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 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.

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.

Linking algal growth inhibition to chemical activity: Excess toxicity below 0.1% of saturation.

Chemical activity quantifies the energetic level of an organic compound relative to its pure liquid [0-1], and several studies have reported that baseline toxicity generally requires chemical activities of 0.01-0.1. The first aim was to challenge this chemical activity range for baseline toxicity. Algal growth inhibition data (median effective concentrations, EC50) were compiled from two recent studies and included 108 compounds categorised as non-polar (mode of toxic action, MOA1) and polar (MOA2) narcotics. These data were linked to chemical activity by (1) plotting them relative to a regression for (subcooled) liquid solubility (SL), which served as visual reference for chemical activity of unity and (2) determining EC50/SL ratios that essentially equal median effective chemical activity (Ea50). Growth inhibition required chemical activity >0.01 for MOA1 and >0.001 for MOA2 compounds. The second aim was to identify compounds exerting excess toxicity, i.e., when growth inhibition occurred at chemical activity <0.001. From a recent review with 2323 data entries, 315 EC50 values passed our selection criteria. 280 of these EC50 values were within or near the baseline toxicity range (Ea50>0.001), and 25 compounds were found to exert excess toxicity (Ea50<0.001). Of these compounds, 16 are pesticides or precursors. Methodologically, this study includes two methods for translating EC50 values into the chemical activity framework, each having advantages and limitations. Scientifically, this study confirms that baseline toxicity generally requires chemical activities of 0.01-0.1 and extends the application of the chemical activity approach beyond baseline toxicity, by demonstrating its utility to identify compounds that exert excess toxicity.

Toxicity of 13 different antibiotics towards freshwater green algae Pseudokirchneriella subcapitata and their modes of action.

Although modes of action (MOAs) play a key role in the understanding of the toxic mechanism of chemicals, the MOAs have not been investigated for antibiotics to green algae. This paper is to discriminate excess toxicity from baseline level and investigate the MOAs of 13 different antibiotics to algae by using the determined toxicity values. Comparison of the toxicities shows that the inhibitors of protein synthesis to bacteria, such as azithromycin, doxycycline, florfenicol and oxytetracycline, exhibit significantly toxic effects to algae. On the other hand, the cell wall synthesis inhibitors, such as cefotaxime and amoxicillin, show relatively low toxic effects to the algae. The concentrations determined by HPLC indicate that quinocetone and amoxicillin can be easily photodegraded or hydrolyzed during the toxic tests. The toxic effects of quinocetone and amoxicillin to the algae are attributed to not only their parent compounds, but also their metabolites. Investigation on the mode of action shows that, except rifampicin, all the tested antibiotics exhibit excess toxicity to Pseudokirchneriella subcapitata (P. subcapitata). These antibiotics can be identified as reactive modes of action to the algae. They act as electrophilic mechanism of action to P. subcapitata. These results are valuable for the understanding of the toxic mechanism to algae.

Discrimination of excess toxicity from baseline level for ionizable compounds: Effect of pH.

The toxic effect can be affected by pH in water through affecting the degree of ionization of ionizable compounds. Wrong classification of mode of action can be made from the apparent toxicities. In this paper, the toxicity data of 61 compounds to Daphnia magna determined at three pH values were used to investigate the effect of pH on the discrimination of excess toxicity. The results show that the apparent toxicities are significantly less than the baseline level. Analysis on the effect of pH on bioconcentration factor (BCF) shows that the log BCF values are significantly over-estimated for the strongly ionizable compounds, leading to the apparent toxicities greatly less than the baseline toxicities and the toxic ratios greatly less than zero. A theoretical equation between the apparent toxicities and pH has been developed basing on the critical body residue (CBR). The apparent toxicities are non-linearly related to pH, but linearly to fraction of unionized form. The determined apparent toxicities are well fitted with the toxicities predicted by the equation. The toxicities in the unionized form calculated from the equation are close to, or greater than the baseline level for almost all the strongly ionizable compounds, which are very different from the apparent toxicities. The studied ionizable compounds can be either classified as baseline, less inert or reactive compounds in D. magna toxicity. Some ionizable compounds do not exhibit excess toxicity at a certain pH, due not to their poor reactivity with target molecules, but because of the ionization in water.

Comparison of toxicity of class-based organic chemicals to algae and fish based on discrimination of excess toxicity from baseline level.

Toxicity data to fish and algae were used to investigate excess toxicity between species. Results show that chemicals exhibiting excess toxicity to fish also show excess toxicity to algae for most of the compounds. This indicates that they share the same mode of action between species. Similar relationships between logKOW and toxicities to fish and algae for baseline and less inert compounds suggest that they have similar critical body residues in the two species. Differences in excess toxicity for some compounds suggest that there is a difference of physiological structure and metabolism between fish and algae. Some reactive compounds (e.g. polyamines) exhibit greater toxic effects for algae than those for fish because of relatively low bio-uptake potential of these hydrophilic compounds in fish as compared with that in algae. Esters exhibiting greater toxicity in fish than that in algae indicate that metabolism can affect the discrimination of excess toxicity from baseline level. Algae growth inhibition is a very good surrogate for fish lethality. This is not only because overall toxicity sensitivity to algae is greater than that to fish, but also the excess toxicity calculated from algal toxicity can better reflect reactivity of compounds with target molecules than fish toxicity.

Discrimination of excess toxicity from narcotic effect: influence of species sensitivity and bioconcentration on the classification of modes of action.

The toxicity data of 2624 chemicals to fish, Daphniamagna, Tetrahymenapyriformis and Vibriofischeri were used to investigate the effects of species sensitivity and bioconcentration on excess toxicity. The results showed that 47 chemical classes were identified as having the same modes of action (MOAs) to all four species, but more than half of the classes were identified as having different MOAs. Difference in chemical MOAs is one of the reasons resulting in the difference in toxic effect to these four species. Other important reasons are the difference in sensitivity and bioconcentration of species. Among the four species, V. fischeri has the most compounds identified as reactive MOA. This may be due to some compounds can be easily absorbed into the bacteria, react with the DNA or proteins, disrupt the normal function of the cell and exhibit significantly greater toxicity to the bacteria. On the other hand, the skin and lipid content of aqueous organisms can strongly inhibit the bio-uptake for some reactive compounds, resulting in a less toxic effect than expected. D. magna is the most sensitive species and T. pyriformis is the least sensitive species of the four species. For a comparison of interspecies toxicity, we need to use the same reference threshold of excess toxicity. However, some reactive compounds may be identified as baseline or less inert compounds for low sensitive species from the threshold developed from high sensitive species. The difference in the discrimination of excess toxicity to different species is not only because of the difference in MOAs for some compounds, but also due to the difference in sensitivity and bioconcentration.

Experimental verification of structural alerts for the protein binding of sulfur-containing compounds.

As often noted by Dr. Gilman Veith, a major barrier to advancing any model is defining its applicability domain. Sulfur-containing industrial organic chemicals can be grouped into several chemical classes including mercaptans (RSH), sulfides (RSR'), disulfides (RSSR'), sulfoxides (RS(=O)R'), sulfones (RS(=O)(=O)R'), sulfonates (ROS(=O)(=O)R') and sulfates (ROS(=O)(=O)OR'). In silico expert systems that predict protein binding reactions from 2D structure sub-divide these chemical classes into a variety of chemical reactive mechanisms and reactions which have toxic consequences. Using the protein binding profilers in version 3.1 of the OECD QSAR Toolbox, a series of sulfur-containing chemicals were profiled for protein binding potential. From these results it was hypothesized which sulfur-containing chemicals would be reactive or non-reactive in an in chemico glutathione assay and whether if reactive they would exhibit toxicity in excess of baseline in the Tetrahymena pyriformis population growth impairment assay. Subsequently, these hypotheses were tested experimentally. The in chemico data show that the in silico profiler predictions were generally correct for all chemical categories, where testing was possible. Mercaptans could not be assessed for GSH reactivity because they react directly with the chromophore 5,5'-dithiobis-(2-nitrobenzoic acid). With some exceptions, the major being disulfides, the in vitro toxicity data supported the in chemico findings.

Discrimination of excess toxicity from narcotic effect: comparison of toxicity of class-based organic chemicals to Daphnia magna and Tetrahymena pyriformis.

The discrimination of excess toxicity from narcotic effect plays a crucial role in the study of modes of toxic action for organic compounds. In this paper, the toxicity data of 758 chemicals to Daphnia magna and 993 chemicals to Tetrahymena pyriformis were used to investigate the excess toxicity. The result showed that mode of toxic action of chemicals is species dependent. The toxic ratio (TR) calculated from baseline model over the experimentally determined values showed that some classes (e.g. alkanes, alcohols, ethers, aldehydes, esters and benzenes) shared same modes of toxic action to both D. magna and T. pyriformis. However, some classes may share different modes of toxic action to T. pyriformis and D. magna (e.g. anilines and their derivatives). For the interspecies comparison, same reference threshold need to be used between species toxicity. The excess toxicity indicates that toxicity enhancement is driven by reactive or specific toxicity. However, not all the reactive compounds exhibit excess toxicity. In theory, the TR threshold should not be related with the experimental uncertainty. The experimental uncertainty only brings the difficulty for discriminating the toxic category of chemicals. The real threshold of excess toxicity which is used to identify baseline from reactive chemicals should be based on the critical concentration difference inside body, rather than critical concentration outside body (i.e. EC50 or IGC50). The experimental bioconcentration factors can be greatly different from predicted bioconcentration factors, resulting in different toxic ratios and leading to mis-classification of toxic category and outliers.