Assessment of toxic mechanisms and mode of action to three different levels of species for 14 antibiotics based on interspecies correlation, excess toxicity, and QSAR.

Antibiotics have received much attention owing to their ecotoxicity toward nontarget aquatic creatures. However, the mode of action (MOA) of toxicity against nontarget organisms is unclear in some aquatic organisms. In this study, the comparison of toxicities through interspecies correlations, excess toxicity calculated from toxicity ratio, and quantitative structure-activity relationship (QSAR) was carried out to investigate the MOAs for 14 antibiotics among Daphnia magna, Vibrio fischeri, and Pseudokirchneriella subcapitata. The results showed that interspecies toxicity correlations were very poor between any two of the three species for the 14 antibiotics. The toxicity ratio revealed that most antibiotics exhibited excess toxicity to algae and Daphnia magna but not to V. fischeri, demonstrating that some antibiotics share the same MOA, but some antibiotics share different MOAs among the three different levels of species. P. subcapitata was the most sensitive species, and V. fischeri was the least sensitive species. This is because of the differences in the biouptake and interactions of antibiotics with the target receptors between the three different trophic levels of the species. Molecular docking simulations suggested that the toxicity of antibiotics depends highly on their interactions with target receptors through hydrogen bonds, electrostatic or polar interactions, π bond interactions, and van der Waals forces. QSAR models demonstrated that hydrogen bonding and electrophilicity/nucleophilicity play key roles in the interaction of antibiotics with different receptors in the three species. The toxic mechanisms of antibiotics are attributed to the interactions between electrophilic antibiotics and biological nucleophiles, and hydrogen-bond interactions. These results are valuable for understanding the toxic mechanisms and MOA of the three different levels of species.

Comparison of modes of toxic action between Rana chensinensis tadpoles and Limnodrilus hoffmeisteri worms based on interspecies correlation, excess toxicity and QSAR for class-based compounds.

Insecticides, fungicides, dinitrobenzenes, resorcinols, phenols and anilines are widely used in agricultural and industrial productions. However, their modes of toxic action are unclear in some nontarget organisms, such as worms and tadpoles. In this study, acute toxicity data was experimentally collected for Limnodrilus hoffmeisteri worms and Rana chensinensis tadpoles, respectively. Interspecies correlation and excess toxicity were calculated to determine modes of action (MOAs) between the two species for class-based compounds. The result showed that, although the interspecies correlation of toxicity between the tadpoles and worms is significant with a coefficient of determination (R2) of 0.83, tadpoles are more sensitive than the worms and toxicity values between these two species are not identical with an overall 0.43 log unit difference. Regression analysis revealed that the toxicity of nonpolar narcotics or baseline compounds is linearly related to hydrophobicity for both the tadpoles and worms and the two baseline models are parallel, suggesting that these nonpolar narcotics share the same MOA between the two species. The difference of baseline toxicities between the two species is attributed to differences in bioconcentration factors. Analysis of the excess toxicity calculated from the toxicity ratio (TR) suggested that phenols and anilines can be classified as polar narcotics, not only to fish, but also to the tadpoles and worms. These compounds are more toxic than the baseline compounds and quantitative structure-activity relationship (QSAR) models show that their toxicity is linearly related to chemical hydrophobicity and polarity. Analysis of the excess toxicity reveals that aminophenols and resorcinols can be classified as reactive compounds, and insecticides and fungicides can be classified as specifically-acting compounds for both species. These compounds exhibited significantly greater toxic effect to both the tadpoles and worms. QSAR models have been developed to describe the toxic mechanisms for nonpolar narcotics, polar narcotics, reactive chemicals and specifically-acting compounds, and a theoretical equation has been derived to explain the effect of bio-uptake and interaction of the chemical with target receptors for both tadpole and worm toxicity. Our study reveals that tadpole toxicity can be estimated from worm toxicity data and the two species can serve as surrogates for each other in the safety evaluation of organic pollutants.

Comparison of modes of action between fish, cell and mitochondrial toxicity based on toxicity correlation, excess toxicity and QSAR for class-based compounds.

Mitochondria are significant targets in cells for many environmental chemicals. Mitochondrial damage and dysfunction can lead to apoptosis and death of fish. The objectives of this study were to compare the modes of action (MOAs) between fish, cell and mitochondrial toxicity. To achieve the goal, toxicity correlation, excess toxicity and quantitative structure-activity relationship (QSAR) were investigated between these three toxicity endpoints for a wide range of compounds. Results showed that fish toxicity is well correlated to cytotoxicity, but overall fish toxicity is relatively greater than the cytotoxicity. On the other hand, fish or cell toxicity is poorly related to mitochondrial toxicity, suggesting some compounds share same toxic mechanism but some not. The excess toxicity calculated from toxicity ratio (TR) shows that specifically-acting compounds in cytotoxicity, such as insecticides, fungicides, herbicides, dyes and medications used to treat cancer, depression, heart failure and blood pressure, are active compounds in mitochondrial toxicity. However, the less inert compounds identified in fish and cell toxicity exhibit greatly mitochondrial toxicity. QSAR models reveal that fish or cell toxicity is closely related to the chemical hydrophobicity, ionization, energy of lowest unoccupied molecular orbital, hydrogen bonding potential and stability. These descriptors reflect chemical bio-uptake, reactivity and interaction with target receptors. On the other hand, binomial model reveals that mitochondrial toxicity is closely related to the chemical hydrophobicity and polarizability/dipolarity, indicating bio-uptake and Van der Waals interaction play key roles in mitochondrial toxicity. Theoretical equations have been used to explain the toxicity correlation, excess toxicity and QSAR for fish, cell and mitochondrial toxicity. Above results suggest that cytotoxicity can serve as a surrogate for fish toxicity and be used in the safety evaluation of organic pollutants in aqueous environment, but not mitochondrial toxicity, although some compounds share same modes of action between fish or cell toxicity and mitochondrial toxicity.

Bio-uptake, tissue distribution and metabolism of a neonicotinoid insecticide clothianidin in zebrafish.

Neonicotinoids have been often detected in aquatic environment with high concentrations; however, little is known about their risk and fate to/in fish. This study systematically investigated the bio-uptake, tissue distribution and metabolism of neonicotinoids in zebrafish, taking clothianidin (CLO) as an example. The results revealed the uptake and elimination kinetics of CLO in whole fish and different tissues was very similar, and its bioconcentration factor (<1) indicates the low bioaccumulation potential in zebrafish. The highest accumulative tissues for CLO were found to be intestine and liver. Eight biotransformation products were identified in intestine and liver, and the metabolic pathways were found to be N-demethylation and nitro-reduction. The metabolic kinetics of two products (desmethyl clothianidin and clothianidin urea) revealed the metabolism of CLO mainly occurred in liver and intestine. This suggested that the hepatobiliary system played an important role in the metabolism and elimination of CLO. This study provides a comprehensive evaluation of the toxicokinetics of CLO in zebrafish, and these results can contribute to its ecological risk assessment.

Behavioral and developmental toxicity assessment of the strobilurin fungicide fenamidone in zebrafish embryos/larvae (Danio rerio).

Strobilurin fungicides are among the most widely used in the world and have characteristics that include high water solubility and toxicity to aquatic organisms. While several studies report on mechanisms of toxicity of strobilurins in fish, there are no data on the sub-lethal toxicity of fish to the fungicide fenamidone. To address this gap, survival and hatch rate, deformities, mitochondrial bioenergetics, expression of oxidative stress and apoptotic genes, and behavior (locomotor activity and anxiolytic-related behaviors) were assessed in zebrafish embryos and larvae following exposure to fenamidone. Fenamidone negatively affected development of zebrafish embryos, causing a delay of hatching time at concentrations of 2.5 and 5 µM. Fenamidone caused morphological deformities in zebrafish, including pericardial edema, yolk sac edema, tail deformities, and spinal curvature. Exposure to 1.5 µM fenamidone reduced surface area of swim bladder in larvae at 6 dpf. Fenamidone significantly reduced oxygen consumption rates of embryos; 5 µM fenamidone decreased basal respiration (~85%), oligomycin induced ATP-linked respiration (~70%), FCCP-induced maximal respiration (~75%) and non-mitochondrial respiration (~90%) compared to controls. Sod2 mRNA levels were decreased by fenamidone in larval fish. Locomotor activity was significantly decreased in zebrafish larvae following exposure to 2 µM fenamidone but there was no evidence for anxiolytic nor anxiety-related behaviors (exposures of 100 nM up to 1.5 µM). This study addresses a data gap for potential risks associated with fenamidone exposure in developing fish.

Exposure to acetochlor impairs swim bladder formation, induces heat shock protein expression, and promotes locomotor activity in zebrafish (Danio rerio) larvae.

Acetochlor is one of the most widely used herbicides in the world, however, there are few data on the sub-lethal effects of acetochlor on early developmental stages of fish. To address this, we measured survival, deformity, swim bladder formation, embryo oxygen consumption rates, reactive oxygen species (ROS) levels, transcripts (related to swim bladder formation, oxidative damage response, and apoptosis) and behavior responses following exposure to acetochlor (0.001 µM up to 125 µM). Exposure to acetochlor at concentrations 50 µM and above exerted 100% mortality after 3 dpf, and significantly reduced the size of the swim bladder (25 µM). In embryos, basal respiration, oligomycin-induced ATP production, and maximal respiration were decreased 30-60% following a 24 h exposure to 125 µM acetochlor. Acetochlor did not affect ROS levels up to 25 µM in larvae with acute exposure. Acetochlor at 25 µM increased mRNA levels of bax1, hsp70, and hsp90a by ~4-fold in larval zebrafish. In both the visual motor response and light-dark preference test, 25 µM acetochlor increased locomotor activity of larval fish. At lower exposure concentrations, 100 and 1000 nM acetochlor increased the mean time spent in the dark zone, suggesting promotion of anxiolytic behavior. This study presents a comprehensive evaluation of sublethal effects of acetochlor, spanning molecular responses to behavior, which can be used to refine risk assessment decisions for aquatic environments.

Discrimination of active and inactive substances in cytotoxicity based on Tox21 10K compound library: Structure alert and mode of action.

In vitro cytotoxicity assay is an ideal alternative method for the in vivo toxicity in the risk assessment of pollutants in environment. However, modes of action (MOAs) of cytotoxicity have not been investigated for a wide range of compounds. In this paper, binomial and recursive partitioning analysis were carried out between the cytotoxicity and molecular descriptors for 8981 compounds. The results showed that cytotoxicity is strongly related to the chemical hydrophobicity and excess molar refraction, indicating the bio-uptake and chemical-receptor interaction through π and n electron pair play important roles in the cytotoxicity. The decision tree derived from recursive partitioning analysis revealed that the studied compounds could be divided into 25 groups and their structural characteristics could be used as structure alert to identify active and inactive compounds in cytotoxicity. The descriptors used in the decision tree revealed that chemical ionization and bioavailability could affect the cytotoxicity for ionizable and highly hydrophobic compounds. Comparison of MOAs based on Verhaar's classification scheme showed that many inert or less inert compounds were inactive substance, and many reactive or specifically-acting compounds were active substances in the cytotoxicity. In vitro toxicity assay instead of in vivo toxicity assay can be used in the environmental hazard and risk assessment of organic pollutants. The descriptors used in the binomial equation and decision tree reveal that chemical hydrophobicity, ionization and solubility play very important roles for identification of active and inactive compounds. The results obtained in this paper are valuable for understanding the modes of action in cytotoxicity and in vivo-in vitro toxicity relationship.

Freshwater quality criteria of four strobilurin fungicides: Interspecies correlation and toxic mechanism.

Strobilurin fungicides are widely used pesticides in the world. They can have toxic effects not only to target organisms, but also to nontarget organisms. To assess their ecological risk, species sensitivity distributions (SSDs) are required for the development of water quality criteria (WQC). In this paper, the acute toxicity of four methoxyacrylate fungicides were experimentally determined and evaluated at 24, 48, 72 and 96 h for the species of Rana chensinensis and Limnodrilus hoffmeisteri, respectively. Acute and chronic HC5 (5% hazard concentration) values and WQC values were calculated from SSDs based on the toxicity values determined in this paper and compiled from literature. SSDs revealed that aquatic animals were relatively sensitive species and aquatic plants are insensitive species for the four fungicides. However, different orders of species sensitivity in the acute and chronic toxicity indicated that these four fungicides had different toxic mechanisms or mode of action (MOA) to different species. According to toxicity correlation and principal component analysis (PCA), the kresoxim-methyl toxicity was very close to trifloxystrobin as compared with others due to that they are neutral compounds with very similar physicochemical properties. Quantitative structure-activity relationship (QSAR) revealed that toxicity of strobilurin fungicides were dependent both on chemical hydrophobicity and hydrogen bond basicity. These two molecular descriptors reflect the bio-uptake process and interaction of compounds with target receptors in an organism. WQC values and interspecies correlation are valuable for assessing water quality and understanding toxic mechanisms to different species.

Identification of active and inactive agonists/antagonists of estrogen receptor based on Tox21 10K compound library: Binomial analysis and structure alert.

Endocrine disrupting chemicals can mimic, block, or interfere with hormones in organisms and subsequently affect their development and reproduction, which has raised significant public concern over the past several decades. To investigate (quantitative) structure-activity relationship, 8280 compounds were compiled from the Tox21 10K compound library. The results show that 50% activity concentrations of agonists are poorly related to that of antagonists because many compounds have considerably different activity concentrations between the agonists and antagonists. Analysis on the chemical classes based on mode of action (MOA) reveals that estrogen receptor (ER) is not the main target site in the acute toxicity to aquatic organisms. Binomial analysis of active and inactive ER agonists/antagonists reveals that ER activity of compounds is dominated by octanol/water partition coefficient and excess molar refraction. The binomial equation developed from the two descriptors can classify well active and inactive ER chemicals with an overall prediction accuracy of 73%. The classification equation developed from the molecular descriptors indicates that estrogens react with the receptor through hydrophobic and π-n electron interactions. At the same time, molecular ionization, polarity, and hydrogen bonding ability can also affect the chemical ER activity. A decision tree developed from chemical structures and their applications reveals that many hormones, proton pump inhibitors, PAHs, progestin, insecticides, fungicides, steroid and chemotherapy medications are active ER agonists/antagonists. On the other hand, many monocyclic/nonaromatic chain compounds and herbicides are inactive ER compounds. The decision tree and binomial equation developed here are valuable tools to predict active and inactive ER compounds.

Relative comparison of strobilurin fungicides at environmental levels: Focus on mitochondrial function and larval activity in early staged zebrafish (Danio rerio).

Strobilurin fungicides are used globally and have been detected in microgram per liter concentrations in aquatic environments. Here, we determined the potential toxicity of four commonly used strobilurins (azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin) on mitochondrial function and locomotor activity of larval zebrafish at an environmentally relevant level. As the mode of action of strobilurins in fungi is binding to cytochrome bc1 in mitochondrial complex III, we evaluated exposure effects on mitochondrial oxidative phosphorylation of zebrafish, by measuring oxygen consumption rates, mitochondria-related enzyme activities, and transcripts levels for genes associated with the electron transfer chain and citric acid cycle. We found that 50 nM pyraclostrobin and trifloxystrobin lowered basal respiration, oligomycin-induced ATP respiration, and maximal respiration of embryos. Dysfunction in mitochondrial bioenergetics was associated with changes in mitochondrial complex III activity and transcripts of oxidative respiration and stress-related genes. Lower activity of complex III, and reduced cytb mRNA levels were hypothesized to contribute to reduced electron supply to complex IV and V. Both coxI and atp6 were up-regulated, suggesting a compensatory response to impaired oxidative respiration. Cluster analysis indicated that strobilurin-induced oxidative stress and cytb transcript were related to impaired oxidative phosphorylation. We also assessed larval behavior responses, as reduced ATP can affect activity. We observed that pyraclostrobin and trifloxystrobin induced hypoactive responses in zebrafish. At 50 nM, azoxystrobin and kresoxim-methyl exerted no effects on mitochondrial function nor locomotion of zebrafish. Studies such as this are important for determining sublethal toxicity to these fungicides, as widespread detection of strobilurins in aquatic environments suggests there is a potential for adverse effects in aquatic organisms.

Evaluation of modes of action of pesticides to Daphnia magna based on QSAR, excess toxicity and critical body residues.

Agricultural pesticides serve as effective controls of unwanted weeds and pests. However, these same chemicals can exert toxic effects in non-target organisms. To determine chemical modes of action, the toxicity ratio (TR) and critical body residues (CBRs) of 57 pesticides were calculated for Daphnia magna. Results showed that the CBR values of inert compounds were close to a constant while the CBR values of pesticides varied over a wider range. Although herbicides are categorized as specifically-acting compounds to plants, herbicides did not exhibit excess toxicity to Daphnia magna and were categorized as inert compounds with an average logTR = 0.41, which was less than a threshold of one. Conversely, fungicides and insecticides exhibited strong potential for toxic effects to Daphnia magna with an average logTR >2. Many of these chemicals act via disruption of the nervous, respiratory, or reproductive system, with high ligand-receptor binding activity which leads to higher toxicity for Daphnia magna. Molecular docking using acetylcholinesterase revealed that fungicides and insecticides bind more easily with the biological macromolecule when compared with inert compounds. Quantitative structure-activity relationship (QSAR) analysis revealed that the toxicity of fungicides was mainly dependent upon the heat of formation and polar surface area, while the toxicity of insecticides was more related to hydrogen-bond properties. This comprehensive analysis reveals that there are specific differences in toxic mechanisms between fungicides and insecticides. These results are useful for determining relative risk associated with pesticide exposure to aquatic crustaceans, such as Daphnia magna.

Comparison of modes of action among different trophic levels of aquatic organisms for pesticides and medications based on interspecies correlations and excess toxicity: Theoretical consideration.

Pesticides and medications have adverse effects in non-target organisms that can lead to different modes of action (MOAs). However, no study has been performed to compare the MOAs between different levels of aquatic species. In this study, theoretical equations of interspecies relationship and excess toxicity have been developed and used to investigate the MOAs among fish, Daphnia magna, Tetrahymena pyriformis and Vibrio fischeri for pesticides and medications. The analysis on the interspecies correlation and excess toxicity suggested that fungicides, herbicides and medications share the similar MOAs among the four species. On the other hand, insecticides share different MOAs among the four species. Exclusion of insecticides from the interspecies correlation can significantly improve regression coefficient. Interspecies relationship is dependent not only on the difference in interaction of chemicals with the target receptor(s), but also on the difference in bio-uptake between two species. The difference in physiological structures will result in the difference in bioconcentration potential between two different trophic levels of organisms. Increasing of molecular size or hydrophobicity will increase the toxicity to higher level of aquatic organisms; on the other hand, chemical ionization will decrease the toxicity to higher level organisms. Hydrophilic compounds can more easily pass through cell membrane than skin or gill, leading to greater excess toxicity to Vibrio fischeri, but not to fish and Daphnia magna.

Theoretical consideration on the prediction of in vivo toxicity from in vitro toxicity: Effect of bio-uptake equilibrium, kinetics and mode of action.

Although in vitro assay is an ideal alternative method for the in vivo toxicity prediction, different in vivo-in vitro correlations have been observed for the toxicity endpoints obtained from different levels of species. In this paper, theoretical in vivo-in vitro toxicity correlations have been developed for cytotoxicity versus human, mammalian and fish toxicity, respectively. These theoretical models were then used to investigate the correlations and the influencing factors between in vivo and in vitro toxicity. Bio-uptake equilibrium theory can well explain why there is a significant correlation between fish and cell toxicity (R2 = 0.70); why human toxicity is very close to fish toxicity; and why hydrophobic compounds exhibit relatively greater toxicity than reactive or specifically-acting compounds to human and fish as compared to cells. The kinetic theory can well explain why there is a very poor relationship between mammal and cell toxicity (R2 = 0.44). This paper reveals that polar and ionized compounds can more easily pass through cell membrane and have greater bioconcentration potential. Increasing of hydrophobicity and ionization can increase the cytotoxicity. Inclusion of descriptors representing hydrophobicity, ionization, acidity and absorption into the correlation equations can significantly improve the correlations of cytotoxicity with human and fish toxicity (R2 > 0.8), but not with mammal toxicity (R2 = 0.49). These descriptors reflect the differences of the toxicodynamics and toxicokinetics between cells and organisms.

Toxicity of some prevalent organic chemicals to tadpoles and comparison with toxicity to fish based on mode of toxic action.

Although mode of action (MOA) plays a key role in the understanding of the toxic mechanism of chemicals, the MOAs of class-based compounds to tadpoles have not been investigated. To explore the MOAs, acute toxicity (expressed as log 1/LC50) to Rana chensinensis tadpoles were determined and molecular descriptors were calculated. Quantitative structure-activity relationship (QSAR) showed that toxicity to tadpoles is closely related to the chemical octanol/water partition coefficient (log KOW), energy of the lowest unoccupied molecular orbital (ELUMO), and number of hydrogen bond donors and acceptors (NHDA), representing the bio-uptake potential in tadpoles, the electrophilicity and hydrogen bonding capacity with target site(s), respectively. Comparison of the toxicity values between tadpoles and fish revealed that there were no significant differences for the overlapping compounds (average residual = 0.29 between tadpole and fish toxicity) with P values of interspecies correlation substantially less than 0.001. Classification of MOAs for the class-based compounds based on the excess toxicity calculated from toxicity ratio suggested that baseline, less inert compounds and some reactive or specifically-acting compounds share same MOAs between tadpoles and fish. Fish and tadpoles can serve as surrogates for each other in the safety evaluation of organic pollutants.

Comparison of modes of action between fish and zebrafish embryo toxicity for baseline, less inert, reactive and specifically-acting compounds.

The mode of action (MOA) plays a key role in the risk assessment of pollutants in water. Although fish is a key model organism used in the risk assessment of pollutants in water, the MOAs have not been compared between fish and embryo toxicity for classified compounds. In this paper, regression analysis was carried out for fish and embryo toxicities against the calculated molecular descriptors and MOAs were evaluated from toxicity ratio. The toxicity significantly related with the chemical hydrophobicity for baseline and less inert compounds, respectively, indicates that these two classes of compounds share the same MOAs between fish and embryos. Comparison of the toxicity ratios shows that reactive compounds exhibit excess toxicity to both fish and embryos. These compounds can react covalently with biologically target molecules through nucleophilic addition reactions, Michael addition oxidation, or amination. Comparing with baseline, less inert and reactive compounds, many specifically-acting compounds have strong docking capacity with protein molecules. Some specifically-acting compounds, such as fungicides, have very similar toxic effect to both fish and embryos. However, insecticides are more toxic to fish than embryos; herbicides and medications are more toxic to embryos than fish. Differences in the interactions of chemicals with target molecules or bioconcentration potentials between fish and embryos may result in the differences in toxic effects. There are some factors that influence the identification of MOAs, such as quality of toxicity data, bioavailability and ionization. These factors should be considered in the identification of MOAs in the risk assessment of organic pollutants.

Fluazinam impairs oxidative phosphorylation and induces hyper/hypo-activity in a dose specific manner in zebrafish larvae.

Fluazinam is a pyridinamine fungicide that induces oxidative stress and mitochondrial damage in cells, and it has been reported to be neurotoxic. To characterize the biological effects of fluazinam, we assessed mitochondrial bioenergetics, dopamine system expression, and behavior of early life staged zebrafish (0.01 µM-0.5 µM). Fluazinam at environmentally-relevant levels did not induce sub-lethal effects in larvae, but at the LC50 (0.5 µM), fluazinam decreased basal and ATP-linked respiration significantly in embryos. As mitochondria are directly related to redox homeostasis and apoptosis, the expression of genes related to oxidative stress and apoptosis were measured. Superoxide dismutase 2 (sod2), heat stock protein 70 (hsp70), bcl2-associated X protein (bax), and caspase 9 (casp9) mRNA levels were up-regulated by 0.5 µM fluazinam. Taken together, there was evidence for mitochondrial dysfunction and oxidative damage at the highest concentration of fluazinam (0.5 µM) tested. As there are reports for fluazinam-induced neurotoxicity in dopamine synthesizing cells, transcriptional targets in the dopamine system were assessed in the zebrafish. Tyrosine hydroxylase 1 (th1) and dopamine receptor 2a (drd2a) mRNA levels were decreased by 0.5 µM fluazinam, suggesting that this fungicide may affect the dopaminergic system. To further assess the potential for fluazinam-mediated neuromodulation, the dark photokinesis response was assessed in larvae following exposure. Larvae exposed to 0.1 µM fluazinam showed hyperactivity, while larvae exposed to 0.2 and 0.3 µM showed hypo-activity. This study demonstrates that fluazinam disrupts mitochondrial bioenergetics in zebrafish, inducing an oxidative stress response, and aberrant behaviors in larvae that are dose dependent.

High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction.

Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the µM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction.

The impact of exposure route for class-based compounds: a comparative approach of lethal toxicity data in rodent models.

Relationships of toxicities from intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) and intragastric (i.g.) exposure routes to mice were investigated. Regression analysis showed that the toxicities from i.v. route is strongly correlated with i.p. and s.c. routes, but poorly with i.g. route. Close toxicities from different routes for some compounds indicate that distribution rate is the determining step and dictates chemical concentration at the target site(s). On the other hand, the absorption rate is the determining step for many compounds, which lead to different toxicities between exposure routes. The classified compounds characterized as having either absorption or distribution rate determining step were based upon the comparison of toxicities from the different routes. We found that some aliphatic acids and benzoic acids have lower toxicity values from i.g. route compared to an i.v. route because of poor absorption. Many compounds show low toxic effects from i.g. route than those from other routes because of the first-pass metabolism in the gastrointestinal tract, resulting in the poor relationship for toxicities between i.g. and i.v. or other routes. Stepwise regression analysis showed that physicochemical properties of a compound, such as molecular volume, polarizability and hydrophobicity, significantly affect adsorption rate, which leads to different toxicities based upon exposure routes. Comparison of the toxicities between mice and rats indicate that toxic effect and the toxicokinetic processes in mice are very similar to that in rats. A universal correlation equation has been developed for the toxicities between rats and mice from different exposure routes, which can be applied to predict toxicities across species.

Mitochondrial bioenergetics and locomotor activity are altered in zebrafish (Danio rerio) after exposure to the bipyridylium herbicide diquat.

Diquat is a non-selective bipyridylium herbicide which has replaced its sister compound paraquat, as paraquat is associated to an increased risk for Parkinson's disease. However, the propensity of diquat to propagate reactive oxygen species ensures that diquat remains an exposure risk in non-target organisms. In this study, zebrafish (Danio rerio) embryos were exposed to diquat (1, 10, 100µM) beginning at ∼6h post fertilization for up to 7days to learn more about the mechanisms underlying diquat toxicity during vertebrate development. Zebrafish embryos exposed to diquat for 96h did not show any significant mortality nor deformity compared to controls. Moreover, there was no difference in the timing of hatch, an indicator of stress, for fish exposed to diquat. To determine whether changes in mitochondrial bioenergetics occurred in early development as a response to diquat exposure, oxygen consumption rate was measured in whole embryos. Basal respiration and ATP production were decreased following a 24h diquat exposure at 100µM, suggesting that diquat negatively affects oxidative phosphorylation. We also assessed locomotor behavior as a sensitive endpoint for impaired activity and neurotoxicity. Seven day old (7 dpf) zebrafish treated with diquat at the highest doses tested (10-100µM) showed an increase (hyper-activity) in total distance travelled, velocity, movement cumulative duration, and overall activity compared to unexposed fish. Lastly, in 7d fish, we measured transcripts related to redox balance and apoptosis as diquat has been reported to induce oxidative stress and can affect mitochondrial bioenergetics. Larvae exposed to 10µM diquat showed higher transcript levels of catalase compared to control fish, implying that reactive oxygen species are produced following diquat exposure. Transcript levels of sod1, sod2, bcl2, bax and caspase 3 however did not vary in abundance among treatments with diquat. This study improves mechanistic understanding of diquat in fish at early stages of development and presents evidence that diquat disrupts mitochondrial bioenergetics and behavior.

Paraquat affects mitochondrial bioenergetics, dopamine system expression, and locomotor activity in zebrafish (Danio rerio).

The dipyridyl herbicide paraquat induces oxidative stress in cells and is implicated in adult neurodegenerative diseases. However, less is known about paraquat toxicity in early stages of vertebrate development. To address this gap, zebrafish (Danio rerio) embryos were exposed to 1, 10 and 100 µM paraquat for 96 h. Paraquat did not induce significant mortality nor deformity in embryos and larvae, but it did accelerate time to hatch. To evaluate whether mitochondrial respiration was related to earlier hatch times, oxygen consumption rate was measured in whole embryos. Maximal respiration of embryos exposed to 100 µM paraquat for 24 h was reduced by more than 70%, suggesting that paraquat negatively impacts mitochondrial bioenergetics in early development. Based upon this evidence for mitochondrial dysfunction, transcriptional responses of oxidative stress- and apoptosis-related genes were measured. Fish exposed to 1 µM paraquat showed higher expression levels of superoxide dismutase 2, heat shock protein 70, Bcl-2-associated X protein, and B-cell CLL/lymphoma 2a compared to control fish. No differences among groups were detected in larvae exposed to 10 and 100 µM paraquat, suggesting a non-monotonic response. We also measured endpoints related to larval behavior and dopaminergic signaling as paraquat is associated with degeneration of dopamine neurons. Locomotor activity was stimulated with 100 µM paraquat and dopamine transporter and dopamine receptor 3 mRNA levels were increased in larvae exposed to 1 µM paraquat, interpreted to be a compensatory response at lower concentrations. This study improves mechanistic understanding into the toxic actions of paraquat on early developmental stages.