Endocrine Disruption Toxicity Research Articles

Overview of deltamethrin residues and toxic effects in the global environment.

Pyrethroids are synthetic organic insecticides. Deltamethrin, as one of the pyrethroids, has high insecticidal activity against pests and parasites and is less toxic to mammals, and is widely used in cities and urban areas worldwide. After entering the natural environment, deltamethrin circulates between solid, liquid and gas phases and enters organisms through the food chain, posing significant health risks. Increasing evidence has shown that deltamethrin has varying degrees of toxicity to a variety of organisms. This review summarized worldwide studies of deltamethrin residues in different media and found that deltamethrin is widely detected in a range of environments (including soil, water, sediment, and air) and organisms. In addition, the metabolism of deltamethrin, including metabolites and enzymes, was discussed. This review shed the mechanism of toxicity of deltamethrin and its metabolites, including neurotoxicity, immunotoxicity, endocrine disruption toxicity, reproductive toxicity, hepatorenal toxicity. This review is aim to provide reference for the ecological security and human health risk assessment of deltamethrin.

Multi-omics reveals the molecular mechanism of the combined toxic effects of PFOA and 4-HBP exposure in MCF-7 cells and the key player: mTORC1

With the discovery of evidence that many endocrine-disrupting chemicals (EDCs) in the environment influence human health, their toxic effects and mechanisms have become a hot topic of research. However, investigations into their endocrine-disrupting toxicity under combined binary exposure, especially the molecular mechanism of combined effects, have rarely been documented. In this study, two typical EDCs, perfluorooctanoic acid (PFOA) and 4-hydroxybenzophenone (4-HBP), were selected to examine their combined effects and molecular mechanism on MCF-7 cell proliferation at environmentally relevant exposure concentrations. We have successfully established a model to evaluate the binary combined toxic effects of endocrine disruptors, presenting combined effects in a simple and direct way. Results indicated that the combined effect changed from additive to synergistic from 1.25 × 10−8 M to 4 × 10−7 M. Metabolomics analyses suggested that exposure to PFOA and 4-HBP caused significant alterations in purine metabolism, arginine, and proline metabolism and had superimposed influences on metabolism. Enhanced combined effects were observed in glycine, serine, and threonine metabolic pathways compared to exposure to PFOS and 4-HBP alone. Additionally, the differentially expressed genes (DEGs) are primarily involved in Biological Processes, especially protein targeting the endoplasmic reticulum, and significantly impact the oxidative phosphorylation and thermogenesis-related KEGG pathway. By integrating metabolome and transcriptome analyses, PFOA and 4-HBP regulate purine metabolism, the TCA cycle, and endoplasmic reticulum protein synthesis in MCF-7 cells via mTORC1, which provides genetic material, protein, and energy for cell proliferation. Furthermore, molecular docking confirmed the ability of PFOA and 4-HBP to stably bind the estrogen receptor, indicating that they have different binding pockets. Collectively, these findings will offer new insights into understanding the mechanisms by which EDCs produce combined toxicity.

Reproductive and endocrine-disrupting toxicity of pyrogallol in catfish (Clariasgariepinus)

Endocrine disruptors are synthetic or natural chemicals that can agonize/antagonize hormone receptors or can interfere with the production and secretion of hormones, leading to altered tissue histology and physiology. Pyrogallol is a contaminant widely distributed in aquatic environments that presents health risks to both humans and animals. However, the potential for endocrine disruption by pyrogallol, particularly in fish, are lacking. The purpose of this study was to shed light on how pyrogallol may affect hormone signalling, histopathology, and reproductive outcomes in African catfish Clarias gariepinus. To investigate this, African catfish were exposed to one sublethal concentration of pyrogallol at either 0, 1, 5 or 10 mg/L for 15 days. We then assessed the effects of pyrogallol on the thyroid gland as well as the reproductive system by measuring sex hormone, seminal quality, gonadal histopathology, and histochemistry. Thyroid stimulating hormone and thyroxine showed notable decreases in catfish, and triiodothyronine was decreased with 10 mg/L pyrogallol. Unlike luteinizing hormone, follicle-stimulating hormone was significantly reduced in fish following exposure to pyrogallol relative to controls. Testosterone was also decreased in fish following pyrogallol exposure, whereas 17β-estradiol increased in catfish exposed to pyrogallol. Additionally, in response to pyrogallol toxicity, sperm quality indices, including count, spermatocrit, motility, and sperm viability were adversely affected in a concentration-dependent manner. Pyrogallol exposure also induced several changes in the gonad following exposure to 1, 5, or 10 mg/L. Deformed tubular structures, vacuolation, thickening of the basement membrane, hypertrophy of the seminiferous tubules, intense melanomacrophage localization, spermatozoa loss, and necrosis were all observed in the testes. In the ovary, atretic follicles, deteriorated mature oocytes, degenerated yolk globules, and an increase in perinucleolar oocytes were observed in catfish exposed to pyrogallol. These findings suggest that pyrogallol may act as endocrine disrupting substance in aquatic environments. Further research on the mechanisms by which pyrogallol impairs endocrine systems, particularly in fish, is recommended.

Acrylamide-Aggravated Liver Injury by Activating Endoplasmic Reticulum Stress in Female Mice with Diabetes.

Acrylamide (ACR) is a common industrial contaminant with endocrine-disrupting toxicity. Numerous studies have indicated that females and diabetics are more sensitive to environmental contaminants. However, it remains unknown whether female diabetics are susceptible to ACR-induced toxicity and its potential mechanisms. Thus, the female ACR-exposure diabetic Balb/c mice model was established to address these issues. Results showed that ACR could induce liver injury in normal mice and cause more serious inflammatory cell infiltration, hepatocyte volume increase, and fusion in diabetic mice liver. Meanwhile, ACR could lead to exacerbation of diabetic symptoms in diabetic mice by disturbing the glucose and lipid metabolism in the liver, which mainly manifests as the accumulation of liver glycogen and liver lipids, the reduction of the activity/content of glycolytic and metabolizing enzyme as well as pentose phosphatase, upregulation of the gene expression in fatty acid transporter and gluconeogenesis, and downregulation of the gene expression in fatty acid synthesis and metabolism. Moreover, ACR exposure could induce oxidative stress, inflammation, and endoplasmic reticulum stress in the liver by a decrease in hepatic antioxidant enzyme activity and antioxidant content, an increase in inflammatory factor levels, and a change in the related protein expression of endoplasmic reticulum stress (ERS) and apoptosis-related pathways in diabetic mice. Statistical analysis results revealed that ACR-induced liver injury was highly correlated with inflammation and oxidative stress, and ERS and diabetic mice had a higher risk of liver injury than normal mice. Overall results suggested that female diabetic mice easily suffer from ACR-induced toxicity, and the reason was that ACR could induce further damage to the liver by worsening the condition of inflammation, oxidative stress, and ERS in the liver.

Endocrine disruptive toxicity of cypermethrin in Labeo catla: Involvement of genes and proteins related to the HPG axis

Cypermethrin (CYP) is a synthetic pyrethroid abundantly used in agriculture and aquaculture. It is an established potent endocrine disruptor to fish, yet the molecular mechanism behind its reproductive toxicity remains unclear. In this study, fish Labeo catla (Catla) was exposed to environmentally relevant concentration of CYP (0.7 μg/L) and 0.14 μg/L for 30 days. The changes in circulating sex steroids, genes, and hormones linked to the hypothalamic-pituitary-gonadal (HPG) axis, stress response and associated histological alterations were studied. Significant decline (P < 0.05) in serum 17 beta (β) estradiol (E2), 11 ketotestosterone (11-KT), and brain (FSH and GnRH) were observed in 0.7 μg/L dose of CYP. These effects may be due to the down-regulated expression of the upstream genes of the HPG axis i.e. Kiss 1 and Kiss 2, which further downregulates the expression of the GnRH gene. The decreased level of E2 and 11-KT also affects the vitellogenin (Vtg) gene expression, reducing the production of Vtg, a crucial protein for ovarian development. Principal component analysis (PCA) revealed the relationship between CYP and the biosynthesis of sex steroids. The toxic effect of CYP was also visible in antioxidant enzyme assay and related histological alterations. Overall, the study elucidated that long-term exposure to CYP, even at an environmentally relevant dose, may affect reproductive potential and fish recruitment. The study provides important insights into molecular mechanisms underlying CYP-induced endocrine disruption in fish, and it also raises questions about CYP's potential toxicity at environmentally relevant concentration in terms of understanding ecological risk.

Endocrine Disruptive Toxicity of Cypermethrin in Labeo Catla: Involvement of Genes and Proteins Related to the Hpg Axis

Endocrine Disruptive Toxicity of Cypermethrin in Labeo Catla: Involvement of Genes and Proteins Related to the Hpg Axis

Molecular Design and Mechanism Analysis of Phthalic Acid Ester Substitutes: Improved Biodegradability in Processes of Sewage Treatment and Soil Remediation.

Phthalic acid esters (PAEs) have the characteristics of environmental persistence. Therefore, improving the biodegradability of PAEs is the key to reducing the extent of ecological harm realized. Firstly, the scoring function values of PAEs docking with various degrading enzymes in sewage treatment were calculated. Based on this, a 3D-quantitative structure-activity relationship (3D-QSAR) model for PAE biodegradability was built, and 38 PAE substitutes were created. By predicting the endocrine-disrupting toxicity and functions of PAE substitutes, two types of PAE substitutes that are easily degraded by microorganisms, have low toxicity, and remain functional were successfully screened. Meanwhile, the differences in the mechanism of molecular degradation difference before and after PAE modification were analyzed based on the distribution characteristics of amino acid residues in the molecular docking complex. Finally, the photodegradability and microbial degradability of the PAE substitutes in the soil environment was evaluated. From the 3D-QSAR model design perspective, the modification mechanism of PAE substitutes suitable for sewage treatment and soil environment degradation was analyzed. We aim to improve the biodegradability of PAEs at the source and provide theoretical support for alleviating the environmental hazards of using PAEs.

A transcriptomic-based analysis predicts the neuroendocrine disrupting effect on adult male and female zebrafish (Danio rerio) following long-term exposure to tetrabromobisphenol A bis(2-hydroxyethyl) ether

Endocrine-disrupting chemicals (EDCs) are now ubiquitously distributed in the environment. Tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE) pollution in environment media poses a significant threat to humans and aquatic organisms as a result of its potential neurotoxicity and endocrine-disrupting effect. The endocrine-disrupting effects of TBBPA-DHEE on aquatic organisms, however, have received limited attention. In this study, the neurotoxicity and reproductive endocrine-disruptive effect of TBBPA-DHEE was evaluated by observing the neurobehavioral changes, vitellogenin (VTG), testosterone, 17β-estradiol and gene expression levels in adult male and female zebrafish exposed to TBBPA-DHEE (0.05, 0.2 and 0.3 mg/L) for 100 days. Furthermore, transcriptomic analysis was conducted to unravel other potential neuroendocrine-disrupting mechanism. Our result showed TBBPA-DHEE significantly (p < 0.05) altered the locomotor behavior and motor coordination abilities in both sexes. Steroid hormone and VTG levels were also altered indicating the neuroendocrine-disrupting effect of TBBPA-DHEE on the hypothalamic–pituitary–gonadal-axis. A total of 1568 genes were upregulated and 542 genes downregulated in males, whereas, 1265 upregulated and 535 downregulated genes were observed in females. The KEGG enrichment analysis showed that cell cycle and p55 signaling pathways were significantly enriched due to TBBPA-DHEE exposure. These pathways and its component genes are potential target of EDCs. The significant upregulation of genes in these pathways could partly explain the neuroendocrine disrupting effect of TBBPA-DHEE. The observed toxic effects of TBBPA-DHEE observed in this study is confirmation of the endocrine-disrupting toxicity of this chemical which would be valuable in biosafety evaluation and biomonitoring of TBBPA-DHEE for public health purposes.

Remediation and toxicity of endocrine disruptors: a review

Remediation and toxicity of endocrine disruptors: a review

Potential Toxicity Risk Assessment and Priority Control Strategy for PAHs Metabolism and Transformation Behaviors in the Environment.

In this study, 16 PAHs were selected as the priority control pollutants to summarize their environmental metabolism and transformation processes, including photolysis, plant degradation, bacterial degradation, fungal degradation, microalgae degradation, and human metabolic transformation. Meanwhile, a total of 473 PAHs by-products generated during their transformation and degradation in different environmental media were considered. Then, a comprehensive system was established for evaluating the PAHs by-products’ neurotoxicity, immunotoxicity, phytotoxicity, developmental toxicity, genotoxicity, carcinogenicity, and endocrine-disrupting effect through molecular docking, molecular dynamics simulation, 3D-QSAR model, TOPKAT method, and VEGA platform. Finally, the potential environmental risk (phytotoxicity) and human health risks (neurotoxicity, immunotoxicity, genotoxicity, carcinogenicity, developmental toxicity, and endocrine-disrupting toxicity) during PAHs metabolism and transformation were comprehensively evaluated. Among the 473 PAH’s metabolized and transformed products, all PAHs by-products excluding ACY, CHR, and DahA had higher neurotoxicity, 152 PAHs by-products had higher immunotoxicity, and 222 PAHs by-products had higher phytotoxicity than their precursors during biological metabolism and environmental transformation. Based on the TOPKAT model, 152 PAH by-products possessed potential developmental toxicity, and 138 PAH by-products had higher genotoxicity than their precursors. VEGA predicted that 247 kinds of PAH derivatives had carcinogenic activity, and only the natural transformation products of ACY did not have carcinogenicity. In addition to ACY, 15 PAHs produced 123 endocrine-disrupting substances during metabolism and transformation. Finally, the potential environmental and human health risks of PAHs metabolism and transformation products were evaluated using metabolic and transformation pathway probability and degree of toxic risk as indicators. Accordingly, the priority control strategy for PAHs was constructed based on the risk entropy method by screening the priority control pathways. This paper assesses the potential human health and environmental risks of PAHs in different environmental media with the help of models and toxicological modules for the toxicity prediction of PAHs by-products, and thus designs a risk priority control evaluation system for PAHs.

Toxicity mechanisms and bioavailability of copper to fish based on an adverse outcome pathway analysis

Copper (Cu) exists in a variety of forms in different aquatic environments, and affects their bioavailability. In this study we provide a systematic review on toxicity of Cu which focuses on identifying evidence in the mechanisms of Cu toxicity, and apply an adverse outcome pathway (AOP) analysis to identify multiple potential mechanisms and their interactions of Cu toxicity to fish. This analysis process included the mechanisms of behavior toxicant, oxidative toxicant, ion regulation disruption toxicity, as well as endocrine disruption toxicity. It was found that at low levels of Cu exposure, swimming, avoid predators, locating prey and other sensory functions will be impaired, and the organism will suffer from metabolic alkalosis and respiratory acidosis following the inhibition of the carbonic anhydrase active. The main pathway of acute toxicity of Cu to fish is the inhibition of the Na+/K+-ATPase enzyme, and lead to reduced intracellular sodium absorption, as well as Cu-induced increased cell permeability, in turn resulting in increased sodium ion loss, leading to cardiovascular collapse and respiratory insufficiency. The endocrine disruption toxicity of Cu to fish caused growth inhibition and reproductive reduction. In addition, there are several key pathways of Cu toxicity that are affected by hardness (e.g., Ca2+) and intracellular DOC concentrations, including inhibiting Cu-induction, improving branchial gas exchange, altering membrane transport functions, decreasing Na+ loss, and increasing Na+ uptake. The results of the AOP analysis will provide a robust framework for future directed research on the mechanisms of Cu toxicity.

Integrated metabolomics and transcriptomics analysis reveals new biomarkers and mechanistic insights on atrazine exposures in MCF‑7 cells

Atrazine (ATZ) is a widely used herbicide worldwide and is a long-suspected endocrine-disrupting chemical. However, most endocrine-disrupting toxicity studies on ATZ have been based on animal models and those investigating inner mechanisms have only focused on a few genes. Therefore, the possible link between ATZ and endocrine-disrupting toxicity is still unclear. In this study, multi-omics and molecular biology techniques were used to elucidate the possible molecular mechanisms underlying the effect of ATZ exposure on MCF-7 proliferation at environmentally relevant concentrations. Our study is the first report on ATZ-induced one carbon pool by folate metabolic disorder in MCF-7 cells. A concentration of 1 μM ATZ yielded the highest cell viability and was selected for further mechanistic studies. A total of 34 significantly changed metabolites were identified based on metabolomic analysis, including vitamins, amino acids, fatty acids, and corresponding derivatives. Folate and pyridoxal have potential as biomarkers of ATZ exposure. One carbon pool by folate metabolic pathway was identified based on metabolic pathway analysis of the significantly altered pathways. Moreover, FTCD and MTHFD related to this pathway were further identified based on transcriptomic analysis and protein assays. Folate and different forms of 5,6,7,8-tetrahydrofolate, which participate in purine synthesis and associate with methyl groups (SOPC, arachidonic acid, and L-tryptophan) in one carbon pool by the folate metabolic pathway, potentially promote MCF-7 cell proliferation. These findings on the key metabolites and regulation of the related differentially expressed genes in folate metabolism will shed light on the mechanism of MCF-7 cell proliferation after ATZ exposure. Overall, this study provides new insights into the mechanistic understanding of toxicity caused by endocrine-disrupting chemicals.

Combined endocrine disruptive toxicity of malathion and cypermethrin to gene transcription and hormones of the HPG axis of male zebrafish (Danio rerio)

Cypermethrin (CYP) and malathion (MAT) have been widely used and are frequently detected in surface waters. The purpose of the present study was to investigate the endocrine disrupting toxicity of CYP, MAT, and CYP + MAT to 5-month-old male zebrafish (Danio rerio). After exposure, the hepatosomatic index (HSI) and gonadosomatic index (GSI) did not change significantly. Following exposure to the combination of 0.1 μg/L CYP +25 μg/L MAT, the E2 and VTG levels of male zebrafish were significantly increased compared to those after individual pesticide treatments. The molecular level of the hypothalamic-pituitary-gonadal (HPG) axis in zebrafish was studied; it was found that the expression of the estrogen-related genes, especially the vtg1 gene, was significantly altered in 0.1 μg/L CYP + 25 μg/L MAT. Overall, our observation indicated that CYP or MAT could disturb the hormonal balance, and their combination of 0.1 μg/L CYP +25 μg/L MAT could significantly enhance the estrogenic effect.

Binding and activation of estrogen related receptor γ as possible molecular initiating events of hydroxylated benzophenones endocrine disruption toxicity

As a class of widely used ultraviolet filters, hydroxylated benzophenones (BPs) have been detected in various environmental and human samples, raising concerns about their ecological and health effects. BPs were found to impair reproductive functions, and their disruption effects on estrogen receptor, G protein-coupled estrogen receptor, and androgen receptor were investigated as possible mechanisms of action. In the present study, we investigated the binding and activation of estrogen related receptor γ (ERRγ) as an alternative mechanism of BPs reproductive toxicity. In competitive fluorescence binding assays, all of the 8 tested BPs were found to bind to the ligand-binding domain of ERRγ but with varying binding affinity. Molecular dock analysis indicated that binding of BPs to ERRγ in the agonistic conformation is preferred. In luciferase reporter gene assays in HepG2 cells, all the 8 BPs activated the transcription of ΕRRγ in a concentration-dependent manner in the range of 3–100 μM,consistent with the docking result. Hazard quotient calculation revealed a high risk on the BP-type personal care products users. The combined in vitro and in silico results suggest that binding and activation of ERRγ might be the molecular initiating events of an alternative mechanism of BPs endocrine disruption toxicity.

Mineralization of high concentrations of the endocrine disruptor dibutyl phthalate by Fusarium culmorum.

Dibutyl phthalate (DBP) is a widely used plasticizer, whose presence in the environment as a pollutant raises concern because of its endocrine-disrupting toxicity. Growth kinetics, glucose uptake, biodegradation constant of DBP (k), half-life of DBP biodegradation (t1/2) and percentage of removal efficiency (%E) were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DBP (500 and 1000mg/l). Intermediate compounds of biodegraded DBP were identified by GC-MS and a novel DBP biodegradation pathway was proposed on the basis of the intermolecular flow of electrons of the intermediates identified using quantum chemical modeling. F. culmorum degraded 99% of both 1000 and 500mg of DBP/l after an incubation period of 168 and 228h, respectively. %E was 99.5 and 99.3 for 1000 and 500mg of DBP/l, respectively. The k was 0.0164 and 0.0231h-1 for 500 and 1000mg of DBP/l, respectively. DBP was fully metabolized to fumaric and malic acids, which are compounds that enter into the Krebs cycle. F. culmorum has a promising ability for bioremediation of environments polluted with DBP because it efficiently degrades DBP and uses high concentrations of this compound as carbon and energy source.

Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish

Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at environmentally relevant levels on the reproductive and endocrine systems of zebrafish are still unknown. The present study investigated the reproductive and endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by short-term exposure (96 h). After exposure, marked histological lesions in the liver or gonads, such as nuclear pyknosis and deformation, were observed, and the fertilization rate and hatchability of eggs spawned from treated females were both significantly lower than they were in females in the control group, suggesting the possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M. aeruginosa exposure decreased the concentration of 17β-estradiol (E2) and testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels increased. The present findings indicate that M. aeruginosa could modulate endocrine function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL) axis-related genes, and impair the reproductive capacity of female zebrafish, suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in Microcystis bloom-contaminated aquatic environments.

Degradation of dimethyl phthalate esters by a filamentous fungus Aspergillus versicolor isolated from deep-sea sediments

AbstractDimethyl phthalate esters (DMPEs), which belong to phthalate esters (PAEs), are widely distributed environmental pollutants with endocrine-disrupting toxicity. The present study focused on a filamentous fungus capable of degrading DMPEs isolated from deep-sea sediments of the Indian Ocean at a water depth of 3300 m using an enrichment culture technique. The filamentous fungus was identified as

Reproductive toxicity of linuron following gestational exposure in rats and underlying mechanisms

Linuron is a widely used herbicide in agriculture; its endocrine disruptive toxicity has recently received public attention. This study was designed to examine the developmental toxicity of linuron on the reproductive system of male offspring following maternal exposure. Mother rats received oral gavages of linuron, once daily, at the dose of 0, 50, 100, 150 or 200mg/kg, from gestational day (GD)13 to GD18; gonadal organs from GD20 fetuses were examined. Data indicated that exposed male offspring had a significantly shortened anogenital distance. Pathological examination further revealed a lack of fusion in the urogenital fold in treated fetuses, the damaged seminiferous tubules, and the injured Leydig cell ultrastructure. Analysis of serum testosterone concentrations at postnatal day (PND)2 showed a significant dose-related reduction (about 33.7–58.75%, r=−0.838, p<0.05) as compared to controls. Immunohistochemical results demonstrated a significantly reduced expression of enzymes pertinent to the testosterone production including P450scc, 3β-HSD, and PCNA in Leydig cells (p<0.05). qPCR studies confirmed decreased levels of mRNAs encoding P450scc, 3β-HSD and PCNA (p<0.05). Taken together, these data suggest that maternal exposure to linuron hampers the male gonadal organ development; this appears to be due to linuron’s direct action on the production of testosterone in fetal and postnatal offspring.

Green microalgae in removal and biotransformation of estradiol and ethinylestradiol

Estrogens, a group of steroid hormones, are serious environmental pollutants due to their persistence, ubiquity, and endocrine-disrupting toxicity. Natural and synthetic estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), respectively, have been found in the influent of municipal wastewater treatment plants. The removal and biotransformation of mixed or single E2 and EE2 by six microalgal species, including three local isolates of Chlamydomonas sp. (WW), Chlorella sp. (2f5aia), and Chlorella sp. (1uoai) and three commercially available species, namely Scenedesmus quadricauda (= Desmodesmus communis) (SQ), Selenastrum capricornutum (SC), and Chlorella vulgaris (CV), were compared. The biological removal of estrogen was incubation time- and species-dependent. Among six species, SC had the highest removal ability for both E2 and EE2; their removal increased with incubation time and achieved 93.9 and 75.3 % removal, respectively, at the end of incubation. After 7-day incubation, the removal percentage of mixed EE2 by SC was significantly higher than that of single EE2 (P ≤ 0.05). Differences in the removal percentages of E2 and EE2 between commercial and local species were not significant, as reflected by the comparable removal efficiency among three Chlorella species, CV, 1uoai, and 2f5aia, with the highest removal of E2, either in mixed or in single treatment, at day 2. In all species, the percentages of E2 or EE2 taken up into cells were very low (0.2–9.6 %) when compared to the respective biological removal percentages. E2 was easily transformed into estrone (E1) and some unknown products by all microalgal species, while the products for EE2 transformation could not be identified due to the limited availability of the standards for metabolites.

Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay.

Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC(50), strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.