The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

The Effect of Pesticide Spray Season and Residential Proximity to Agriculture on Glyphosate Exposure among Pregnant People in Southern Idaho, 2021.

BACKGROUND: Glyphosate is one of the most heavily used pesticides in the world, but little is known about sources of glyphosate exposure in pregnant people living in agricultural regions. OBJECTIVE: Our objective was to evaluate glyphosate exposure during pregnancy in relation to residential proximity to agriculture as well as agricultural spray season. METHODS: We quantified glyphosate concentrations in 453 urine samples collected biweekly from a cohort of 40 pregnant people in southern Idaho from February through December 2021. We estimated each participant's glyphosate exposure as the geometric mean (GM) of glyphosate concentrations measured in all samples (average n=11 samples/participant), as well as the GM of samples collected during the pesticide "spray season" (defined as those collected 1 May-15 August; average n=5 samples/participant) and the "nonspray season" (defined as those collected before 1 May or after 15 August; average n=6 samples/participant). We defined participants who resided <0.5km from an actively cultivated agriculture field to live "near fields" and those residing ≥0.5km from an agricultural field to live "far from fields" (n=22 and 18, respectively). RESULTS: Among participants living near fields, urinary glyphosate was detected more frequently and at significantly increased GM concentrations during the spray season in comparison with the nonspray season (81% vs. 55%; 0.228µg/L vs. 0.150µg/L, p<0.001). In contrast, among participants who lived far from fields, neither glyphosate detection frequency nor GMs differed in the spray vs nonspray season (66% vs. 64%; 0.154µg/L vs. 0.165µg/L, p=0.45). Concentrations did not differ by residential proximity to fields during the nonspray season (0.154µg/L vs. 0.165µg/L, for near vs. far, p=0.53). DISCUSSION: Pregnant people living near agriculture fields had significantly increased urinary glyphosate concentrations during the agricultural spray season than during the nonspray season. They also had significantly higher urinary glyphosate concentrations during the spray season than those who lived far from agricultural fields at any time of year, but concentrations did not differ during the nonspray season. These findings suggest that agricultural glyphosate spray is a source of exposure for people living near fields. https://doi.org/10.1289/EHP12768.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of ß-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling ßAS3-globin transgene is potentially curative for all ß-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for ßAS3-globin addition and evaluates strategies for an increased ß-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-ßAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior ß-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent ßAS3-globin expression. LVs were initially compared for their ability to achieve high ß-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that ß-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased ß-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with ßAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-ßAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-ßAS3 for the treatment of severe ß-hemoglobinopathies.

Effect of perinatal exposure to glyphosate and its mixture with 2,4-D and dicamba on rat dam kidney and thyroid function and offspring's health.

The increasing use of the herbicide mixture of glyphosate, dicamba and 2-4-D to deal with glyphosate-resistant weeds raises concerns regarding human health and environmental risks. This study aimed to evaluate the effects of developmental exposure to glyphosate and a herbicide mixture containing glyphosate, dicamba and 2-4-D on rat dams' kidney and thyroid function and offspring's health. Pregnant Wistar rats were exposed from day-6 of gestation till weaning to regulatory relevant doses of glyphosate corresponding to the European Union (EU) acceptable daily intake (ADI; 0.5 mg/kg bw/day), and the no-observed-adverse-effect level (NOAEL; 50 mg/kg bw/day), and to a mixture of glyphosate, dicamba and 2,4-D all at the EU ADI (0.5, 0.002 and 0.3 mg/kg bw/day) respectively. After weaning the dams were sacrificed and blood and organs were collected. The pups' health was assessed by measuring viability, gestational and anogenital indices. Perinatal exposure to GLY alone and the herbicide mixture resulted in anti-androgenic effects in male offspring. In dams, exposure to glyphosate resulted in kidney glomerular and tubular dysfunction as well as increased thyroid hormone levels in a dose-dependent manner. Furthermore, exposure to the herbicide mixture resulted in effects similar to those observed with glyphosate at the NOAEL, suggesting at least an additive effect of the herbicide mixture at doses individually considered safe for humans.

Urinary Glyphosate Concentrations among Pregnant Participants in a Randomized, Crossover Trial of Organic and Conventional Diets.

BACKGROUND: Consumption of an organic diet reduces exposure to a range of agricultural pesticides. Only three studies have examined the effect of an organic diet intervention on exposure to the herbicide glyphosate, the most heavily used agricultural chemical in the world. Despite its widespread use, the primary sources of glyphosate exposure in humans are poorly understood. OBJECTIVE: Our objective was to examine the effect of an organic diet intervention on urinary glyphosate concentrations among pregnant individuals. METHODS: We conducted a 2-wk randomized crossover trial in which 39 pregnant participants living near (≤0.5km) and far (>0.5km) from agricultural fields received a 1-wk supply of conventional groceries and 1 wk of organic groceries, randomized to order. We collected daily first morning void urine samples and analyzed composite samples from each week for glyphosate. We examined differences in urinary glyphosate concentrations between the conventional week and the organic week among all participants and stratified by residential proximity to an agricultural field. RESULTS: Median specific gravity-adjusted glyphosate concentrations were 0.19µg/L and 0.16µg/L during the conventional and organic weeks, respectively. We observed modest decreases in urinary glyphosate concentrations from the conventional to organic week among far-field participants, but no difference among near-field participants. In secondary analyses excluding participants who did not meet a priori criteria of compliance with the intervention, we observed significant decreases in urinary glyphosate concentrations, particularly among far-field participants (p<0.01-0.02, depending on exclusion criteria). DISCUSSION: This trial is the first to examine the effect of an organic diet intervention on glyphosate among people living near and far from agricultural fields. Our results suggest that diet is an important contributor to glyphosate exposure in people living >0.5km from agricultural fields; for people living near crops, agriculture may be a dominant exposure source during the pesticide spray season. https://doi.org/10.1289/EHP12155.

No impacts of glyphosate or Crithidia bombi, or their combination, on the bumblebee microbiome.

Pesticides are recognised as a key threat to pollinators, impacting their health in many ways. One route through which pesticides can affect pollinators like bumblebees is through the gut microbiome, with knock-on effects on their immune system and parasite resistance. We tested the impacts of a high acute oral dose of glyphosate on the gut microbiome of the buff tailed bumblebee (Bombus terrestris), and glyphosate's interaction with the gut parasite (Crithidia bombi). We used a fully crossed design measuring bee mortality, parasite intensity and the bacterial composition in the gut microbiome estimated from the relative abundance of 16S rRNA amplicons. We found no impact of either glyphosate, C. bombi, or their combination on any metric, including bacterial composition. This result differs from studies on honeybees, which have consistently found an impact of glyphosate on gut bacterial composition. This is potentially explained by the use of an acute exposure, rather than a chronic exposure, and the difference in test species. Since A. mellifera is used as a model species to represent pollinators more broadly in risk assessment, our results highlight that caution is needed in extrapolating gut microbiome results from A. mellifera to other bee species.

Comparison of transcriptome alterations induced by pendimethalin or its commercial formulation Stomp Aqua in human MCF-7, MCF-10 A and MCF-12 A mammary epithelial cells.

OBJECTIVE: The toxicology of herbicides, which are currently in use is under-explored. One highly used but under investigated herbicide is pendimethalin. Here we mined high-throughput data from the US National Toxicology Program (NTP) to identify whether pendimethalin possesses an estrogenic capability in human cells. We also evaluated effects of pendimethalin and its reference commercial formulated herbicide Stomp Aqua on the transcriptome profile of three human mammary epithelial cell lines, cancerous MCF-7 and non-cancerous MCF-10 A and MCF-12 A to see whether this compound could have endocrine disrupting effects and if co-formulants present in the commercial formulation could amplify its toxicity. RESULTS: The data mined from the US NTP database suggests that pendimethalin activates estrogen receptors at a concentration of approximately 10?M. MCF-7, MCF-10A and MCF-12A cells were exposed to 10 ?M pendimethalin and Stomp Aqua at an equivalent concentration. Transcriptome analysis showed changes in gene expression patterns implying that pendimethalin affected ubiquitin-mediated proteolysis and the function of the spliceosome. The formulated pendimethalin product Stomp Aqua gave comparable effects suggesting pendimethalin was responsible for the observed transcriptome alterations. Given the lack of information on the exposure to this pesticide, our study prompts the need for biomonitoring studies, especially under occupational use scenarios, to understand if low level exposure to pendimethalin could have endocrine disrupting effects on populations exposed to this compound. A deeper understanding of the exposure and mechanisms of action of this endocrine-disrupting pesticide is needed.

Evaluation of perinatal exposure of glyphosate and its mixture with 2,4-D and dicamba οn liver redox status in Wistar rats.

Wide-scale emergence of glyphosate-resistant weeds has led to an increase in the simultaneous application of herbicide mixtures exacerbated by the introduction of crops tolerant to glyphosate plus dicamba or glyphosate plus 2,4-D. This raises serious concerns regarding the environmental and health risks resulting from increased exposure to a mixture of herbicide active ingredients. We evaluated hepatotoxic effects following perinatal exposure to glyphosate alone or in combination with 2,4-D and dicamba from gestational day-6 until adulthood in Wistar rats. Animals were administered with glyphosate at the European Union (EU) acceptable daily intake (ADI; 0.5 mg/kg bw/day) and no-observed-adverse-effect level (NOAEL; 50 mg/kg bw/day). A mixture of glyphosate with 2,4-D (0.3 mg/kg bw/day) and dicamba (0.02 mg/kg bw/day) with each at their EU ADI was evaluated. Redox status was determined by measuring levels of reduced glutathione, decomposition rate of Η2Ο2, glutathione reductase, glutathione peroxidase, total antioxidant capacity, thiobarbituric reactive substances, and protein carbonyls. Gene expression analysis of Nr1d1, Nr1d2, Clec2g, Ier3, and Gadd45g associated with oxidative damage to DNA, was also performed. Analysis of liver samples showed that exposure to the mixture of the three herbicides induced a marked increase in the concentration of glutathione and malondialdehyde indicative of a disturbance in redox balance. Nevertheless, the effect of increased lipid peroxidation was not discernible following a 3-month recuperation period where animals were withdrawn from pesticide exposure post-weaning. Interestingly, toxic effects caused by prenatal exposure to the glyphosate NOAEL were present after the same 3-month recovery period. No statistically significant changes in the expression of genes linked with genotoxicity were observed. Our findings reinforce the importance of assessing the combined effects of chemical pollutants at doses that are asserted by regulatory agencies to be safe individually.

Unusual Suspects: Bone and Cartilage ECM Proteins as Carcinoma Facilitators.

The extracellular matrix (ECM) is the complex three-dimensional network of fibrous proteins and proteoglycans that constitutes an essential part of every tissue to provide support for normal tissue homeostasis. Tissue specificity of the ECM in its topology and structure supports unique biochemical and mechanical properties of each organ. Cancers, like normal tissues, require the ECM to maintain multiple processes governing tumor development, progression and spread. A large body of experimental and clinical evidence has now accumulated to demonstrate essential roles of numerous ECM components in all cancer types. Latest findings also suggest that multiple tumor types express, and use to their advantage, atypical ECM components that are not found in the cancer tissue of origin. However, the understanding of cancer-specific expression patterns of these ECM proteins and their exact roles in selected tumor types is still sketchy. In this review, we summarize the latest data on the aberrant expression of bone and cartilage ECM proteins in epithelial cancers and their specific functions in the pathogenesis of carcinomas and discuss future directions in exploring the utility of this selective group of ECM components as future drug targets.

Cytotoxicity Mechanisms of Eight Major Herbicide Active Ingredients in Comparison to Their Commercial Formulations.

Commercial pesticide formulations contain co-formulants, which are generally considered as having no toxic effects in mammals. This study aims to compare the toxicity of 8 major herbicide active ingredients-namely glyphosate, dicamba, 2,4-D, fluroxypyr, quizalofop-p-ethyl, pendimethalin, propyzamide and metazachlor-with a typical commercial formulation of each active ingredient. Cytotoxicity and oxidative stress capability was assessed in human hepatoma HepG2 cells. Using an MTT assay, formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) were more toxic than the active ingredient alone. Metazachlor and its formulation Sultan had similar cytotoxicity profiles. Cytotoxicity profiles were comparable in immortalised human fibroblasts. Toxilight necrosis assays showed the formulation of metazachlor (Sultan50C) resulted in significant membrane disruption compared to the active ingredient. Generation of reactive oxygen species was detected for glyphosate, fluroxypyr, pendimethalin, quizalofop-p-ethyl, the formulation of 2,4-D (Anti-Liserons), and dicamba and its formulation Hunter. Further testing of quizalofop-p-ethyl and its formulation Targa Super in the ToxTracker assay system revealed that both products induced oxidative stress and an unfolded protein response. In conclusion, these results show that most herbicide formulations tested in this study are more toxic than their active ingredients in human tissue culture cell model systems. The results add to a growing body of evidence, which implies that commercial herbicide formulations and not just their active ingredients should be evaluated in regulatory risk assessment of pesticides.

Glyphosate and its formulations Roundup Bioflow and RangerPro alter bacterial and fungal community composition in the rat caecum microbiome.

The potential health consequences of glyphosate-induced gut microbiome alterations have become a matter of intense debate. As part of a multifaceted study investigating toxicity, carcinogenicity and multigenerational effects of glyphosate and its commercial herbicide formulations, we assessed changes in bacterial and fungal populations in the caecum microbiota of rats exposed prenatally until adulthood (13 weeks after weaning) to three doses of glyphosate (0.5, 5, 50 mg/kg body weight/day), or to the formulated herbicide products Roundup Bioflow and RangerPro at the same glyphosate-equivalent doses. Caecum bacterial microbiota were evaluated by 16S rRNA sequencing whilst the fungal population was determined by ITS2 amplicon sequencing. Results showed that both fungal and bacterial diversity were affected by the Roundup formulations in a dose-dependent manner, whilst glyphosate alone significantly altered only bacterial diversity. At taxa level, a reduction in Bacteroidota abundance, marked by alterations in the levels of Alloprevotella, Prevotella and Prevotellaceae UCG-003, was concomitant to increased levels of Firmicutes (e.g., Romboutsia, Dubosiella, Eubacterium brachy group or Christensenellaceae) and Actinobacteria (e.g., Enterorhabdus, Adlercreutzia, or Asaccharobacter). Treponema and Mycoplasma also had their levels reduced by the pesticide treatments. Analysis of fungal composition indicated that the abundance of the rat gut commensal Ascomycota Kazachstania was reduced while the abundance of Gibberella, Penicillium, Claviceps, Cornuvesica, Candida, Trichoderma and Sarocladium were increased by exposure to the Roundup formulations, but not to glyphosate. Altogether, our data suggest that glyphosate and its Roundup RangerPro and Bioflow caused profound changes in caecum microbiome composition by affecting the fitness of major commensals, which in turn reduced competition and allowed opportunistic fungi to grow in the gut, in particular in animals exposed to the herbicide formulations. This further indicates that changes in gut microbiome composition might influence the long-term toxicity, carcinogenicity and multigenerational effects of glyphosate-based herbicides.

Glyphosate exposure in early pregnancy and reduced fetal growth: a prospective observational study of high-risk pregnancies.

BACKGROUND: Prenatal glyphosate (GLY) exposure is associated with adverse reproductive outcomes in animal studies. Little is known about the effects of GLY exposure during pregnancy in the human population. This study aims to establish baseline urine GLY levels in a high-risk and racially diverse pregnancy cohort and to assess the relationship between prenatal GLY exposure and fetal development and birth outcomes. METHODS: Random first trimester urine specimens were collected from high risk pregnant women between 2013 and 2016 as part of the Indiana Pregnancy Environmental Exposures Study (PEES). Demographic and clinical data were abstracted from mother and infant medical records. Urine glyphosate levels were measured as a proxy for GLY exposure and quantified using liquid chromatography-tandem mass spectrometry. Primary outcome variables included gestation-adjusted birth weight percentile (BWT%ile) and neonatal intensive care unit (NICU) admission. Relationships between primary outcome variables and GLY exposure were assessed using univariate and multivariate linear and logistic regression models. RESULTS: Urine GLY levels above the limit of detection (0.1 ng/mL) were found in 186 of 187 (99%) pregnant women. Further analyses were limited to 155 pregnant women with singleton live births. The mean age of participants was 29 years, and the majority were non-Hispanic white (70%) or non-Hispanic Black (21%). The mean (± SD) urine GLY level was 3.33 ± 1.67 ng/mL. Newborn BWT%iles were negatively related to GLY (adjusted slope ± SE = -0.032 + 0.014, p = 0.023). Infants born to women living outside of Indiana's large central metropolitan area were more likely to have a lower BWT%ile associated with mother's first trimester GLY levels (slope ± SE = -0.064 ± 0.024, p = 0.007). The adjusted odds ratio for NICU admission and maternal GLY levels was 1.16 (95% CI: 0.90, 1.67, p = 0.233). CONCLUSION: GLY was found in 99% of pregnant women in this Midwestern cohort. Higher maternal GLY levels in the first trimester were associated with lower BWT%iles and higher NICU admission risk. The results warrant further investigation on the effects of GLY exposure in human pregnancies in larger population studies.

The surfactant co-formulant POEA in the glyphosate-based herbicide RangerPro but not glyphosate alone causes necrosis in Caco-2 and HepG2 human cell lines and ER stress in the ToxTracker assay.

The toxicity of co-formulants present in glyphosate-based herbicides (GBHs) has been widely discussed leading to the European Union banning the polyoxyethylene tallow amine (POEA). We identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used US GBH RangerPro. Cytotoxicity assays using human intestinal epithelial Caco-2 and hepatocyte HepG2 cell lines showed that RangerPro and POE-15 are far more cytotoxic than glyphosate alone. RangerPro and POE-15 but not glyphosate caused cell necrosis in both cell lines, and that glyphosate and RangerPro but not POE-15 caused oxidative stress in HepG2 cells. We further tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their capability for inducing DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15 but not glyphosate gave rise to ER stress. We conclude that the toxicity resulting from RangerPro exposure is thus multifactorial involving ER stress caused by POE-15 along with oxidative stress caused by glyphosate. Our observations reinforce the need to test both co-formulants and active ingredients of commercial pesticides to inform the enactment of more appropriate regulation and thus better public and environmental protection.

Impacts of dietary exposure to pesticides on faecal microbiome metabolism in adult twins.

BACKGROUND: Dietary habits have a profound influence on the metabolic activity of gut microorganisms and their influence on health. Concerns have been raised as to whether the consumption of foodstuffs contaminated with pesticides can contribute to the development of chronic disease by affecting the gut microbiome. We performed the first pesticide biomonitoring survey of the British population, and subsequently used the results to perform the first pesticide association study on gut microbiome composition and function from the TwinsUK registry. METHODS: Dietary exposure of 186 common insecticide, herbicide, or fungicide residues and the faecal microbiome in 65 twin pairs in the UK was investigated. We evaluated if dietary habits, geographic location, or the rural/urban environment, are associated with the excretion of pesticide residues. The composition and metabolic activity of faecal microbiota was evaluated using shotgun metagenomics and metabolomics respectively. We performed a targeted urine metabolomics analysis in order to evaluate whether pesticide urinary excretion was also associated with physiological changes. RESULTS: Pyrethroid and/or organophosphorus insecticide residues were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that residues from organophosphates were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and faecal metabolite concentrations were detected. Glyphosate excretion was positively associated with an overall increased bacterial species richness, as well as to fatty acid metabolites and phosphate levels. The insecticide metabolite Br2CA, reflecting deltamethrin exposure, was positively associated with the phytoestrogens enterodiol and enterolactone, and negatively associated with some N-methyl amino acids. Urine metabolomics performed on a subset of samples did not reveal associations with the excretion of pesticide residues. CONCLUSIONS: The consumption of conventionally grown fruit and vegetables leads to higher ingestion of pesticides with unknown long-term health consequences. Our results highlight the need for future dietary intervention studies to understand effects of pesticide exposure on the gut microbiome and possible health consequences.

Comparative Toxicogenomics of Glyphosate and Roundup Herbicides by Mammalian Stem Cell-Based Genotoxicity Assays and Molecular Profiling in Sprague-Dawley Rats.

Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remain controversial. As GBHs are more cytotoxic than glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs, namely MON 52276 (European Union), MON 76473 (United Kingdom), and MON 76207 (United States) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (at 0.5, 50, and 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased miR-30, whereas miR-10 levels were increased. DNA methylation profiling of liver revealed 5727 and 4496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals, respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate.

Genotoxicity evaluation of 2,4-D, dicamba and glyphosate alone or in combination with cell reporter assays for DNA damage, oxidative stress and unfolded protein response.

The current generation of carcinogenicity tests is often insufficient to predict cancer outcomes from pesticide exposures. In order to facilitate health risk assessment, The International Agency for Research on Cancer identified 10 key characteristics which are commonly exhibited by human carcinogens. The ToxTracker panel of six validated GFP-based mouse embryonic stem reporter cell lines is designed to measure a number of these carcinogenic properties namely DNA damage, oxidative stress and the unfolded protein response. Here we present an evaluation of the carcinogenic potential of the herbicides glyphosate, 2,4-D and dicamba either alone or in combination, using the ToxTracker assay system. The pesticide 2,4-D was found to be a strong inducer of oxidative stress and an unfolded protein response. Dicamba induced a mild oxidative stress response, whilst glyphosate did not elicit a positive outcome in any of the assays. The results from a mixture of the three herbicides was primarily an oxidative stress response, which was most likely due to 2,4-D with dicamba or glyphosate only playing a minor role. These findings provide initial information regarding the risk assessment of carcinogenic effects arising from exposure to a mixture of these herbicides.

Alterations in small RNA profiles in liver following a subchronic exposure to a low-dose pesticide mixture in Sprague-Dawley rats.

Small RNAs have emerged as a promising new type of biomarker to monitor health status and track the development of diseases. Here we report changes in the levels of small RNAs in the liver of rats exposed to a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole). Multivariate analysis with OPLS-DA methods showed that small RNA profiles can discriminate samples from pesticide treated rats from their concurrent controls. A total of 9 miRNAs were found to have their levels altered in the liver of the pesticide-treated rats in comparison to the controls, which included 7 that were downregulated (miR-22-5p, miR-193a-3p, miR-32-5p, miR-33-5p, miR-122-5p, miR-22-3p, miR-130a-3p) and 2 that were upregulated (miR-486-5p, miR-146a-5p). These miRNAs were predicted to regulate genes, which were found to have their expression altered by the pesticide mixture and have known health implications in the regulation of hepatic metabolism. This supports and extends our recent conclusions that high- throughput 'omics' analyses can reveal molecular perturbations, which can potentially act as sensitive and accurate markers of health risks arising from exposure to environmental pollutants such as pesticides.

Commentary: Novel strategies and new tools to curtail the health effects of pesticides.

BACKGROUND: Flaws in the science supporting pesticide risk assessment and regulation stand in the way of progress in mitigating the human health impacts of pesticides. Critical problems include the scope of regulatory testing protocols, the near-total focus on pure active ingredients rather than formulated products, lack of publicly accessible information on co-formulants, excessive reliance on industry-supported studies coupled with reticence to incorporate published results in the risk assessment process, and failure to take advantage of new scientific opportunities and advances, e.g. biomonitoring and "omics" technologies. RECOMMENDED ACTIONS: Problems in pesticide risk assessment are identified and linked to study design, data, and methodological shortcomings. Steps and strategies are presented that have potential to deepen scientific knowledge of pesticide toxicity, exposures, and risks. We propose four solutions: (1) End near-sole reliance in regulatory decision-making on industry-supported studies by supporting and relying more heavily on independent science, especially for core toxicology studies. The cost of conducting core toxicology studies at labs not affiliated with or funded directly by pesticide registrants should be covered via fees paid by manufacturers to public agencies. (2) Regulators should place more weight on mechanistic data and low-dose studies within the range of contemporary exposures. (3) Regulators, public health agencies, and funders should increase the share of exposure-assessment resources that produce direct measures of concentrations in bodily fluids and tissues. Human biomonitoring is vital in order to quickly identify rising exposures among vulnerable populations including applicators, pregnant women, and children. (4) Scientific tools across disciplines can accelerate progress in risk assessments if integrated more effectively. New genetic and metabolomic markers of adverse health impacts and heritable epigenetic impacts are emerging and should be included more routinely in risk assessment to effectively prevent disease. CONCLUSIONS: Preventing adverse public health outcomes triggered or made worse by exposure to pesticides will require changes in policy and risk assessment procedures, more science free of industry influence, and innovative strategies that blend traditional methods with new tools and mechanistic insights.

Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats.

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.