Embryotoxicity Induced by Triclopyr in Zebrafish (Danio rerio) Early Life Stage.

Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 µM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.

Diuron and its metabolites induce mitochondrial dysfunction-mediated cytotoxicity in urothelial cells.

In the environment, or during mammalian metabolism, the diuron herbicide (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is transformed mainly into 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous research suggests that such substances are toxic to the urothelium of Wistar rats where, under specific exposure conditions, they may induce urothelial cell degeneration, necrosis, hyperplasia, and eventually tumors. However, the intimate mechanisms of action associated with such chemical toxicity are not fully understood. In this context, the purpose of the current in vitro study was to analyze the underlying mechanisms involved in the urothelial toxicity of those chemicals, addressing cell death and the possible role of mitochondrial dysfunction. Thus, human 1T1 urothelial cells were exposed to six different concentrations of diuron, DCA, and DCPMU, ranging from 0.5 to 500 µM. The results showed that tested chemicals induced oxidative stress and mitochondrial damage, cell cycle instability, and cell death, which were more expressive at the higher concentrations of the metabolites. These data corroborate previous studies from this laboratory and, collectively, suggest mitochondrial dysfunction as an initiating event triggering urothelial cell degeneration and death.

Toxic Effects Induced by Diuron and Its Metabolites in Caenorhabditis elegans.

The toxicity of diuron herbicide and its metabolites has been extensively investigated; however, their precise toxic mechanisms have yet to be fully appreciated. In this context, we evaluated the toxic mechanism of diuron, 3,4-dichloroaniline (DCA) and 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), using Caenorhabditis elegans (C. elegans) in the L1 larval stage. For this purpose, worms were acutely exposed to the test chemicals with a preliminary concentration range of 0.5 to 500 µM and first analyzed for lethality (%). Next, the highest concentration (500 µM) was considered for survival (%), reactive oxygen and nitrogen species (RONS), glutathione (GSH) and ATP levels, autophagy index, behavior, and dopaminergic neurodegeneration parameters. Interestingly, increased lethality (%) was found for all chemicals at the higher concentrations tested (100 and 500 µM), with significant differences at 500 µM DCA (p < 0.05). A decrease in the median survival was observed mainly for DCA. Although no changes were observed in RONS production, GSH levels were significantly increased upon diuron and DCA treatment, likely reflecting an attempt to restore the redox status. Moreover, diuron and its metabolites impaired ATP levels, suggesting an alteration in mitochondrial function. The latter may trigger autophagy as an adaptive survival mechanism, but this was not observed in C. elegans. Dopaminergic neurotoxicity was observed upon treatment with all the tested chemicals, but only diuron induced alterations in the worms' locomotor behavior. Combined, these results indicate that exposure to high concentrations of diuron and its metabolites elicit distinct adverse outcomes in C. elegans, and DCA in particular, plays an important role in the overall toxicity observed in this experimental model.

Molecular signatures associated with diuron exposure on rat urothelial mitochondria.

Diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is a worldwide used herbicide whose biotransformation gives rise to the metabolites, 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous studies indicate that diuron and/or its metabolites are toxic to the bladder urothelium of the Wistar rats where, under certain conditions of exposure, they may induce successively urothelial cell degeneration, necrosis, hyperplasia and eventually tumors. The hypothesis was raised that the molecular initiating event (MIE) of this Adverse Outcome Pathway is the mitochondrial toxicity of those compounds. Therefore, this study aimed to investigate in vitro the metabolic alterations resulting from urothelial mitochondria isolated from male Wistar rats exposure to diuron, DCPMU and DCA at 10 and 100 µM. A non-targeted metabolomic analysis using mass spectrometry showed discriminative clustering among groups and alterations in the intensity abundance of membrane-associated molecules phosphatidylcholine, phosphatidylinositol and phosphatidylserine, in addition to methylhexanoyl-CoA and, particularly for diuron 100 µM, dehydro-L-gulonate, all of them involved in critical mitochondrial metabolism. Collectively, these data indicate the mitochondrial dysfunction as an MIE that triggers cellular damage and death observed in previous studies.

Rupture of Brumadinho dam (Minas Gerais, Brazil): embryotoxicity in zebrafish induced by metal mixture-contaminated water.

Rupture of dam B-1 at the Córrego do Feijão mine in Brumadinho, state of Minas Gerais, Brazil (25/Jan/2019) contaminated the Paraopeba River with mine waste. To identify the adverse effects of this event on public and environmental health, we conducted the Fish Embryo Acute Toxicity (FET) test (OECD No. 236). During the tests, zebrafish embryos were exposed for 144 hours to solutions containing realistic concentrations of dissolved iron and aluminum and total manganese at the following analysis points upstream or downstream of the dam: 10 km, upstream; 19.7 km, downstream, at the point where water for consumption is collected; 24.5 km, downstream, in the city of Mário Franco; and 59 km, downstream, on the border between the towns of Juatuba and Betim. Metal concentrations were taken from September 2019 report No. 53 released by IGAM. Mortality was high at all exposure points and reached 93% at the Juatuba/Betim point. We also detected lethal, sublethal and teratogenic effects, such as non-hatching, non-inflation of the swim bladder, pericardial edema and scoliosis, affecting up to 25% of embryos at the other analysis points. The results highlight the need for continuous monitoring of the water quality of the Paraopeba River.

Immunocytochemistry Analysis of HepG2 Cell 3D Culture Encapsulated as Spheroids in Alginate Beads.

3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.

In Vitro Apoptotic Cell Death Assessment.

Chemical compounds induce cytotoxicity by various mechanisms, including interference in membrane integrity, metabolism, cellular component degradation or release, and cell division. Between the classic death pathways, namely, autophagy, apoptosis, and necrosis, apoptosis have been in the focus for the last several years as an important pathway for the toxicity of different types of xenobiotics. Because of that, having the tools to evaluate it is key for understanding and explaining the toxicodynamics of different classes of substances. Here, we describe a wide array of classic assays that can be easily implemented to evaluate apoptosis induction.

Do trifluralin and tebuthiuron impair isolated rat liver mitochondria?

Emerging contaminants, such as the herbicides trifluralin and tebuthiuron, comprise a class of compounds for which toxicological data are lacking, especially data regarding their harmful effects and biomarkers of exposure. Their potential damage to the environment and non-target organisms makes understanding their toxic mechanisms an urgent matter. Mitochondria, which exert an energy production function, play a vital role in maintaining many cellular activities and therefore are reliable predictors of substance toxicity. This study evaluates whether the herbicides trifluralin and tebuthiuron (at concentrations ranging from 1 to 100 µM) affect isolated rat liver mitochondria. The herbicides were analyzed according to their ability to interact with the mitochondrial membrane and induce swelling, lipoperoxidation, ROS formation, and NAD(P)H oxidation; dissipate the membrane potential; dysregulate calcium homeostasis; and alter ATP and GSH/GSSG levels. Tebuthiuron does not disrupt the mitochondrial biochemistry at any of the tested concentrations. In contrast, trifluralin can disturb the mitochondrial respiration, especially at the highest concentration, but it cannot induce oxidative stress. These results suggest that the aforementioned effects can occur as toxic mechanisms of trifluralin in non-target organisms, as well.

Niacin prevents mitochondrial oxidative stress caused by sub-chronic exposure to methylmercury.

Humans and animals can be exposed to different chemical forms of mercury (Hg) in the environment. For example, methylmercury (MeHg)-contaminated fish is part of the basic diet of the riparian population in the Brazilian Amazon Basin, which leads to high total blood and plasma Hg levels in people living therein. Hg induces toxic effects mainly through oxidative stress. Different compounds have been used to prevent the damage caused by MeHg-induced reactive oxygen species (ROS). This study aims to investigate the in vivo effects of sub-chronic exposure to low MeHg levels on the mitochondrial oxidative status and to evaluate the niacin protective effect against MeHg-induced oxidative stress. For this purpose, Male Wistar rats were divided into four groups: control group, treated with drinking water on a daily basis; group exposed to MeHg at a dose of 100 µg/kg/day; group that received niacin at a dose of 50 mg/kg/day in drinking water, with drinking water being administered by gavage; group that received niacin at a dose of 50 mg/kg/day in drinking water as well as MeHg at a dose of 100 µg/kg/day. After 12 weeks, the rats, which weighed 500-550 g, were sacrificed, and their liver mitochondria were isolated by standard differential centrifugation. Sub-chronic exposure to MeHg (100 µg/kg/day for 12 weeks) led to mitochondrial swelling (p < 0.05) and induced ROS overproduction as determined by increased DFCH oxidation (p < 0.05), increased gluthatione oxidation (p < 0.05), and reduced protein thiol content (p < 0.05). In contrast, niacin supplementation inhibited oxidative stress, which counteracted and minimized the toxic MeHg effects on mitochondria.

Mitochondrial damage and apoptosis: Key features in BDE-153-induced hepatotoxicity.

Brominated flame retardants are used in consumer goods to increase product resistance to fire and/or high temperatures. Polybrominated diphenyl ethers (PBDEs) are the most commonly employed class of brominated flame retardants because they are inexpensive and can effectively prevent flame from spreading. PBDEs are persistent, can bioaccumulate, are transported over long distances, and display toxicity. However, their toxic mechanisms of action have not been well established. Because mitochondria are recognized as the main energy-producing cell organelle and play a vital role in cellular function maintenance, here we apply mitochondria as an experimental model to evaluate the toxic effects of the PBDE congener BDE-153 (Hexa-BDE) at concentrations ranging from 0.1 to 25 µM. We also assess BDE-153 cytotoxicity to HepG2 cells in order to elucidate its mechanisms of toxicity. Exposure to BDE-153 affects isolated mitochondria: this congener can interact with the mitochondrial membrane, to dissipate the membrane potential and to induce significant ATP depletion. Furthermore, BDE-153 can diminish MTT reduction and cell proliferation and can interfere in cell cycle, as evaluated in cell cultures. These cytotoxic effects are related to mitochondrial dysfunction due to mitochondrial membrane potential dissipation and reactive oxygen species accumulation. These effects result in apoptotic cell death, as demonstrated by phosphatidylserine maintenance on the cell membrane external surface, nuclear condensation and fragmentation, and presence of pro-apoptotic factors such as cytochrome c and Apoptosis-inducing Factor (AIF) plus caspase 3 activation in the cytosol. Together, our results show PBDEs can induce cytotoxicity, reinforcing the idea that these compounds pose a risk to the exposed population.

Mechanistic insights into functional characteristics of native crotamine.

The chemical composition of snake venoms is a complex mixture of proteins and peptides that can be pharmacologically active. Crotamine, a cell-penetrating peptide, has been described to have antimicrobial properties and it exerts its effects by interacting selectively with different structures, inducing changes in the ion flow pattern and cellular responses. However, its real therapeutic potential is not yet fully known. Bearing in mind that crotamine is a promising molecule in therapeutics, this study investigated the action of purified molecule in three aspects: I) antibacterial action on different species of clinical interest, II) the effect of two different concentrations of the molecule on platelet aggregation, and III) its effects on isolated mitochondria. Crotamine was purified to homogeneity in a single step procedure using Heparin Sepharose. The molecular mass of the purified enzyme was 4881.4 Da, as determined by mass spectrometry. To assess antibacterial action, changes in the parameters of bacterial oxidative stress were determined. The peptide showed antibacterial activity on Escherichia coli (MIC: 2.0 µg/µL), Staphylococcus aureus (MIC: 8-16 µg/µL) and methicillin-resistant Staphylococcus aureus (MIC: 4.0-8.0 µg/µL), inducing bacterial death by lipid peroxidation and oxidation of target proteins, determined by thiobarbituric acid reactive substances and sulfhydryl groups, respectively. Crotamine induced increased platelet aggregation (IPA) at the two concentrations analyzed (0.1 and 1.4 µg/µL) compared to ADP-induced aggregation of PRP. Mitochondrial respiratory parameters and organelle structure assays were used to elucidate the action of the compound in this organelle. The exposure of mitochondria to crotamine caused a decrease in oxidative phosphorylation and changes in mitochondrial permeability, without causing damage in the mitochondrial redox state. Together, these results support the hypothesis that, besides the antimicrobial potential, crotamine acts on different molecular targets, inducing platelet aggregation and mitochondrial dysfunction.

A perspective of mitochondrial dysfunction in rats treated with silver and titanium nanoparticles (AgNPs and TiNPs).

Nanotechnology is a growing branch of science that deals with the development of structural features bearing at least one dimension in the nano range. More specifically, nanomaterials are defined as objects with dimensions that range from 1 to 100 nm, which give rise to interesting properties. In particular, silver and titanium nanoparticles (AgNPs and TiNPs, respectively) are known for their biological and biomedical properties and are often used in consumer products such as cosmetics, food additives, kitchen utensils, and toys. This situation has increased environmental and occupational exposure to AgNPs and TiNPs, which has placed demand for the risk assessment of NPs. Indeed, the same properties that make nanomaterials so attractive could also prove deleterious to biological systems. Of particular concern is the effect of NPs on mitochondria because these organelles play an essential role in cellular homeostasis. In this scenario, this work aimed to study how AgNPs and TiNPs interact with the mitochondrial respiration chain and to analyze how this interaction interferes in the bioenergetics and oxidative state of the organelles after sub-chronic exposure. Mitochondria were exposed to the NPs by gavage treatment for 21 days to check whether co-exposure of the organelles to the two types of NPs elicited any mitochondrion-NP interaction. More specifically, male Wistar rats were randomly assigned to four groups. Groups I, II, III, and IV received mineral oil, TiNPs (100 µg/kg/day), AgNPs (100 µg/kg/day), and TiNPs + AgNPs (100 µg/kg/day), respectively, by gavage. The liver was immediately removed, and the mitochondria were isolated and used within 3 h. Exposure of mitochondria to TiNPs + AgNPs lowered the respiratory control ratio, causing an uncoupling effect in the oxidative phosphorylation system. Moreover, both types of NPs induced mitochondrial swelling. Extended exposure of mitochondria to the NPs maintained increased ROS levels and depleted the endogenous antioxidant system. The AgNPs and TiNPs acted synergistically-the intensity of the toxic effect on the mitochondrial redox state was more significant in the presence of both types of NPs. These findings imply that the action of the NPs on mitochondria underlie NP toxicity, so future application of NPs requires special attention.

The herbicides trifluralin and tebuthiuron have no genotoxic or mutagenic potential as evidenced by genetic tests.

Brazil has been the largest world consumer of pesticides since 2008, followed by the USA. The herbicides trifluralin and tebuthiuron have been widely applied in agriculture. These herbicides are selective for some plant species, and their use brings various benefits. However, the genotoxic and mutagenic effects of tebuthiuron on non-target organisms are poorly known, and in addition, the effects of trifluralin must be better investigated. Therefore, this study employed genetic tests including the comet assay and micronucleus test to evaluate the genotoxic effects of trifluralin and tebuthiuron on HepG2 cells. In addition, we have used the Ames test to assess the mutagenic effects of the herbicides on the TA97a, TA98, TA100, and TA1535 strains of Salmonella typhimurium. On the basis of the comet assay and the micronucleus test, trifluralin did not cause genetic damage to HepG2 cells. In addition, trifluralin did not impact the tested S. typhimurium strains. Regarding tebuthiuron, literature has shown that this herbicide damaged DNA in Oreochromis niloticus. Nevertheless, we have found that tebuthiuron was not genotoxic to either HepG2 cells or the S. typhimurium strains. Therefore, neither trifluralin nor tebuthiuron exerted genotoxic or mutagenic potential at the tested conditions.

Exposure to BDE-153 induces autophagy in HepG2 cells.

Autophagy is a pro-survival process that occurs under stressful "life-threatening" conditions. This process clears the cells of damaged organelles, long-lived proteins, and/or misfolded proteins. Under stressful conditions, activation of the autophagic process leads to cell death and acts as a protective mechanism against xenobiotic, which is the most widely accepted mechanism in the literature. Exposure to flame retardants and other pollutants is associated with several diseases, during which cell death and mitochondrial damage takes place. Although a body of research has aimed to understand the toxicity mechanism of flame retardants better, risk evaluation and the consequences of exposure to these toxicants have been poorly described. In this work, we have found that the BDE-153 congener (representant of flame retardants) induces autophagy after 24 and 48h (0.1-25µM). The autophagic process is associated with accumulation of lysosomes, and process triggering is evident from the levels of autophagy-related proteins such as p62 and LC3. Mitophagy (an autophagic process that specifically involves damaged mitochondria) may be involved, as judged from the decreased amount of mitochondrial DNA. Taken together, our results point out that induction of autophagy upon cell should contribute to better understanding of the consequences of human exposure to this class of environmental contaminants.

An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

To reduce flammability and meet regulatory requirements, Brominated Flame Retardants (BFRs) are added to a wide variety of consumer products including furniture, textiles, electronics, and construction materials. Exposure to polybrominated phenyl ethers (PBDEs) adversely affects the human health. Bearing in mind that (i) PBDEs are potentially toxic, (ii) the mechanism of PBDE toxicity is unclear, and (iii) the importance of the autophagy to the field of toxicology is overlooked, this study investigates whether an autophagic process is activated in HepG2 cells (human hepatoblastoma cell line) to mediate BDE-100-induced toxicity. HepG2 cells were exposed with BDE-100 at three concentrations (0.1, 5, and 25µM), selected from preliminary toxicity tests, for 24 and 48h. To assess autophagy, immunocytochemistry was performed after exposure of HepG2 cells to BDE-100. Labeling of HepG2 cells with 100nM LysoTracker Red DND-99 aided examination of lysosome distribution. Proteins that are key to the autophagic process (p62 and LC3) were evaluated by western blotting. DNA was isolated and quantified to assess mitochondrial DNA copy number by qPCR on the basis of the number of DNA copies of a mitochondrial encoded gene normalized against a nuclear encoded gene. Conversion of LC3-I to LC3-II increased in HepG2 cells. Pre-addition of 100nM wortmannin decreased the amount of LC3 in the punctuate form and increased nuclear fragmentation (apoptotic feature). HepG2 cells exposed to BDE-100 presented increased staining with the lysosomal dye and had larger LC3 and p62 content after pre-treatment with ammonium chloride. The mitochondrial DNA copy number decreased, which probably constituted an attempt of the cell to manage mitochondrial damage by selective mitochondrial degradation (mitophagy). In conclusion, an autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

Comparative Study of Genotoxicity Induced by Six Different PBDEs.

Indiscriminate use of synthetic substances has led to environmental contamination and increasing human and animal exposure to harmful chemicals. Polybrominated flame retardants (PBDEs), which serve as non-covalent additives that enhance the safety of a variety of commercial and consumer goods, are an important class among potentially damaging synthetic substances. Its use is very common in developing countries, including Brazil. In theory, 209 different PBDE congeners exist, and many are currently being used during the manufacture of several products. Unfortunately, PBDEs are easily released from the original products, promptly reaching the environment. Knowledge about the toxicological power of these substances is still limited, which has prevented environmental and regulatory authorities from conducting adequate risk assessments. This research addresses the genotoxic and mutagenic potential of PBDEs. The effects of HepG2 cells and Salmonella typhimurium exposure to six main representatives of PBDEs, namely tetrabromodiphenyl ether (BDE-47), pentabromodiphenyl ether (BDE-99 and BDE-100), hexabromodiphenyl ether (BDE-153 and BDE-154) and decabromodiphenyl ether (BDE-209), were evaluated. The comet assay revealed that all the assessed BDEs exerted genotoxic effects but induced no micronuclei formation in HepG2 cells. These BDEs had no significant mutagenic effects on the Salmonella typhimurium strains TA98 and TA100. Taken together, the results of the genomic instability assays showed that PBDEs can represent a risk to the health of directly and indirectly exposed population, because the assessed BDEs induce genotoxic effects in the HepG2 cell line.

A perspective on the potential risks of emerging contaminants to human and environmental health.

Technological, agricultural, and medical advances have improved the lifestyle of humankind. However, these advances have caused new problems that affect the environment and future generations. Emerging contaminants display properties such as low degradation potential and environmental persistence. In addition, most contaminants are lipophilic, which culminates in high bioaccumulation. The disposal of pharmaceuticals and personal care products into the environment underlies microbial and bacterial resistance. Plasticizers change several characteristics of industrialized materials, such as flexibility, but they are potentially carcinogenic and disrupt the endocrine system. Pesticides prevent the propagation of numerous kinds of pests; nevertheless, they exert neurotoxic and mutagenic effects, and they impact the environment negatively. Addition of flame retardants to a number of materials prevents flame propagation; however, after their release into the environment, these chemicals may bioaccumulate in organisms and disrupt the endocrine system, too. Surfactants can change the surface and interfacial properties of liquids, but their presence in the environment can interfere with countless enzymes and can even impair the endocrine system of various organisms and induce the feminization of species. Hence, gaining knowledge about emerging contaminants is increasingly important to minimize future damage and enable proper monitoring of each class of compounds in the environment which will help to improve legislation on this matter.

Toxicity of brominated flame retardants, BDE-47 and BDE-99 stems from impaired mitochondrial bioenergetics.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants, and they have been detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies report liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99.

BDE-154 induces mitochondrial permeability transition and impairs mitochondrial bioenergetics.

Brominated flame retardants are used in various consumer goods to make these materials difficult to burn. Polybrominated diphenyl ethers (PBDE), which are representative of this class of retardants, consist of two benzene rings linked by an oxygen atom, and contain between 1 and 10 bromine atoms in their chemical structure, with the possibility of up to 209 different congeners. Among these congeners, BDE-154 (hexa-BDE) is persistent in the environment and easy to detect in the biota, but no apparent information regarding the mechanism underlying action and toxicity is available. Mitochondria, as the main energy-producing organelles, play an important role in the maintenance of various cellular functions. Therefore, mitochondria were used in the present study as an experimental model to determine the effects of BDE-154 congener at concentrations ranging from 0.1 µM to 50 µM. Our results demonstrated that BDE-154 interacts with the mitochondrial membrane, preferably by inserting into the hydrophobic core of the mitochondrial membrane, which partially inhibits respiration, dissipates Δψ, and permeabilizes the inner mitochondrial membrane to deplete ATP. These effects are more pronounced at concentrations equal to or higher than 10 µM. Results also showed that BDE-154 did not induce reactive oxygen species (ROS) accumulation within the mitochondria, indicating the absence of oxidative stress. Therefore, BDE-154 impairs mitochondrial bioenergetics and permeabilizes the mitochondrial membrane, potentially leading to cell death but not via mechanisms involving oxidative stress.

Polybrominated diphenyl ether congener (BDE-100) induces mitochondrial impairment.

Brominated flame retardants are used in various consumer products to increase their resistance to fire and/or high temperatures. Polybrominated diphenyl ethers (PBDEs) are representatives of this class and among the most widely used congeners, and BDE-100 is produced on a large scale. There is a lack of toxicological data about these compounds, which has recently become a matter of concern to the scientific community. The mitochondria are recognized as the main energy-producing organelles, as well as playing a vital role in the maintenance of many cell functions. Therefore, mitochondria were used in the present work as an experimental model to evaluate the effects of the BDE-100 congeners at concentrations ranging from 0.1 µM to 50 µM. The results showed that high concentrations of BDE-100 were able to induce mitochondrial alterations. It was observed that the substance had an affinity for the hydrophilic portion of the mitochondrial membrane, as monitored by ANS, inhibiting the glutamate + malate-stimulated mitochondrial respiration and also inducing dissipation of the mitochondrial membrane potential, deregulation of calcium homoeostasis and mitochondrial swelling, the latter being insensitive to cyclosporin A (CsA) but partially inhibited by Ruthenium Red and N-ethyl maleimide. In addition, a significant reduction in mitochondrial ATP content was found, but on the other hand, no oxidative stress was observed after exposure of the mitochondria to BDE-100. These results show the key role of mitochondria in the cytotoxicity induced by BDE-100.