidic emissions, evaluation of stand edge monitoring locations began in 2008. Monitoring of a targeted suite of indicators began in 2012 at 25 jack pine stand edge monitoring sites. This special issue presents the results derived from biophysical sampling campaigns (1998 to 2013), coupled with ongoing ambient atmospheric, deposition and epiphytic lichen monitoring (data through 2017) and source apportionment studies, as well as papers contributed by others engaged in regional research and monitoring programs. The Forest Health Monitoring Program provides data supportive of regulatory and stakeholder evaluations of environmental quality, and is adaptive to new needs, extreme environmental events and technological development while providing continuity of monitoring.

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6-17.9% for DHEA-S and cortisol, recoveries of 102.4-109.5% for DHEA-S and 94.6-98.3% for cortisol, and assay linearity with R2 = 0.9964 for DHEA-S (1.0-25.0 ng/mL) and R2 = 0.997 (1.0-25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R2 = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS - 0.7355) for cortisol, and R2 = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3-5.9 ng/mL for females and 0.1-5.6 ng/mL for males for salivary cortisol, and 0.6-7.4 ng/mL for females and 0.6-10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.

Liquid chromatography-tandem mass spectrometry analysis of 17-hydroxyprogesterone in dried blood spots revealed matrix effect on immunoassay.

Immunoassays for measuring 17-hydroxyprogesterone (17-OHP) produce high rates of false positives that impact the identification of congenital adrenal hyperplasia (CAH) in neonates. A confirmatory test with high analytical specificity employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology is needed in newborn screening for CAH. 17-OHP and cortisol were extracted from dried blood spot (DBS) samples, resolved on a C18 column, and measured using tandem mass spectrometry. The results were compared with those determined using the AutoDELFIA immunoassay. The LC-MS/MS method had a limit of quantitation of 10.0 and 5.0 ng/mL for 17-OHP and cortisol, respectively. The method characteristics showed coefficient variation (%CV) ≤ 11.9% for both 17-OHP and cortisol, recoveries ranging from 83.1 to 101.5% for 17-OHP and from 95.1 to 102.8% for cortisol, and linearity with R2 = 0.9994 for 17-OHP and R2 = 0.9996 for cortisol, clinical sensitivity of 100.0% and a specificity of 96.4% as obtained by receiver operating characteristic analysis on 45 patient samples when 17-OHP > 39.1 ng/mL was selected as the cutoff value. Comparison between the LC-MS/MS and the AutoDELFIA immunoassay methods revealed a poor correlation for patient DBS samples (R2 = 0.6784); however, an excellent correlation was obtained for QC and proficiency test (PT) DBS samples (R2 = 0.9797). The LC-MS/MS method produced reliable results for 17-OHP and cortisol for the diagnosis of CAH. The AutoDELFIA immunoassay appears to be subject to matrix effects in the analysis for 17-OHP in DBS patient samples. The DBS samples of non-patient origin may not be suitable for assessing analytical accuracy of immunoassays.

Alternate approaches for assessing impacts of oil sands development on air quality: A case study using the First Nation Community of Fort McKay.

Previous analyses of continuously measured compounds in Fort McKay, an indigenous community in the Athabasca Oil Sands, have detected increasing concentrations of nitrogen dioxide (NO2) and total hydrocarbons (THC), but not of sulfur dioxide (SO2), ozone (O3), total reduced sulfur compounds (TRS), or particulate matter (aerodynamic diameter <2.5 µm; PM2.5). Yet the community frequently experiences odors, dust, and reduced air quality. The authors used Fort McKay's continuously monitored air quality data (1998-2014) as a case study to assess techniques for air quality analysis that make no assumptions regarding type of change. Linear trend analysis detected increasing concentrations of higher percentiles of NO2, nitric oxide (NO), and nitrogen oxides (NOx), and THC. However, comparisons of all compounds between an early industrial expansion period (1998-2001) and current day (2011-2014) show that concentrations of NO2, SO2, THC, TRS, and PM2.5 have significantly increased, whereas concentrations of O3 are significantly lower. An assessment of the frequency and duration of periods when concentrations of each compound were above a variety of thresholds indicated that the frequency of air quality events is increasing for NO2 and THC. Assessment of change over time with odds ratios of the 25th, 50th, 75th, and 90th percentile concentrations for each compound compared with an estimate of natural background variability showed that concentrations of TRS, SO2, and THC are dynamic, higher than background, and changes are nonlinear and nonmonotonic. An assessment of concentrations as a function of wind direction showed a clear and generally increasing influence of industry on air quality. This work shows that evaluating air quality without assumptions of linearity reveals dynamic changes in air quality in Fort McKay, and that it is increasingly being affected by oil sands operations. IMPLICATIONS: Understanding the nature and types of air quality changes occurring in a community or region is essential for the development of appropriate air quality management policies. Time-series trending of air quality data is a common tool for assessing air quality changes and is often used to assess the effectiveness of current emission management programs. The use of this tool, in the context of oil sands development, has significant limitations, and alternate air quality change analysis approaches need to be applied to ensure that the impact of this development on air quality is fully understood so that appropriate emission management actions can be taken.

An enhanced approach for the use of satellite-derived leaf area index values in dry deposition modeling in the Athabasca oil sands region.

In the Athabasca oil sands region (AOSR) of Northern Alberta, the dry deposition of sulphur and nitrogen compounds represents a major fraction of total (wet plus dry) deposition due to oil sands emissions. The leaf area index (LAI) is a critical parameter that affects the dry deposition of these gaseous and particulate compounds to the surrounding boreal forest canopy. For this study, LAI values based on Moderate Resolution Imaging Spectroradiometer satellite imagery were obtained and compared to ground-based measurements, and two limitations with the satellite data were identified. The satellite LAI data firstly represents one-sided LAI values that do not account for the enhanced LAI associated with needle leaf geometry, and secondly, underestimates LAI in winter-time northern latitude regions. An approach for adjusting satellite LAI values for different boreal forest cover types, as a function of time of year, was developed to produce more representative LAI values that can be used by air quality sulphur and nitrogen deposition models. The application of the approach increases the AOSR average LAI for January from 0.19 to 1.40, which represents an increase of 637%. Based on the application of the CALMET/CALPUFF model system, this increases the predicted regional average dry deposition of sulphur and nitrogen compounds for January by factors of 1.40 to 1.30, respectively. The corresponding AOSR average LAI for July increased from 2.8 to 4.0, which represents an increase of 43%. This increases the predicted regional average dry deposition of sulphur and nitrogen compounds for July by factors of 1.28 to 1.22, respectively. These findings reinforce the importance of the LAI metric for predicting the dry deposition of sulphur and nitrogen compounds. While satellite data can provide enhanced spatial and temporal resolution, adjustments are identified to overcome associated limitations. This work is considered to have application for other deposition model studies where dry deposition represents a significant fraction of total deposition.

Twelve-year trends in ambient concentrations of volatile organic compounds in a community of the Alberta Oil Sands Region, Canada.

Environmental exposure to volatile organic compounds (VOCs) in ambient air is one of a number of concerns that the First Nation Community of Fort McKay, Alberta has related to development of Canada's oil sands. An in-depth investigation of trends in ambient air VOC levels in Fort McKay was undertaken to better understand the role and possible significance of emissions from Alberta's oil sands development. A non-parametric trend detection method was used to investigate trends in emissions and ambient VOC concentrations over a 12-year (2001-2012) period. Relationships between ambient VOC concentrations and production indicators of oil sands operations around Fort McKay were also examined. A weak upward trend (significant at 90% confidence level) was found for ambient concentrations of total VOCs based on sixteen detected species with an annual increase of 0.64µg/m(3) (7.2%) per year (7.7µg/m(3) increase per decade). Indicators of production (i.e., annual bitumen production and mined oil sands quantities) were correlated with ambient total VOC concentrations. Only one of 29 VOC species evaluated (1-butene) showed a statistically significant upward trend (p=0.05). Observed geometric (arithmetic) mean and maximum ambient concentrations of selected VOCs of public health concern for most recent three years of the study period (2010-2012) were below chronic and acute health risk screening criteria of the U.S. Agency for Toxic Substances and Disease Registry and U.S. Environmental Protection Agency. Thirty-two VOCs are recommended for tracking in future air quality investigations in the community to better understand whether changes are occurring over time in relation to oil sands development activities and to inform policy makers about whether or not these changes warrant additional attention.

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer.

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.

Measurement of unconjugated estriol in serum by liquid chromatography-tandem mass spectrometry and assessment of the accuracy of chemiluminescent immunoassays.

BACKGROUND: Unconjugated estriol (uE3) is routinely analyzed in clinical laboratories as risk assessment for Down syndrome. Immunoassays of various types are the most commonly used methods. The accuracies of RIAs and ELISAs for uE3 have been questioned, and to date there have been no independent studies investigating the accuracy of the relatively new chemiluminescent immunoassays. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for uE3 measurements in serum. METHODS: Serum samples from patients in the second trimester of pregnancy were used, and uE3 concentrations were measured by LC-MS/MS and the Beckman Coulter Access® 2 and Siemens IMMULITE 2000 automatic chemiluminescent immunoassay analyzers. RESULTS: The LC-MS/MS method was validated and showed limit of detection 0.05 ng/mL; limit of quantification 0.2 ng/mL; linearity of response to 32 ng/mL; total imprecision of 16.2%, 10.4%, and 8.2% for uE3 at 1.10, 4.18, and 8.32 ng/mL, respectively; and analytical recoveries of 95.9%-104.2%. ANOVA of the correlation for LC-MS/MS results vs chemiluminescent immunoassays results showed R(2) = 0.9678 (Access 2 = 0.9305 LC-MS/MS + 0.2177, Sy|x = 0.1786, P < 0.0001), and R(2) = 0.9663 (IMMULITE 2000 = 0.8849 LC-MS/MS - 0.0403, Sy|x = 0.1738, P < 0.0001). Bland-Altman plots of uE3 results revealed concentration-dependent immunoassay biases. Mock risk analysis for Down syndrome showed no apparent difference in the risk assessment outcomes if the adjusted method-specific multiples of the median were used, and the assay imprecision was <10% CV. CONCLUSIONS: Standardization of immunoassay methods for uE3 analysis is needed to improve the accuracy of the measurements.

Expression of the aryl hydrocarbon receptor is not required for the proliferation, migration, invasion, or estrogen-dependent tumorigenesis of MCF-7 breast cancer cells.

The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of two MCF-7-derived sublines: AH(R100) , which express minimal AhR, and AhR(exp) , which overexpress AhR. Quantitative PCR, Western immunoblots, 17ß-estradiol (E2 ) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack of AhR expression and AhR-regulated CYP1 expression in AH(R100) cells, and enhanced AhR and CYP1 expression in AhR(exp) cells. In the presence of 1 nM E2 , rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF-7 and AhR(exp) cells, AH(R100) cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AH(R100) cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2 , producing mean tumor volumes comparable to those observed with MCF-7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation of MCF-7 cells do not require the AhR.

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.