A pilot study of chlormequat in food and urine from adults in the United States from 2017 to 2023.

Chlormequat chloride is a plant growth regulator whose use on grain crops is on the rise in North America. Toxicological studies suggest that exposure to chlormequat can reduce fertility and harm the developing fetus at doses lower than those used by regulatory agencies to set allowable daily intake levels. Here we report, the presence of chlormequat in urine samples collected from people in the U.S., with detection frequencies of 69%, 74%, and 90% for samples collected in 2017, 2018-2022, and 2023, respectively. Chlormequat was detected at low concentrations in samples from 2017 through 2022, with a significant increase in concentrations for samples from 2023. We also observed high detection frequencies of chlormequat in oat-based foods. These findings and chlormequat toxicity data raise concerns about current exposure levels, and warrant more expansive toxicity testing, food monitoring, and epidemiological studies to assess health effects of chlormequat exposures in humans. IMPACT: This study reports the detection of chlormequat, an agricultural chemical with developmental and reproductive toxicity, in the U.S. population and U.S. food supplies for the first time. While similar levels of the chemical were found in urine sampled from 2017 to 2022, markedly increased levels were found in samples from 2023. This work highlights the need for more expansive monitoring of chlormequat in U.S. foods and in human specimens, as well as toxicological and epidemiological study on chlormequat, as this chemical is an emerging contaminant with documented evidence of low-dose adverse health effects in animal studies.

Volatile organic compounds emitted by conventional and "green" cleaning products in the U.S. market.

Exposure to cleaning products has been associated with harm to the respiratory system, neurotoxicity, harm to the reproductive system, and elevated risk of cancer, with greatest adverse impacts for workers exposed in an occupational setting. Social and consumer interest in cleaning products that are safer for health created a market category of "green" products defined here as products advertised as healthier, non-toxic, or free from harmful chemicals as well as products with a third-party certification for safety or environmental features. In the present study we examined the air quality impacts of cleaning products and air fresheners, measuring the number, concentrations, and emission factors of volatile organic compounds (VOCs) in an air chamber following product application. Across seven common product categories, 30 products were tested overall including 14 conventional, 9 identified as "green" with fragrance, and 7 identified as "green" and fragrance-free. A total of 530 unique VOCs were quantified with 205 additional VOCs detected below the limits of quantification. Of the quantifiable VOCs, 193 were considered hazardous according to either the California's Department of Toxic Substances Control Candidate Chemicals List or the European Chemical Agency's Classification and Labeling Inventory. The total concentration of VOCs and total emission factors across all products with detections ranged from below limits of detection to 18,708 µg/m3, 38,035 µg/g product and 3803 µg/application. Greater total concentration, total emission factors, and numbers of VOCs were generally observed in conventional cleaning products compared to products identified as "green", particularly compared to fragrance-free products. A hazard index approach was utilized to assess relative risk from measured VOC emissions. The five products with the highest hazard indices were conventional products with emissions of 2-butoxyethanol, isopropanol, toluene and chloroform. Overall, this analysis suggests that the use of "green" cleaning products, especially fragrance-free products, may reduce exposure to VOC emissions.

Discussion. Has the human population become a sentinel for the adverse effects of PFAS contamination on wildlife health and endangered species?

Global contamination with per- and polyfluoroalkyl substances (PFAS) poses a threat to both human health and the environment, with significant implications for ecological conservation policies. A growing list of peer-reviewed publications indicates that PFAS can harm wildlife health and that the adverse effects associated with PFAS exposure in wildlife are in concordance with human epidemiological studies. The correlation of cross-species data supports a unique perspective that humans can be regarded as a sentinel for PFAS effects in other species. The health harms due to PFAS are potentially most concerning for populations of endangered and threatened species that are simultaneously exposed to PFAS and other toxic pollutants, and also face threats to their survival due to habitat loss, degradation of ecosystems, and over-harvesting. Human epidemiological studies on the PFAS doses associated with health harm present a rich source of information about potential impacts on wildlife health due to PFAS. Our analysis suggests that national and international efforts to restrict the discharges of PFAS into the environment and to clean up PFAS-contaminated sites present an opportunity to protect wildlife from chemical pollution and to advance species conservation worldwide.

Racial and social disparities in Ventura County, California related to agricultural pesticide applications and toxicity.

Application of agricultural pesticides poses health concerns for farmworkers and for local communities due to pesticide drift from spraying or fumigation, pesticide volatilization into the air, contamination of household dust, as well as direct exposure for people who work in agriculture and their families. In this analysis of pesticide use records for Ventura County, California (USA) from 2016 to 2018, we identified the most prevalent toxicological effects of the pesticides applied. We also developed a cumulative toxicity index that incorporates specific toxicity endpoints for individual pesticides, the severity and strength of association for each endpoint, and the reliability of the data sources. Combining the toxicity index for each pesticide with the pounds applied within each square mile section in Ventura County, we calculated the total toxicity-weighted pesticide use and identified pesticides associated with higher potential risk to health. Analysis of U.S. Census data for Ventura County found a greater percentage of Hispanic/Latino, African American and Asian community members in township sections with a greater volume of pesticides applied and higher toxicity-weighted pesticide use. Similarly, areas with limited economic and social resources had elevated pesticide application overall and elevated toxicity-weighted pesticide use. The combination of toxicological and demographic analyses presented in this study provides information that can support the development of policies to protect public health from excessive exposure to pesticides and better environmental health protection for socially vulnerable populations.

Laboratory testing of sunscreens on the US market finds lower in vitro SPF values than on labels and even less UVA protection.

BACKGROUND: New research has attributed increased significance to the causal link between ultraviolet A (UVA) radiation and immunosuppression and carcinogenesis. In the United States, sunscreens are labeled with only their sun protection factor (SPF) and an imprecise term "broad-spectrum protection." Sunscreen marketing and efficacy evaluations continue to be based primarily on skin redness (sunburn) or erythema. We sought to evaluate the ultraviolet (UV) protection offered by common sunscreen products on the US market using laboratory-measured UV-absorption testing and comparing with computer-modeled protection and the labeled SPF values. This approach enables an investigation of the relationship between the labeled SPF and measured UVA protection, a factor that is ignored in current regulations. METHODS: Fifty-one sunscreen products for sale in the United States with SPF values from 15 to 110 and labeled as providing broad-spectrum protection were tested using a commercial laboratory. All products were evaluated using the ISO 24443:2012 method for sunscreen effectiveness. The final absorbance spectra were used for analysis of in vitro UV protection. RESULTS: In vitro SPF values from laboratory-measured UV absorption and computer modeling were on average just 59 and 42 percent of the labeled SPF. The majority of products provided significantly lower UVA protection with the average unweighted UVA protection factor just 24 percent of the labeled SPF. CONCLUSION: Regulations and marketplace forces promote sunscreens that reduce sunburn instead of products that provide better, more broad-spectrum UV protection. The production and use of products with broad spectrum UV protection should be incentivized, removing the emphasis on sunburn protection and ending testing on people.

Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food.

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

Application of the Key Characteristics of Carcinogens to Per and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) constitute a large class of environmentally persistent chemicals used in industrial and consumer products. Human exposure to PFAS is extensive, and PFAS contamination has been reported in drinking water and food supplies as well as in the serum of nearly all people. The most well-studied member of the PFAS class, perfluorooctanoic acid (PFOA), induces tumors in animal bioassays and has been associated with elevated risk of cancer in human populations. GenX, one of the PFOA replacement chemicals, induces tumors in animal bioassays as well. Using the Key Characteristics of Carcinogens framework for cancer hazard identification, we considered the existing epidemiological, toxicological and mechanistic data for 26 different PFAS. We found strong evidence that multiple PFAS induce oxidative stress, are immunosuppressive, and modulate receptor-mediated effects. We also found suggestive evidence indicating that some PFAS can induce epigenetic alterations and influence cell proliferation. Experimental data indicate that PFAS are not genotoxic and generally do not undergo metabolic activation. Data are currently insufficient to assess whether any PFAS promote chronic inflammation, cellular immortalization or alter DNA repair. While more research is needed to address data gaps, evidence exists that several PFAS exhibit one or more of the key characteristics of carcinogens.

Increased adiposity, inflammation, metabolic disruption and dyslipidemia in adult male offspring of DOSS treated C57BL/6 dams.

Evidence indicates that obesity can be promoted by chemical 'obesogens' that drive adiposity, hunger, inflammation and suppress metabolism. Dioctyl sodium sulfosuccinate (DOSS), a lipid emulsifier and candidate obesogen in vitro, is widely used in processed foods, cosmetics and as stool softener medicines commonly used during pregnancy. In vivo testing of DOSS was performed in a developmental origins of adult obesity model. Pregnant mice were orally administered vehicle control or DOSS at times and doses comparable to stool softener use during human pregnancy. All weaned offspring consumed only standard diet. Adult male but not female offspring of DOSS-treated dams showed significantly increased body mass, overall and visceral fat masses, and decreased bone area. They exhibited significant decreases in plasma adiponectin and increases in leptin, glucose intolerance and hyperinsulinemia. Inflammatory IL-6 was elevated, as was adipose Cox2 and Nox4 gene expressions, which may be associated with promoter DNA methylation changes. Multiple significant phospholipid/sterol lipid increases paralleled profiles from long-term high-fat diet induced obesity in males. Collectively, developmental DOSS exposure leads to increased adult adiposity, inflammation, metabolic disorder and dyslipidemia in offspring fed a standard diet, suggesting that pharmaceutical and other sources of DOSS taken during human pregnancy might contribute to long-term obesity-related health concerns in offspring.

The commonly used nonionic surfactant Span 80 has RXRα transactivation activity, which likely increases the obesogenic potential of oil dispersants and food emulsifiers.

Obesity has reached pandemic proportions, and there is mounting evidence that environmental exposures to endocrine disrupting chemicals known as "obesogens" may contribute to obesity and associated medical conditions. The Deepwater Horizon (DWH) oil spill resulted in a massive environmental release of crude oil and remediation efforts applied large quantities of Corexit dispersants to the oil spill. The Corexit-enhanced Water Accommodated Fraction (CWAF) of DWH crude oil contains PPARγ transactivation activity, which is attributed to dioctyl sodium sulfosuccinate (DOSS), a probable obesogen. In addition to its use in oil dispersants, DOSS is commonly used as a stool softener and food additive. Because PPARγ functions as a heterodimer with RXRα to transcriptionally regulate adipogenesis we investigated the potential of CWAF to transactivate RXRα and herein demonstrated that the Corexit component Span 80 has RXRα transactivation activity. Span 80 bound to RXRα in the low micromolar range and promoted adipocyte differentiation of 3T3-L1 preadipocytes. Further, the combination of DOSS and Span 80 increased 3T3-L1 adipocyte differentiation substantially more than treatment with either chemical individually, likely increasing the obesogenic potential of Corexit dispersants. From a public health standpoint, the use of DOSS and Span 80 as food additives heightens concerns regarding their use and mandates further investigations.

Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen.

BACKGROUND: The obesity pandemic is associated with multiple major health concerns. In addition to diet and lifestyle, there is increasing evidence that environmental exposures to chemicals known as obesogens also may promote obesity. OBJECTIVES: We investigated the massive environmental contamination resulting from the Deepwater Horizon (DWH) oil spill, including the use of the oil dispersant COREXIT in remediation efforts, to determine whether obesogens were released into the environment during this incident. We also sought to improve the sensitivity of obesogen detection methods in order to guide post-toxicological chemical assessments. METHODS: Peroxisome proliferator-activated receptor gamma (PPARγ) transactivation assays were used to identify putative obesogens. Solid-phase extraction (SPE) was used to sub-fractionate the water-accommodated fraction generated by mixing COREXIT, cell culture media, and DWH oil (CWAF). Liquid chromatography-mass spectrometry (LC-MS) was used to identify components of fractionated CWAF. PPAR response element (PPRE) activity was measured in PPRE-luciferase transgenic mice. Ligand-binding assays were used to quantitate ligand affinity. Murine 3T3-L1 preadipocytes were used to assess adipogenic induction. RESULTS: Serum-free conditions greatly enhanced the sensitivity of PPARγ transactivation assays. CWAF and COREXIT had significant dose-dependent PPARγ transactivation activities. From SPE, the 50:50 water:ethanol volume fraction of CWAF contained this activity, and LC-MS indicated that major components of COREXIT contribute to PPARγ transactivation in the CWAF. Molecular modeling predicted several components of COREXIT might be PPARγ ligands. We classified dioctyl sodium sulfosuccinate (DOSS), a major component of COREXIT, as a probable obesogen by PPARγ transactivation assays, PPAR-driven luciferase induction in vivo, PPARγ binding assays (affinity comparable to pioglitazone and arachidonic acid), and in vitro murine adipocyte differentiation. CONCLUSIONS: We conclude that DOSS is a putative obesogen worthy of further study, including epidemiological and clinical investigations into laxative prescriptions consisting of DOSS. CITATION: Temkin AM, Bowers RR, Magaletta ME, Holshouser S, Maggi A, Ciana P, Guillette LJ, Bowden JA, Kucklick JR, Baatz JE, Spyropoulos DD. 2016. Effects of crude oil/dispersant mixture and dispersant components on PPARγ activity in vitro and in vivo: identification of dioctyl sodium sulfosuccinate (DOSS; CAS #577-11-7) as a probable obesogen. Environ Health Perspect 124:112-119; http://dx.doi.org/10.1289/ehp.1409672.

Induced pluripotent stem cell technology and aquatic animal species.

Aquatic animal species are the overall leaders in the scientific investigation of tough but important global health issues, including environmental toxicants and climate change. Historically, aquatic animal species also stand at the forefront of experimental biology, embryology and stem cell research. Over the past decade, intensive and high-powered investigations principally involving mouse and human cells have brought the generation and study of induced pluripotent stem cells (iPSCs) to a level that facilitates widespread use in a spectrum of species. A review of key features of these investigations is presented here as a primer for the use of iPSC technology to enhance ongoing aquatic animal species studies. iPSC and other cutting edge technologies create the potential to study individuals from "the wild" closer to the level of investigation applied to sophisticated inbred mouse models. A wide variety of surveys and hypothesis-driven investigations can be envisioned using this new capability, including comparisons of organism-specific development and exposure response and the testing of fundamental dogmas established using inbred mice. However, with these new capabilities, also come new criteria for rigorous baseline assessments and testing. Both the methods for inducing pluripotency and the source material can negatively impact iPSC quality and bourgeoning applications. Therefore, more rigorous strategies not required for inbred mouse models will have to be implemented to approach global health issues using individuals from "the wild" for aquatic animal species.

Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

HDAC inhibitors and decitabine are highly synergistic and associated with unique gene-expression and epigenetic profiles in models of DLBCL.

Interactions between histone deacetylase inhibitors (HDACIs) and decitabine were investigated in models of diffuse large B-cell lymphoma (DLBCL). A number of cell lines representing both germinal center B-like and activated B-cell like DLBCL, patient-derived tumor cells and a murine xenograft model were used to study the effects of HDACIs and decitabine in this system. All explored HDACIs in combination with decitabine produced a synergistic effect in growth inhibition and induction of apoptosis in DLBCL cells. This effect was time dependent, mediated via caspase-3 activation, and resulted in increased levels of acetylated histones. Synergy in inducing apoptosis was confirmed in patient-derived primary tumor cells treated with panobinostat and decitabine. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. We analyzed the molecular basis for this synergistic effect by evaluating gene-expression and methylation patterns using microarrays, with validation by bisulfite sequencing. These analyses revealed differentially expressed genes and networks identified by each of the single treatment conditions and by the combination therapy to be unique with few overlapping genes. Among the genes uniquely altered by the combination of panobinostat and decitabine were VHL, TCEB1, WT1, and DIRAS3.