The human health effects of unconventional oil and gas (UOG) chemical exposures: a scoping review of the toxicological literature.

Many chemicals associated with unconventional oil and natural gas (UOG) are known toxicants, leading to health concerns about the effects of UOG. Our objective was to conduct a scoping review of the toxicological literature to assess the effects of UOG chemical exposures in models relevant to human health. We searched databases for primary research studies published in English or French between January 2000 and June 2023 on UOG-related toxicology studies. Two reviewers independently screened abstracts and full texts to determine inclusion. Seventeen studies met our study inclusion criteria. Nine studies used solely in vitro models, while six conducted their investigation solely in animal models. Two studies incorporated both types of models. Most studies used real water samples impacted by UOG or lab-made mixtures of UOG chemicals to expose their models. Most in vitro models used human cells in monocultures, while all animal studies were conducted in rodents. All studies detected significant deleterious effects associated with exposure to UOG chemicals or samples, including endocrine disruption, carcinogenicity, behavioral changes and metabolic alterations. Given the plausibility of causal relationships between UOG chemicals and adverse health outcomes highlighted in this review, future risk assessment studies should focus on measuring exposure to UOG chemicals in human populations.

Trace elements alone or in mixtures associated with unconventional natural gas exploitation affect rat fetal steroidogenesis and testicular development in vitro.

Biomonitoring studies have shown that pregnant women living in regions of unconventional natural gas (UNG) exploitation have higher levels of trace elements. Whether developmental endocrine disruption can be expected at these exposure levels during pregnancy is unclear. In this study, we aimed to test the impact of five trace elements alone or in mixtures using in vitro cell- and tissue-based assays relevant to endocrine disruption and development. Manganese, aluminum, strontium, barium, and cobalt were tested at concentrations including those representatives of human fetal exposure. Using transactivation assays, none of the tested elements nor their mixture altered the human estrogen receptor 1 or androgen receptor genomic signalling. In the rat fetal testis assay, an organ culture system, cobalt (5 µg/l), barium (500 µg/l) and strontium (500 µg/l) significantly increased testosterone secretion. Cobalt and strontium were associated with hyperplasia and/or hypertrophy of fetal Leydig cells. Mixing the five elements at concentrations where none had an effect individually stimulated testosterone secretion by the rat fetal testis paralleled by the significant increase of 3ß-hydroxysteroid dehydrogenase protein level in comparison to the vehicle control. The mechanisms involved may be specific to the fetal testis as no effect was observed in the steroidogenic H295R cells. Our data suggest that some trace elements in mixture at concentrations representative of human fetal exposure can impact testis development and function. This study highlights the potential risk posed by UNG operations, especially for the most vulnerable populations, pregnant individuals, and their fetus.

Altered oxidative stress and antioxidant biomarkers concentrations in pregnant individuals exposed to oil and gas sites in northeastern British Columbia.

Northeastern British Columbia is a region of prolific unconventional oil and gas activity (UOG). UOG activity can release volatile organic compounds (VOCs) which can elevate oxidative stress and disrupt antioxidant activity in exposed pregnant individuals, potentially increasing the risk of adverse pregnancy outcomes. This study measured biomarkers of oxidative stress and antioxidant activity in pooled urine samples of 85 pregnant individuals living in Northeastern British Columbia, to analyze associations between indoor air VOCs, oil and gas well density and proximity metrics, and biomarker concentrations. Concentrations of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), total antioxidant capacity (TAC), 6-hydroxymelatonin sulfate (aMT6s), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and 8-isoprostane (8-IP) were measured using assay kits. Associations between exposure metrics and biomarker concentrations were determined using multiple linear regression models adjusted for biomarker-specific covariables. UOG proximity was associated with decreased SOD and 8-OHdG. Decreased 8-OHdG was associated with increased proximity to all wells. Decreased aMT6s was observed with increased indoor air hexanal concentrations. MDA was negatively associated with indoor air 1,4-dioxane concentrations. No statistically significant associations were found between other biomarkers and exposure metrics. While some associations linked oil and gas activity to altered oxidative stress and antioxidant activity, the possibility of chance findings due to the large number of tests can not be discounted. This study shows that living near UOG wells may alter oxidative stress and antioxidant activity in pregnant individuals. More research is needed to elucidate underlying mechanisms and to what degree UOG activity affects oxidative stress and antioxidant activity.

The human health effects of unconventional oil and gas development (UOGD): A scoping review of epidemiologic studies.

OBJECTIVE: Unconventional oil and gas development (UOGD, sometimes termed "fracking" or "hydraulic fracturing") is an industrial process to extract methane gas and/or oil deposits. Many chemicals used in UOGD have known adverse human health effects. Canada is a major producer of UOGD-derived gas with wells frequently located in and around rural and Indigenous communities. Our objective was to conduct a scoping review to identify the extent of research evidence assessing UOGD exposure-related health impacts, with an additional focus on Canadian studies. METHODS: We included English- or French-language peer-reviewed epidemiologic studies (January 2000-December 2022) which measured exposure to UOGD chemicals directly or by proxy, and where health outcomes were plausibly caused by UOGD-related chemical exposure. Results synthesis was descriptive with results ordered by outcome and hierarchy of methodological approach. SYNTHESIS: We identified 52 studies from nine jurisdictions. Only two were set in Canada. A majority (n = 27) used retrospective cohort and case-control designs. Almost half (n = 24) focused on birth outcomes, with a majority (n = 22) reporting one or more significant adverse associations of UOGD exposure with: low birthweight; small for gestational age; preterm birth; and one or more birth defects. Other studies identified adverse impacts including asthma (n = 7), respiratory (n = 13), cardiovascular (n = 6), childhood acute lymphocytic leukemia (n = 2), and all-cause mortality (n = 4). CONCLUSION: There is a growing body of research, across different jurisdictions, reporting associations of UOGD with adverse health outcomes. Despite the rapid growth of UOGD, which is often located in remote, rural, and Indigenous communities, Canadian research on its effects on human health is remarkably sparse. There is a pressing need for additional evidence.

RéSUMé: OBJECTIF: L'exploitation pétrolière et gazière non conventionnelle (EPGNC, parfois appelée « fracturation ¼ ou « fracturation hydraulique ¼) est un processus industriel d'extraction du méthane et/ou de gisements de pétrole. De nombreux produits chimiques utilisés dans l'EPGNC ont des effets indésirables connus sur la santé humaine. Le Canada est un grand producteur de gaz dérivé de l'EPGNC, dont les puits sont souvent situés à l'intérieur et autour de communautés rurales et autochtones. Nous avons mené une étude de champ pour déterminer l'étendue des données de recherche évaluant les effets sur la santé de l'exposition à l'EPGNC, en nous concentrant plus particulièrement sur les études canadiennes. MéTHODE: Nous avons inclus des études épidémiologiques en anglais ou en français évaluées par les pairs (janvier 2000 à décembre 2022) qui mesuraient l'exposition directe ou indirecte aux produits chimiques de l'EPGNC et dans lesquelles les résultats cliniques étaient plausiblement causés par l'exposition aux produits chimiques liés à l'EPGNC. La synthèse des résultats est descriptive, et les résultats sont ordonnés selon les résultats cliniques et l'approche méthodologique. SYNTHèSE: Nous avons identifié 52 études menées dans neuf juridictions. Deux seulement étaient canadiennes. La majorité (n = 27) faisaient appel à des cohortes rétrospectives ou étaient des études cas-témoins. Près de la moitié (n = 24) portaient sur les issues de la grossesse, et la majorité (n = 22) déclaraient une ou plusieurs associations indésirables significatives entre l'exposition à l'EPGNC et : l'insuffisance de poids à la naissance; la petite taille du bébé pour son âge gestationnel; la naissance avant terme; et une ou plusieurs anomalies congénitales. D'autres études faisaient état d'effets indésirables, dont l'asthme (n = 7), les troubles respiratoires (n = 13), les troubles cardiovasculaires (n = 6), la leucémie aiguë lymphoblastique infantile (n = 2) et la mortalité toutes causes confondues (n = 4). CONCLUSION: Il existe dans différents pays un corpus croissant d'études qui font état d'associations entre l'EPGNC et des résultats sanitaires indésirables. Malgré la croissance rapide de l'EPGNC, souvent présente dans des communautés éloignées, rurales et autochtones, la recherche canadienne sur ses effets sur la santé humaine est remarquablement clairsemée. Il y a un besoin urgent de recueillir d'autres données probantes à ce sujet.

Modelling spatial & temporal variability of air pollution in an area of unconventional natural gas operations.

Despite the growing unconventional natural gas production industry in northeastern British Columbia, Canada, few studies have explored the air quality implications on human health in nearby communities. Researchers who have worked with pregnant women in this area have found higher levels of volatile organic compounds (VOCs) in the indoor air of their homes associated with higher density and closer proximity to gas wells. To inform ongoing exposure assessments, this study develops land use regression (LUR) models to predict ambient air pollution at the homes of pregnant women by using natural gas production activities as predictor variables. Using the existing monitoring network, the models were developed for three temporal scales for 12 air pollutants. The models predicting monthly, bi-annual, and annual mean concentrations explained 23%-94%, 54%-94%, and 73%-91% of the variability in air pollutant concentrations, respectively. These models can be used to investigate associations between prenatal exposure to air pollutants associated with natural gas production and adverse health outcomes in northeastern British Columbia.

Density and proximity of oil and gas wells and concentrations of trace elements in urine, hair, nails and tap water samples from pregnant individuals living in Northeastern British Columbia.

BACKGROUND: Oil and gas exploitation can release several contaminants in the environment, including trace elements, with potentially deleterious effects on exposed pregnant individuals and their developing fetus. Currently, there is limited data on pregnant individuals' exposure to contaminants associated with oil and gas activity. OBJECTIVES: We aimed to 1)measure concentrations of trace elements in biological and tap water samples collected from pregnant individuals participating in the EXPERIVA study; 2)compare with reference populations and health-based guidance values; 3)assess correlations across matrices; and 4)evaluate associations with the density/proximity of oil and gas wells. METHODS: We collected tap water, hair, nails, and repeated urine samples from 85pregnant individuals, and measured concentrations of 21trace elements. We calculated oil and gas well density/proximity (Inverse Distance Weighting [IDW]) for 4buffer sizes (2.5 km, 5 km, 10 km, no buffer). We performed Spearman's rank correlation analyses to assess the correlations across elements and matrices. We used multiple linear regression models to evaluate the associations between IDWs and concentrations. RESULTS: Some study participants had urinary trace element concentrations exceeding the 95th percentile of reference values; 75% of participants for V, 29% for Co, 22% for Ba, and 20% for Mn. For a given trace element, correlation coefficients ranged from -0.23 to 0.65 across matrices; correlations with tap water concentrations were strongest for hair, followed by nails, and urine. Positive (e.g., Cu, Cr, Sr, U, Ga, Ba, Al, Cd) and negative (e.g., Fe) associations were observed between IDW metrics and the concentrations of certain trace elements in water, hair, and nails. SIGNIFICANCE: Our results suggest that pregnant individuals living in an area of oil and gas activity may be more exposed to certain trace elements (e.g., Mn, Sr, Co, Ba) than the general population. Association with density/proximity of wells remains unclear.

Estimation of exposure to particulate matter in pregnant individuals living in an area of unconventional oil and gas operations: Findings from the EXPERIVA study.

Northeastern British Columbia (Canada) is an area of oil and gas exploitation, which may result in release of fine (PM2.5) and inhalable (PM10) particulate matter. The aims of this study were to: 1) apply extrapolation methods to estimate exposure to PM2.5 and PM10 concentrations among EXPERIVA (Exposures in the Peace River Valley study) participants using air quality data archives; and 2) conduct exploratory analyses to investigate correlation between PM exposure and metrics of oil and gas wells density, proximity, and activity. Gestational exposure to PM2.5 and PM10 of the EXPERIVA participants (n = 85) was estimated by averaging the concentrations measured at the closest or three closest air monitoring stations during the pregnancy period. Drilling metrics were calculated based upon the density and proximity of conventional and unconventional oil and gas wells to each participant's residence. Phase-specific metrics were determined for unconventional wells. The correlations (ρ) between exposure to PM2.5 and PM10 and metrics of well density/proximity were determined using Spearman's rank correlation test. Estimated PM ambient air concentrations ranged between 4.73 to 12.13 µg/m3 for PM2.5 and 7.14 to 26.61 µg/m3 for PM10. Conventional wells metrics were more strongly correlated with PM10 estimations (ρ between 0.28 and 0.79). Unconventional wells metrics for all phases were positively correlated with PM2.5 estimations (ρ between 0.23 and 0.55). These results provide evidence of a correlation between density and proximity of oil and gas wells and estimated PM exposure in the EXPERIVA participants.

Assessing gestational exposure to trace elements in an area of unconventional oil and gas activity: comparison with reference populations and evaluation of variability.

BACKGROUND: Located in Northeastern British Columbia, the Montney formation is an important area of unconventional oil and gas exploitation, which can release contaminants like trace elements. Gestational exposure to these contaminants may lead to deleterious developmental effects. OBJECTIVES: Our study aimed to (1) assess gestational exposure to trace elements in women living in this region through repeated urinary measurements; (2) compare urinary concentrations to those from North American reference populations; (3) compare urinary concentrations between Indigenous and non-Indigenous participants; and (4) evaluate inter- and intra-individual variability in urinary levels. METHODS: Eighty-five pregnant women participating in the Exposures in the Peace River Valley (EXPERIVA) study provided daily spot urine samples over 7 consecutive days. Samples were analyzed for 20 trace elements using inductively-coupled mass spectrometry (ICP-MS). Descriptive statistics were calculated, and inter- and intra-individual variability in urinary levels was evaluated through intraclass correlation coefficient (ICC) calculation for each trace element. RESULTS: When compared with those from North American reference populations, median urinary levels were higher in our population for barium (~2 times), cobalt (~3 times) and strontium (~2 times). The 95th percentile of reference populations was exceeded at least 1 time by a substantial percentage of participants during the sampling week for barium (58%), cobalt (73%), copper (29%), manganese (28%), selenium (38%), strontium (60%) and vanadium (100%). We observed higher urinary manganese concentrations in self-identified Indigenous participants (median: 0.19 µg/g creatinine) compared to non-Indigenous participants (median: 0.15 µg/g of creatinine). ICCs varied from 0.288 to 0.722, indicating poor to moderate reliability depending on the trace element. SIGNIFICANCE: Our results suggest that pregnant women living in this region may be more exposed to certain trace elements (barium, cobalt, copper, manganese, selenium, strontium, and vanadium), and that one urine spot sample could be insufficient to adequately characterize participants' exposure to certain trace elements. IMPACT STATEMENT: Unconventional oil and gas (UOG) is an important industry in the Peace River Valley region (Northeastern British Columbia, Canada). Information on the impacts of this industry is limited, but recent literature emphasizes the risk of environmental contamination. The results presented in this paper highlight that pregnant women living near UOG wells in Northeastern British Columbia may be more exposed to some trace elements known to be related to this industry compared to reference populations. Furthermore, our results based on repeated urinary measurements show that one urine sample may be insufficient to adequately reflect long-term exposure to certain trace elements.

Proximity and density of unconventional natural gas wells and mental illness and substance use among pregnant individuals: An exploratory study in Canada.

BACKGROUND: Hydraulic fracturing (fracking) is a method used to extract unconventional natural gas (UNG). Living near UNG operations has been associated with various health outcomes, but few have explored the association between UNG and mental health and substance use. Our objective was to evaluate the association between metrics of residential UNG well density/proximity and mental illness and substance use among pregnant individuals in Northeastern British Columbia, Canada. METHODS: Individuals who gave birth at the Fort St John hospital between December 30, 2006 and December 29, 2016 (n = 6278) were included in the study. Exposure was determined using inverse distance weighting (IDW) to calculate the density and proximity of UNG wells to the postal code centroid ofindividual's residential address at delivery. Four exposure metrics, categorized by quartiles, were calculated based on 50, 10, 5 and 2.5 km buffer zones around each postal code centroid. Logistic regression was used to separately evaluate associations between IDW quartiles of each metric and diagnosis of depression and anxiety prior to or during pregnancy, and self-reported substance use during pregnancy, controlling for relevant and available confounders. RESULTS: The second and third quartile (Q) of the 10 km IDW were associated with greater odds of depression (Q2: adjusted (aOR) 1.30, 95% (confidence interval) CI 1.03-1.64; Q3: aOR 1.35, 95% CI 1.07-1.70) compared to the first quartile, but not the fourth. Using the 5 km IDW, we observed a suggestive positive association with depression in the second and third quartile (aOR Q2: 1.21, 95% CI 0.96-1.53; aOR Q3: 1.24, 95% CI 0.98-1.57) compared to the first quartile. No statistically significant association was observed using the 2.5 km IDW exposure metric. CONCLUSION: We observed some evidence of greater odds of mental illness prior to or during pregnancy, and substance use during pregnancy in pregnant individuals living in postal codes with increased UNG well density/proximity, although associations were not observed in smaller buffer zones. This study adds to the growing literature on the adverse health outcomes surrounding living in proximity to UNG operations.

Volatile organic compounds (VOCs) in indoor air and tap water samples in residences of pregnant women living in an area of unconventional natural gas operations: Findings from the EXPERIVA study.

BACKGROUND: Northeastern British Columbia (Canada) is an area of unconventional natural gas (UNG) exploitation by hydraulic fracturing, which can release several contaminants, including volatile organic compounds (VOCs). To evaluate gestational exposure to contaminants in this region, we undertook the Exposures in the Peace River Valley (EXPERIVA) study. OBJECTIVES: We aimed to: 1) measure VOCs in residential indoor air and tap water from EXPERIVA participants; 2) compare concentrations with those in the general population and explore differences related to sociodemographic and housing characteristics; and 3) determine associations between VOC concentrations and density/proximity to UNG wells. METHODS: Eighty-five pregnant women participated. Passive air samplers were analyzed for 47 VOCs, and tap water samples were analyzed for 44 VOCs. VOC concentrations were compared with those from the Canadian Health Measure Survey (CHMS). We assessed the association between different metrics of well density/proximity and indoor air and tap water VOC concentrations using multiple linear regression. RESULTS: 40 VOCs were detected in >50% of air samples, whereas only 4 VOCs were detected in >50% of water samples. We observed indoor air concentrations >95th percentile of CHMS in 10-60% of samples for several compounds (acetone, 2-methyl-2-propanol, chloroform, 1,4-dioxane, hexanal, m/p-xylene, o-xylene, styrene, decamethylcyclopentasiloxane, dodecane and decanal). Indoor air levels of chloroform and tap water levels of total trihalomethanes were higher in Indigenous participants compared to non-Indigenous participants. Indoor air levels of chloroform and acetone, and tap water levels of total trihalomethanes were positively associated with UNG wells density/proximity metrics. Indoor air BTEX (benzene, toluene, ethylbenzene, xylenes) levels were positively correlated with some well density/proximity metrics. CONCLUSION: Our results suggest higher exposure to certain VOCs in pregnant women living in an area of intense unconventional natural gas exploitation compared with the general Canadian population, and that well density/proximity is associated with increased exposure to certain VOCs.

Exposure to benzene, toluene and polycyclic aromatic hydrocarbons in Nunavimmiut aged 16 years and over (Nunavik, Canada) - Qanuilirpitaa 2017 survey.

There are numerous volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) that Inuit may be exposed to from combustion, cooking, heating, vehicle exhaust, active and passive smoking and other local sources of contaminants such as oil spills or open-air burning in landfills. To better assess the levels of exposure to these non-persistent chemicals, we measured a suite of benzene, toluene (two VOCs) and PAHs metabolites in pooled urine samples from youth and adults aged 16 years old and over who participated in the Qanuilirpitaa? 2017 Inuit Health Survey (Q2017), a population health survey conducted in Nunavik. A cost-effective pooling strategy was established and 30 different pools from individual urine samples (n = 1266) were created by grouping individual urine samples by sex, age groups and regions. To assess smoking and exposure to second-hand smoke, cotinine levels were measured in individual urine samples. We found that benzene, toluene, all detected PAHs metabolites and cotinine levels were significantly higher in Q2017 compared to adults in the Canadian Health Measure Survey Cycle 4 (2014-2015) or the general U.S population (2015-2016). Moreover, mean levels of one benzene metabolite, S-phenylmercapturic acid, and several PAHs metabolites, 1-naphthol, 2-and 3-hydroxyfluorene, and 4- and 9-hydroxyphenanthrene, known to be associated with smoking habits, were higher in Q2017 compared to reference values (RV95) established for non-smokers in the general Canadian population. Furthermore, benzene and PAHs metabolites were all correlated with cotinine levels. Our results suggest that the high smoking prevalence in Nunavik is an important contributor to the elevated benzene and PAHs exposure. Other local sources may add to that exposure, although we were not able to account for their contribution. These data highlight the importance of regional and community efforts for reducing smoking and to encourage smoke-free homes in Nunavik, while continuing to investigate and reduce other possible local sources of exposure to benzene, toluene and PAHs.

Effects of Neonicotinoid Pesticides on Promoter-Specific Aromatase (CYP19) Expression in Hs578t Breast Cancer Cells and the Role of the VEGF Pathway.

BACKGROUND: Aromatase (CYP19) is a key enzyme in estrogens biosynthesis. In the mammary gland, CYP19 gene is expressed at low levels under the regulation of its I.4 promoter. In hormone-dependent breast cancer, fibroblast cells surrounding the tumor express increased levels of CYP19 mRNA due to a decrease of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter activity. Little is known about the effects of environmental chemicals on the promoter-specific CYP19 expression. OBJECTIVE: We aimed to determine the effects of two neonicotinoids (thiacloprid and imidacloprid) on promoter-specific CYP19 expression in Hs578t breast cancer cells and understand the signaling pathways involved. METHODS: Hs578t cells were exposed to various signaling pathway stimulants or neonicotinoids for 24 h. Promoter-specific expression of CYP19 was determined by real-time quantitative polymerase chain reaction and catalytic activity of aromatase by tritiated water release assay. RESULTS: To our knowledge, we are the first to demonstrate that the normal I.4 promoter and the breast cancer-relevant PII, I.3, and I.7 promoters of CYP19 are active in these cells. We found that the expression of CYP19 via promoters PII, I.3, and I.7 in Hs578t cells was, in part, dependent on the activation of two VEGF signaling pathways: mitogen-activated protein kinase (MAPK) 1/3 and phospholipase C (PLC). Exposure of Hs578t cells to environmental concentrations of imidacloprid and thiacloprid resulted in a switch in CYP19 promoter usage, involving inhibition of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter-mediated CYP19 expression and aromatase catalytic activity. Greater effects were seen at lower concentrations. Our results suggest that thiacloprid and imidacloprid exert their effects at least partially by inducing the MAPK 1/3 and/or PLC pathways. CONCLUSIONS: We demonstrated in vitro that neonicotinoids may stimulate a change in CYP19 promoter usage similar to that observed in patients with hormone-dependent breast cancer. https://doi.org/10.1289/EHP2698.

The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production.

Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy.

Effects of Neonicotinoids on Promoter-Specific Expression and Activity of Aromatase (CYP19) in Human Adrenocortical Carcinoma (H295R) and Primary Umbilical Vein Endothelial (HUVEC) Cells.

The enzyme aromatase (CYP19; cytochrome P450 19) in humans undergoes highly tissue- and promoter-specific regulation. In hormone-dependent breast cancer, aromatase is over-expressed via several normally inactive promoters (PII, I.3, I.7). Aromatase biosynthesizes estrogens, which stimulate breast cancer cell proliferation. The placenta produces estrogens required for healthy pregnancy and the major placental CYP19 promoter is I.1. Exposure to certain pesticides, such as atrazine, is associated with increased CYP19 expression, but little is known about the effects of neonicotinoid insecticides on CYP19. We developed sensitive and robust RT-qPCR methods to detect the promoter-specific expression of CYP19 in human adrenocortical carcinoma (H295R) and primary umbilical vein endothelial (HUVEC) cells, and determined the potential promoter-specific disruption of CYP19 expression by atrazine and the commonly used neonicotinoids imidacloprid, thiacloprid, and thiamethoxam. In H295R cells, atrazine concentration-dependently increased PII- and I.3-mediated CYP19 expression and aromatase catalytic activity. Thiacloprid and thiamethoxam induced PII- and I.3-mediated CYP19 expression and aromatase activity at relatively low concentrations (0.1-1.0 µM), exhibiting non-monotonic concentration-response curves with a decline in gene induction and catalytic activity at higher concentrations. In HUVEC cells, atrazine slightly induced overall (promoter-indistinct) CYP19 expression (30 µM) and aromatase activity (≥ 3 µM), without increasing I.1 promoter activity. None of the neonicotinoids increased CYP19 expression or aromatase activity in HUVEC cells. Considering the importance of promoter-specific (over)expression of CYP19 in disease (breast cancer) or during sensitive developmental periods (pregnancy), our newly developed RT-qPCR methods will be helpful tools in assessing the risk that neonicotinoids and other chemicals may pose to exposed women.