Automated Image-Based Fluorescence Screening of Mitochondrial Membrane Potential in Daphnia magna: An Advanced Ecotoxicological Testing Tool.

This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 µM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations.

To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

Altered gut microbiota community structure and correlated immune system changes in dibutyl phthalate exposed mice.

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health.

Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3ß2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

Correction to: Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) induce epigenetic alterations and promote human breast cell carcinogenesis in vitro.

The original article can be found online.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) induce epigenetic alterations and promote human breast cell carcinogenesis in vitro.

Gene-environment interactions are involved in the development of breast cancer, the tumor type that accounts for the majority of the cancer-related deaths among women. Here, we demonstrate that exposure to PFOS (10 µM) and PFOA (100 µM)-two contaminants ubiquitously found in human blood-for 72 h induced breast epithelial cell (MCF-10A cell line) proliferation and alteration of regulatory cell-cycle proteins (cyclin D1, CDK6, p21, p53, p27, ERK 1/2 and p38) that persisted after a multitude of cell divisions. The contaminants also promoted cell migration and invasion by reducing the levels of E-cadherin, occludin and ß-integrin in the unexposed daughter cells. The compounds further induced an increase in global DNA methylation and differentially altered histone modifications, epigenetic mechanisms implicated in tumorigenesis. This mechanistic evidence for PFOS- and PFOA-induced malignant transformation of human breast cells supports a role of these abundant contaminants in the development and progression of breast cancer. Increased knowledge of contaminant-induced effects and their contribution to breast tumorigenesis is important for a better understanding of gene-environment interactions in the etiology of breast cancer.

The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) targets the olfactory bulb region.

Olfactory dysfunction is implicated in neurodegenerative disorders and typically manifests years before other symptoms. The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) is suggested as a risk factor for neurodegenerative disease. Detection of BMAA in air filters has increased the concern that aerosolization may lead to human BMAA exposure through the air. The aim of this study was to determine if BMAA targets the olfactory system. Autoradiographic imaging showed a distinct localization of radioactivity in the right olfactory mucosa and bulb following a unilateral intranasal instillation of 3H-BMAA (0.018 µg) in mice, demonstrating a direct transfer of BMAA via the olfactory pathways to the brain circumventing the blood-brain barrier, which was confirmed by liquid scintillation. Treatment of mouse primary olfactory bulb cells with 100 µM BMAA for 24 h caused a disruption of the neurite network, formation of dendritic varicosities and reduced cell viability. The NMDA receptor antagonist MK-801 and the metabotropic glutamate receptor antagonist MCPG protected against the BMAA-induced alterations, demonstrating the importance of glutamatergic mechanisms. The ionotropic non-NMDA receptor antagonist CNQX prevented the BMAA-induced decrease of cell viability in mixed cultures containing both neuronal and glial cells, but not in cultures with neurons only, suggesting a role of neuron-glial interactions and glial AMPA receptors in the BMAA-induced toxicity. The results show that the olfactory region may be a target for BMAA following inhalation exposure. Further studies on the relations between environmental olfactory toxicants and neurodegenerative disorders are warranted.

The environmental neurotoxin ß-N-methylamino-L-alanine inhibits melatonin synthesis in primary pinealocytes and a rat model.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) is a glutamate receptor agonist that can induce oxidative stress and has been implicated as a possible risk factor for neurodegenerative disease. Detection of BMAA in mussels, crustaceans, and fish illustrates that the sources of human exposure to this toxin are more abundant than previously anticipated. The aim of this study was to determine uptake of BMAA in the pineal gland and subsequent effects on melatonin production in primary pinealocyte cultures and a rat model. Autoradiographic imaging of 10-day-old male rats revealed a high and selective uptake in the pineal gland at 30 minutes to 24 hours after 14 C-L-BMAA administration (0.68 mg/kg). Primary pinealocyte cultures exposed to 0.05-3 mmol/L BMAA showed a 57%-93% decrease in melatonin synthesis in vitro. Both the metabotropic glutamate receptor 3 (mGluR3) antagonist Ly341495 and the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate prevented the decrease in melatonin secretion, suggesting that BMAA inhibits melatonin synthesis by mGluR3 activation and PKC inhibition. Serum analysis revealed a 45% decrease in melatonin concentration in neonatal rats assessed 2 weeks after BMAA administration (460 mg/kg) and confirmed an inhibition of melatonin synthesis in vivo. Given that melatonin is a most important neuroprotective molecule in the brain, the etiology of BMAA-induced neurodegeneration may include mechanisms beyond direct excitotoxicity and oxidative stress.

PFOS induces proliferation, cell-cycle progression, and malignant phenotype in human breast epithelial cells.

Perfluorooctanesulfonic acid (PFOS) is a synthetic fluorosurfactant widely used in the industry and a prominent environmental toxicant. PFOS is persistent, bioaccumulative, and toxic to mammalian species. Growing evidence suggests that PFOS has the potential to interfere with estrogen homeostasis, posing a risk of endocrine-disrupting effects. Recently, concerns about a potential link between PFOS and breast cancer have been raised, but the mechanisms underlying its actions as a potential carcinogen are unknown. By utilizing cell proliferation assays, flow cytometry, immunocytochemistry, and cell migration/invasion assays, we examined the potentially tumorigenic activity of PFOS (100 nM-1 mM) in MCF-10A breast cell line. The results showed that the growth of MCF-10A cells exposed to 1 and 10 µM PFOS was higher compared to that of the control. Mechanistic studies using 10 µM PFOS demonstrated that the compound promotes MCF-10A proliferation through accelerating G0/G1-to-S phase transition of the cell cycle after 24, 48, and 72 h of treatment. In addition, PFOS exposure increased CDK4 and decreased p27, p21, and p53 levels in the cells. Importantly, treatment with 10 µM PFOS for 72 h also stimulated MCF-10A cell migration and invasion, illustrating its capability to induce neoplastic transformation of human breast epithelial cells. Our experimental results suggest that exposure to low levels of PFOS might be a potential risk factor in human breast cancer initiation and development.

Perfluorooctanoic acid (PFOA) exposure promotes proliferation, migration and invasion potential in human breast epithelial cells.

Despite significant advances in early detection and treatment, breast cancer remains a major cause of morbidity and mortality. Perfluorooctanoic acid (PFOA) is a suspected endocrine disruptor and a common environmental pollutant associated with various diseases including cancer. However, the effects of PFOA and its mechanisms of action on hormone-responsive cells remain unclear. Here, we explored the potential tumorigenic activity of PFOA (100 nM-1 mM) in human breast epithelial cells (MCF-10A). MCF-10A cells exposed to 50 and 100 µM PFOA demonstrated a higher growth rate compared to controls. The compound promoted MCF-10A proliferation by accelerating G0/G1 to S phase transition of the cell cycle. PFOA increased cyclin D1 and CDK4/6 levels, concomitant with a decrease in p27. In contrast to previous studies of perfluorooctane sulfate (PFOS), the estrogen receptor antagonist ICI 182,780 had no effect on PFOA-induced cell proliferation, whereas the PPARα antagonist GW 6471 was able to prevent the MCF-10A proliferation, indicating that the underlying mechanisms involve PPARα-dependent pathways. Interestingly, we also showed that PFOA is able to stimulate cell migration and invasion, demonstrating its potential to induce neoplastic transformation of human breast epithelial cells. These results suggest that more attention should be paid to the roles of PFOA in the development and progression of breast cancer.