Combined Exposures and Mixtures Research: An Enduring NIEHS Priority.

BACKGROUND: The National Institute of Environmental Health Sciences (NIEHS) continues to prioritize research to better understand the health effects resulting from exposure to mixtures of chemical and nonchemical stressors. Mixtures research activities over the last decade were informed by expert input during the development and deliberations of the 2011 NIEHS Workshop "Advancing Research on Mixtures: New Perspectives and Approaches for Predicting Adverse Human Health Effects." NIEHS mixtures research efforts since then have focused on key themes including a) prioritizing mixtures for study, b) translating mixtures data from in vitro and in vivo studies, c) developing cross-disciplinary collaborations, d) informing component-based and whole-mixture assessment approaches, e) developing sufficient similarity methods to compare across complex mixtures, f) using systems-based approaches to evaluate mixtures, and g) focusing on management and integration of mixtures-related data. OBJECTIVES: We aimed to describe NIEHS driven research on mixtures and combined exposures over the last decade and present areas for future attention. RESULTS: Intramural and extramural mixtures research projects have incorporated a diverse array of chemicals (e.g., polycyclic aromatic hydrocarbons, botanicals, personal care products, wildfire emissions) and nonchemical stressors (e.g., socioeconomic factors, social adversity) and have focused on many diseases (e.g., breast cancer, atherosclerosis, immune disruption). We have made significant progress in certain areas, such as developing statistical methods for evaluating multiple chemical associations in epidemiology and building translational mixtures projects that include both in vitro and in vivo models. DISCUSSION: Moving forward, additional work is needed to improve mixtures data integration, elucidate interactions between chemical and nonchemical stressors, and resolve the geospatial and temporal nature of mixture exposures. Continued mixtures research will be critical to informing cumulative impact assessments and addressing complex challenges, such as environmental justice and climate change. https://doi.org/10.1289/EHP14340.

Herbicides and pesticides synergistically interact at low concentrations in complex mixtures.

Assessing a complex mixture of pesticides at the impacted sites has been challenging for risk assessors for 50 years. The default assumption is that at low concentrations, pesticides interact additively with one another; thus, the risk posed by each component of a complex mixture could be simply added up. The EPA interaction-based hazard index (HIInteraction) modifies this assumption using a binary weight-of-evidence (BINWOE). However, data gaps often preclude HIInteraction use at most sites. This study evaluated these assumptions using the BINWOE to estimate the hazard index (HI) of select pesticide mixtures. The lack of in vivo binary interaction data led us to use a cell line, SH-SY5Y, to obtain the data necessary for the BINWOE approach. In the risk assessment, we considered the most active exposure scenario inhaling a mixture of volatile pesticides from contaminated soil and groundwater. The potential interactions between pesticides in 15 binary mixtures were investigated using the MTT assay in SH-SY5Y cells. Our findings showed that 60% of the binary mixtures elicited synergism (in at least one concentration), 27% displayed antagonism, and 13% showed additive effects in SH-SY5Y cells. Combining human safety data with in vitro interaction data indicated that adults and toddlers were at the highest risk when considering industrial and commercial land use, respectively, compared to other subpopulations. Incorporating interaction data into the risk assessment either increased the risk by up to 20% or decreased the risk by 2%, depending on the mixture. Our results demonstrate the predominant synergistic interactions, even at low concentrations, altered risk characterization at the complex operating site. Most concerning, organochlorine pesticides with the same mechanism of action did not follow dose additivity when evaluated by SH-SY5Y cell lines. Based on our observations, we caution that current HI methods based on additivity assumptions may underestimate the risk of organochlorine mixtures.

Differential neuroprotective effect of curcuminoid formulations in aluminum chloride-induced Alzheimer's disease.

Alzheimer's disease (AD) is a slowly progressive brain degenerative disorder which gradually impairs memory, thinking, and ability to perform easy routine tasks. This degenerative disorder mainly targets the elderly people and has imposed an endemic burden on society. Hence, there is a crucial need to investigate the efficacious herbal pharmacotherapies that can effectively mitigate and prevent the pathological hallmarks of AD. The current study aims to explore the potential efficacy of curcuminoid-rich extract (CRE) and its ternary complex (TC). Experimental rodents were administered with AlCl3 (300 mg/kg) to induce AD and treated with rivastigmine, curcuminoid crude extract, CRE, and TC orally for three consecutive weeks. Neurobehavioral, biochemical, and histopathological studies were performed from the last week of the study period. The mRNA expression of different pathological biomarkers was estimated by RT-qPCR analysis. The results of the study suggested that CRE and TC significantly improved the behavioral, biochemical parameters and acetylcholinesterase inhibitory activity in treatment groups. Histological analysis was also carried out indicating that the neurodegenerative changes and neuronal loss were stabilized by CRE and TC supplementation. CRE and TC supplementation remarkably downregulated the interleukin-1α, tumor necrosis factor-α, interleukin-1ß, acetylcholinesterase, and ß-secretase pathological gene expression. Hence, it was concluded that CRE and TC may act as promising candidates in the prevention of AD via numerous underlying signaling pathways.

Associations of a metal mixture with iron status in U.S. adolescents: Evidence from the National Health and Nutrition Examination Survey.

Iron is needed for normal development in adolescence. Exposure to individual environmental metals (e.g., lead) has been associated with altered iron status in adolescence, but little is known about the cumulative associations of multiple metals with Fe status. We used data from the 2017-2018 National Health and Nutrition Examination Survey (NHANES) to examine associations between a metal mixture (lead, manganese, cadmium, selenium) and iron status in 588 U.S. adolescents (12-17 years). We estimated cumulative and interactive associations of the metal mixture with five iron status metrics using Bayesian Kernel Machine Regression (BKMR). Higher concentrations of manganese and cadmium were associated with lower log-transformed ferritin concentrations. Interactions were observed between manganese, cadmium, and lead for ferritin and the transferrin receptor, where iron status tended to be worse at higher concentrations of all metals. These results may reflect competition between environmental metals and iron for cellular uptake. Mixed metal exposures may alter normal iron function, which has implications for adolescent development.

Skin irritation and potential antioxidant, anti-collagenase, and anti-elastase activities of edible insect extracts.

This study aimed to investigate antioxidant, anti-aging, and irritation properties of Thai edible insect extracts, including Bombyx mori, Omphisa fuscidentalis, Euconocephalus sp., Patanga succincta, Acheta domesticus, and Lethocerus indicus. Insects were extracted by 2 different methods, including maceration using ethanol or hexane and digestion using DI water. Then the extracts were determined for protein content using bicinchoninic acid assay and antioxidant activities using 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and ferric thiocyanate assays. Anti-aging activities were investigated by determination of collagenase and elastase inhibitory activities using spectrophotometric assay. Maceration by hexane yielded the highest extract content, whereas aqueous extract from digestion possessed the significantly highest protein content and biological activities (p < 0.05). Interestingly, aqueous extracts of A. domesticus possessed the significantly highest biological activities (p < 0.05) with Trolox equivalent antioxidant capacity value of 8.8 ± 0.1 mmol Trolox/mg, DPPH· inhibition of 19.5 ± 3.8%, equivalent concentration of 12.1 ± 0.7 µM FeSO4/mg, lipid peroxidation inhibition of 31.3 ± 2.4%, collagenase inhibition of 60.8 ± 2.1%, elastase inhibition of 17.0 ± 0.1%, and no irritation effect on chorioallantoic membrane and volunteers. Therefore, aqueous extract of A. domesticus would be suggested for further topical product development.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Mutagenicity and genotoxicity assessments of some industrially processed meat products in Algeria.

Processed meat products are presumptive sources of mutagens and genotoxins for consumers. Several epidemiological studies have reported that these products' high intakes have a positive link with cancer incidence. In Algeria, industrially processed meat products are widely consumed. However, there are no earlier studies involving their genotoxic activity. For this end, the current study aimed at evaluating the mutagenicity and the genotoxicity of some representative industrially processed meat products sold in popular supermarkets. All samples were extracted by established method, using both polar and non-polar solvents. The meat extracts mutagenicity was assessed by Ames test, using four strains of Salmonella typhimurium in the presence and absence of metabolic activation, and subsequently by treat and wash assay for extracts showing biologically significant results. The genotoxicity was determined in TK6 human lymphoblastoid cells using the in vitro micronucleus assay in micromethod. The results showed that all extracts analyzed induce no mutagenic activity. However, one of these extracts induced a biologically significant increase in the number of micronucleated cells. Our findings indicate the importance of the genetic damage detection for taking measures to suppress or reduce the exposure to harmful contaminants and encourage further research investigating genotoxic effects of industrially processed meat worldwide.

Acaricidal activity of Furcraea foetida leaf extract against engorged female Rhipicephalus (Boophilus) microplus ticks

The Rhipicephalus (Boophilus) microplus tick is a major concern for the livestock market worldwide, as it causes serious economic damage. Plant-derived acaricides are an attractive alternative to control this ectoparasite and limit the development of resistance. Therefore, the aim of this study was to evaluate the acaricidal activity of Furcraea foetida leaf extract against engorged female R. (B.) microplus ticks. Our in vitro bioassays showed that the crude extract of leaves from F. foetida caused hemorrhagic swelling and skin lesions in the ticks, and three days of treatment caused 100% mortality. Dose-response assay indicated that this toxicity effect was dose-dependent. Similar effects were observed when the crude extract from F. foetida leaves was denatured by boiling at 100°C. These results suggest that the toxicity of the leaf extract might be associated with thermostable biomolecules. Together, our results show for the first time that the crude extract of F. foetida leaves has acaricidal activity against engorged female R. (B.) microplus ticks and it acts in a dose-dependent manner.

Sub-acute oral toxicity study of aqueous extract of tobacco leaves (Nicotiana tabacum L.) on lipid profile, the tissue, and serum of the liver and kidney of male Wistar rats.

CONTEXT: Tobacco consumption may pose a very serious threat to the physiological state of the body; yet, fewer records have been documented in that regard. OBJECTIVE: We investigated the impact of aqueous extract of tobacco leaves on the lipid profile, the tissue, and serum levels of the liver and kidney of male Wister rats. MATERIALS AND METHODS: Rats (n = 52; weight = 33 - 47 g; ∼ 2½ weeks old) were acclimatised for 7 days and administered aqueous extract of tobacco leaves at 100, 200, 400, 0 mg/kg of body weight (to group A, B, C, D) for 30 days. RESULTS: Compared with the control group, the kidney tissue and serum (i.e., urea and creatinine) were not influenced, in contrast, indices of the liver such as AST, ALT, and ALP, dose-dependently increased. Changes such as coagulative necrosis resulted in the infiltration of mononuclear inflammatory cells and the vacuolar degeneration of the liver. Beside the reduction in the high-density lipoprotein of the rats, there was an increase in the concentration of triglycerides, very low-density lipoprotein, low-density lipoprotein, and the total cholesterol. CONCLUSION: Thus, extract of tobacco leaves can greatly influence the body lipid profile, beside the serum and tissues of the liver.

Characterization of Macroscopic Colony-Forming Filamentous Cyanobacteria from Okinawan Coasts as Potential Sources of Bioactive Compounds.

Marine macroscopic colony-forming filamentous (MMCFF) cyanobacteria are considered as prolific producers of bioactive compounds. Thus, knowledge of the diversity of MMCFF cyanobacteria as related to bioactive compound production has become very important. However, basic taxonomic studies of MMCFF cyanobacteria are lacking. Many cyanobacterial taxa are still misidentified or undescribed. In this study, a total of 32 cyanobacterial colonies from nine coastal regions of Okinawa Prefecture were investigated for a diversity assessment. A polyphasic approach including morphological and molecular studies based on 16S rRNA gene sequences was performed to characterize Okinawan MMCFF cyanobacteria. Both morphological and molecular phylogenetic results showed that MMCFF cyanobacteria from Okinawan coasts are very diverse. We found morphotypes of Lyngbya-like, Phormidium-like, and Leptolyngbya-like groups among Okinawan cyanobacterial samples. Genetically, samples were distributed in various clades in the phylogenetic tree, including within Moorena, Okeania, Caldora, Neolyngbya, Dapis, as well as several unknown clades. In addition, cytotoxic activities of three samples from Kiyan coast were tested against HeLa cells. All three crude extracts of these samples showed strong cytotoxic activity with IC50 < 1 µg/ml.

Developmental exposure to a mixture of unconventional oil and gas chemicals: A review of experimental effects on adult health, behavior, and disease.

Unconventional oil and natural gas extraction (UOG) combines directional drilling and hydraulic fracturing and produces billions of liters of wastewater per year. Herein, we review experimental studies that evaluated the potential endocrine-mediated health impacts of exposure to a mixture of 23 UOG chemicals commonly found in wastewater. The purpose of this manuscript is to synthesize and summarize a body of work using the same UOG-mix but with different model systems and physiological endpoints in multiple experiments. The studies reviewed were conducted in laboratory animals (mice or tadpoles) and human tissue culture cells. A key feature of the in vivo studies was the use of four environmentally relevant doses spanning three orders of magnitude ranging from concentrations found in surface and ground water in UOG dense areas to concentrations found in UOG wastewater. This UOG-mix exhibited potent antagonist activity for the estrogen, androgen, glucocorticoid, progesterone, and thyroid receptors in human tissue culture cells. Subsequently, pregnant mice were administered the UOG-mix in drinking water and offspring were examined in adulthood or to tadpoles. Developmental exposure profoundly impacted pituitary hormone concentrations, reduced sperm counts, altered folliculogenesis, and increased mammary gland ductal density and preneoplastic lesions in mice. It also altered energy expenditure, exploratory and risk-taking behavior, the immune system in three immune models in mice, and affected basal and antiviral immunity in frogs. These findings highlight the diverse systems affected by developmental EDC exposure and the need to examine human and animal health in UOG regions.

Defining embryonic developmental effects of chemical mixtures using the embryonic stem cell test.

The embryonic stem cell test (EST) was applied to evaluate dose addition in combined exposures of teratogenic compounds in the EFSA-defined cumulative assessment group "craniofacial malformations", which was one of the selected cases in the EU-H2020 project "EuroMix". Test compounds were selected through reported effects in rodents, and represented a wide variety of chemical families and modes of action (MOA), including triazoles to inhibit CYP26; (synthetic) retinoids, to activate RAR/RXR; valproic acid, to inhibit histone deacetylase; dithiocarbamates, to disrupt extracellular matrix formation; dioxin (-like) compounds, to activate the aryl hydrocarbon receptor; 17alpha-ethynylestradiol, to activate the estrogen receptor; 5-fluorouracil, to disrupt DNA-synthesis; MEHP and PFOS, to activate peroxisome proliferation activated receptors; and methyl mercury, to induce oxidative stress and inhibit protein function. The EST appeared particularly useful to evaluate differentiation-inhibiting effects of compounds targeting early processes in craniofacial development, possibly related to the early fate of neural crest cells. Mixtures, designed as equipotent concentrations of two compounds with similar or dissimilar MOA, and single compounds showed overlapping dose-responses. This observation is consistent with dose addition in the EST in all studied binary mixtures, irrespective of MOA, and thereby supports the application of dose-addition as a default in cumulative risk assessment.

The Cumulative Risk Assessment of Hepatotoxic Chemicals: A Hepatic Histopathology Perspective.

The increased concern on the consequence of exposure to multiple chemical combinations has led national regulatory authorities to develop different concepts to conduct risk assessments on chemical mixtures. Pesticide residues were identified as "problem formulation" in the respective European regulations and in this context, the European Food and Safety Authority has suggested to group pesticidal active ingredients (AIs) into cumulative assessment groups (CAGs) based on the toxicological properties of each AI. One proposed CAG, on the liver, currently consists of 15 subgroups, each representing a specific hepatotoxic effect observed in toxicity studies. Dietary cumulative risk assessments would then have to be conducted assuming dose additivity of all members of each CAG subgroup. The purpose of this publication is to group AIs based upon the knowledge of the pathogenesis of liver effects to discriminate between primary end points (direct consequence of chemical interaction with a biological target) and secondary end points (which are a consequence of, or that arise out of, a previous pathological change). Focusing on the relevant primary end points strengthens and simplifies the selection of compounds for cumulative risk assessment regarding the liver and better rationalizes the basis for chemical grouping. Relevant dose additivity is to be expected at the level of the primary/leading pathological end points and not at the level of the secondary end points. We recognize, however, that special consideration is needed for substances provoking neoplasia, and this category is included in the group of primary end points for which chemicals inducing them are grouped for risk assessment. Using the pathological basis for defining the respective CAGs, 6 liver subgroups and 2 gallbladder/bile duct groups are proposed. This approach simplifies the cumulative assessment calculation without obviously affecting consumer safety.

Metabolism and genotoxicity of polycyclic aromatic hydrocarbons in human skin explants: mixture effects and modulation by sunlight.

Cutaneous exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH) occurs frequently in the industrialized workplace. In the present study, we addressed this topic in a series of experiments using human skin explants and organic extracts of relevant industrial products. PAH mixtures were applied topically in volumes containing either 10 or 1 nmol B[a]P. We first observed that although mixtures were very efficient at inducing expression of CYP450 1A1, 1A2, and 1B1, formation of adducts of PAH metabolites to DNA, like those of benzo[a]pyrene diol epoxide (BPDE), was drastically reduced as the complexity of the surrounding matrix increased. Interestingly, observation of a nonlinear, dose-dependent response with the least complex mixture suggested the existence of a threshold for this inhibitory effect. We then investigated the impact of simulated sunlight (SSL) on the effects of PAH in skin. SSL was found to decrease the expression of CYP450 genes when applied either after or more efficiently before PAH treatment. Accordingly, the level of DNA-BPDE adducts was reduced in skin samples exposed to both PAH and SSL. The main conclusion of our work is that both increasing chemical complexity of the mixtures and co-exposure to UV radiation decreased the production of adducts between DNA and PAH metabolites. Such results must be taken into account in risk management.

Osmoregulatory disturbance in Neotropical fish exposed to the crude extracts of the cyanobacterium, Radiocystis fernandoi.

Blooms of cyanobacteria, a common event in eutrophic environments, result in the release of potentially toxic substances into the water. The cyanobacterium Radiocystis fernandoi produces microcystin (MC) and other peptides that may disturb homeostasis. This study evaluated the effect of intraperitoneal injections containing the crude extract (CE) of R. fernandoi strain R28 on the gills and kidneys of neotropical fish, Piaractus mesopotamicus, 3, 6 and 24 h post-injection. CE contained MC-RR, MC-YR and minor other oligopeptides. Plasma ions and the activities of the enzymes PP1 and PP2A, Na+/K+-ATPase (NKA), H+-ATPase (HA) and carbonic anhydrase (CA) were determined and morphological changes in both the gills and kidneys were characterized. Compared to controls, the concentration of Na+ within the plasma of P. mesopotamicus decreased after treatment with CE 3 h post treatment and increased after 24 h; the concentration of K+ decreased after 6 h. The activity of the endogenous PP1 and PP2A was unchanged in the gills and was inhibited in the kidneys 6 h after i.p. injection. In the gills, NKA activity increased after 3 h and decreased 6 h post i.p. exposure. Further, NKA activity did not differ from the controls 24-h post injection. In the kidneys, NKA, HA and CA activities were unaffected by treatment. The mitochondria-rich cell (MRC) density in the gills decreased after 3 h in the filament and 3 and 6 h in the lamellae and was restored to the control levels 24 h post-exposure. Filament epithelial hyperplasia and hypertrophy, lamellar atrophy and rupture of the lamellar epithelium were the most common effects of treatment in the gills. No histopathological changes occurred in the kidneys. This study demonstrates that a single dose of toxic CE from R. fernandoi can cause a transitory ion imbalance in P. mesopotamicus which is related to the changes in MRC levels and NKA activity. Ionic balance was recovered 24 h post i.p. injection, however, morphological changes that occurred in the gills took a longer amount of time to return to normal. To conclude, the effects of components contained within the CE of R. fernandoi may be harmful to P. mesopotamicus. In particular, the recovery of ionic regulation depends on MRC responses and histopathological changes produced by CE may affect gas exchange and other gill functions.

Hazard assessment of air pollutants: The transforming ability of complex pollutant mixtures in the Bhas 42 cell model.

The use of in vitro alternative methods is a promising approach to characterize the hazardous properties of environmental chemical mixtures, including urban airborne particulate matter (PM). The aim of this study was to examine seasonal differences in the toxic and transforming potential of PM samples, by using the in vitro cell transformation assay in Bhas 42 cells for the prediction of potential carcinogenic effects. Bhas 42 cells are already initiated, and the v-Ha-ras transfection, together with genetic modification following the immortalization process, makes them a valuable model to study the late steps of cellular transformation leading to the acquisition of the malignant phenotype. Exposure to organic extracts of PM1 and PM2.5 induced dose-related effects. The transforming and cytotoxic properties are related to the amount of PM collected during the sampling campaign and associated with the concentrations of polycyclic aromatic hydrocarbons (PAHs) in the samples. All the samples induced cell transformation following prolonged exposure of 2 weeks. Our results support the utility of the in vitro top-down approach to characterise the toxicity of real mixtures, thereby supporting regulators in the decision-making process. The results also identify the need for appropriate assay selection within the in vitro testing strategy to address the complexity of the final adverse outcomes.

Neurotoxic Effect of Ethanolic Extract of Propolis in the Presence of Copper Ions is Mediated through Enhanced Production of ROS and Stimulation of caspase-3/7 Activity.

Elevated amounts of copper are considered to be contributing factor in the progression of neurodegenerative diseases as they promote oxidative stress conditions. The aim of our study was to examine the effects of ethanolic extract of propolis (EEP) against copper-induced neuronal damage. In cultured P19 neuronal cells, EEP exacerbated copper-provoked neuronal cell death by increasing the generation of reactive oxygen species (ROS) and through the activation of caspase-3/7 activity. EEP augmented copper-induced up-regulation of p53 and Bax mRNA expressions. Neurotoxic effects of EEP were accompanied by a strong induction of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and decrease in the expression of c-fos mRNA. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK) prevented detrimental effects of EEP, whereas SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), exacerbated EEP-induced neuronal cell death. Quercetin, a polyphenolic nutraceutical, which is usually present in propolis, was also able to exacerbate copper-induced neuronal death. Our data indicates a pro-oxidative and apoptotic mode of EEP action in the presence of excess copper, wherein ROS/p53/p38 interactions play an important role in death cascades. Our study also pointed out that detailed pharmacological and toxicological studies must be carried out for propolis and other dietary supplements in order to fully recognize the potential adverse effects in specific conditions.

Compounds of PAH mixtures dependent interaction between multiple signaling pathways in granulosa tumour cells.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Investigation of pesticide exposure by genotoxicological, biochemical, genetic polymorphic and in silico analysis.

Soybean farmers are exposed to various types of pesticides that contain in their formulations a combination of chemicals with genotoxic and mutagenic potential. Therefore, the objective of this paper was to evaluate the genetic damages caused by this pesticide exposure to soybean producers in the state of Mato Grosso (Brazil), regarding biochemical, genetic polymorphic and in silico analyses. A total of 148 individuals were evaluated, 76 of which were occupationally exposed and 72 were not exposed at all. The buccal micronucleus cytome assay (BMCyt) detected in the exposed group an increase on DNA damage and cell death. No inhibition of butyrylcholinesterase (BchE) was observed within the exposed group. The detection of inorganic elements was made through the particle-induced X-ray emission technique (PIXE), which revealed higher concentrations of Bromine (Br), Rubidium (Rb) and Lead (Pb) in rural workers. A molecular model using in silico analysis suggests how metal ions can cause both DNA damage and apoptosis in the exposed cells. Analysis of the compared effect of X-ray Repair Cross-complement Protein 1 (XRCC1) and Paraoxonase 1 (PON1) genotypes in the groups demonstrated an increase of binucleated cells (exposed group) and nuclear bud (non-exposed group) in individuals with the XRCC1 Trip/- and PON1 Arg/- genes. There was no significant difference in the telomere (TL) mean value in the exposed group in contrast to the non-exposed group. Our results showed that soybean producers showed genotoxic effect and cell death, which may have been induced by exposure to complex mixtures of agrochemicals and fertilizers. In addition, XRCC1 Arg/Arg could, in some respects, provide protection to individuals.

Toxic effects of single animal hormones and their mixtures on the growth of Chlorella vulgaris and Scenedesmus armatus.

In their environments, aquatic organisms are simultaneously exposed to mixtures of several endocrine disrupting compounds, including hormones. However, most of the toxicity studies so far focused on effects of single contaminants. The available information on the potential toxicity of combined hormones on microalgae is extremely limited. For these reasons the aim of this study was to evaluate the individual and mixture effect of estrone (E1), ß-estradiol (E2), estriol (E3), 17-α-ethinylestradiol (EE2), progesterone (PRO), 5-pregnen-3ß-ol-20-one (PRE), levonorgestrel (LG) and testosterone (TST) on Chlorella vulgaris and Scenedesmus armatus. Green algae cells were exposed to different concentrations (0.1-100 mg L-1) of hormones for 14 days. Biomass in the form of dry weight and chlorophyll a was examined. The decreasing order of toxicity (based on EC50, 14d) to Chlorella vulgaris and Scenedesmus armatus was: EE2>PRO > E2>PRE > TST > E3>LG > E1 and EE2>PRO > TST > E2>PRE > LG > E1>E3, respectively. Chlorella vulgaris was more sensitive to the effects of hormones than Scenedesmus armatus. Although mixed hormones were more toxic to green algae than single hormones, in the ecosystem mixtures can pose higher ecological risk than single pollutants. Therefore, data on the toxicology of both single and mixed hormones is very valuable for assessment of the possibility of adverse ecological effects caused by these pollutants. Furthermore, these results suggest that environmental exposure to hormone mixtures may cause toxicity levels different to the sum of those of the single hormones and provides a basic understanding of their toxic effect on algae.