Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel mammalian findings, although select trends were conserved. Escitalopram showed greater activity than citalopram, duloxetine was more potent than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing but may serve as a rapid, low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/7408242b076aab3aeac08c8cd5d820f5.png"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs initially dissolved in different solvents. A) Responsiveness to SSRIs with drug stocks originally dissolved in water (n=4). B) Responsiveness to citalopram or escitalopram when their drug stocks are dissolved in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with drug stocks originally dissolved in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine when their drug stocks are dissolved in DMSO (n=4).
","imageTitle":"Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were either dissolved in water, DMSO, or ethanol starting between 20-50 mM and diluted down to 4 mM to start serial dilutions on a 96-well plate. Serial dilution drug exposures were performed in water rather than in buffering solutions to maximize solubility of the compounds tested since some have difficulty solubilizing in water. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles were prodded with a platinum rod and the number of responsive and nonresponsive animals were scored. These data were then expressed as % responsive (number of responsive/ (number of responsive+number of nonresponsive)). These data were then analyzed by Graphpad Prism and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"Caenorhabditis (C.) elegans is an invertebrate model organism that has been used in research to explore areas as diverse as development (Pincus and Slack 2010) to stress responses (Rodriguez, Snoek et al. 2013). This organism is advantageous in these studies for a number of reasons, most notably its relatively short maturation cycle (from egg to fertile adult in about 72 hours), their relative ease of cultivation, and its short lifespans (Tejeda-Benitez and Olivero-Verbel 2016). Furthermore, they can also be cultivated in liquid or on agar plates (Boyd, Smith et al. 2012), which allows for a higher degree of flexibility in their use. These nematodes also have proteins that are homologous to their mammalian counterparts (Lai, Chou et al. 2000). For example, they also have the serotonin transporter mod-4, which is analogous to that found in mammals (Yu, Vogt et al. 2023). Although C. elegans lack a norepinephrine transporter or the neurotransmitter norepinephrine itself, they utilize the neurotransmitter octopamine instead (Chase and Koelle 2007).
One underexploited avenue that involves C. elegans is that of drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt 2017), these animals have rarely been part of the normal drug development procedures (Letizia, Cornaglia et al. 2018), even though these medications have targets within C. elegans (Hobert 2013). This is most likely because C. elegans do not have some key systems that mammalian model organisms have such as a circulatory system and conscious behavioral characteristics and abilities (Giunti, Andersen et al. 2021). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale 2015), potentially the nonmammalian C. elegans can offer a cheaper alternative that will not replace the required animal tests prior to drug approval but may give guidance as to the drug design and development process.
In this study, we evaluated, in C. elegans, the toxicity of reuptake inhibitors, selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), in drug solutions within a 96-well plate exposure format. These drugs were selected because these medications are prescribed for mental health disorders (Machado and Einarson 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang, Han et al. 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham 1994). None have been tested in C. elegans even though these drugs may target homologous reuptake transporters in this animal. Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs with their original stock solution dissolved in water (FIG 1A). We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+ 0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LD50 where sertraline is least toxic (Davies and Kluwe 1998). For citalopram and escitalopram, we also tried to dissolve their original stock solution in DMSO or ethanol (FIG 1B). When dissolved in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram in DMSO, but not citalopram, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez, Bøgesø et al. 2004). This effect was like that observed when the original stock of citalopram and escitalopram was dissolved in ethanol (escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were dissolved in water (FIG 1C). We observed toxicity for only duloxetine (duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. This may be due to the absence of a norepinephrine transporter. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For example, in mammals, duloxetine has some additional affinity toward its dopamine transporters (Bymaster, Dreshfield-Ahmad et al. 2001). For milnacipran and levomilnacipran, their lack of toxicity may be because of their more selective affinity to the norepinephrine transporter compared to the dopamine transporter (Bymaster, Dreshfield-Ahmad et al. 2001). For venlafaxine, we dissolved its original drug stock in DMSO and like that observed in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors. Nevertheless, it does provide some indication of what is observed in mammalian models such as the fact that milnacipran did not demonstrate toxicity in C. elegans since this organism lacks its main target. Other indicators of C. elegans output may be better gauges of drug toxicology such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the initial solvent used to dissolve the drug. This indicates that the method of stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans cannot replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Pincus Z, Slack FJ. 2010. Developmental biomarkers of aging in Caenorhabditis elegans. Developmental Dynamics 239: 1306-1314.
","pubmedId":"","doi":"10.1002/dvdy.22224"},{"reference":"Rodriguez M, Snoek LB, De Bono M, Kammenga JE. 2013. Worms under stress: C. elegans stress response and its relevance to complex human disease and aging. Trends in Genetics 29: 367-374.
","pubmedId":"","doi":"10.1016/j.tig.2013.01.010"},{"reference":"Tejeda-Benitez L, Olivero-Verbel J. 2016. Caenorhabditis elegans, a Biological Model for Research in Toxicology. Reviews of Environmental Contamination and Toxicology,Reviews of Environmental Contamination and Toxicology Volume 237 : 1-35.
","pubmedId":"","doi":"10.1007/978-3-319-23573-8_1"},{"reference":"Boyd WA, Smith MV, Freedman JH. 2012. Caenorhabditis elegans as a Model in Developmental Toxicology. Methods in Molecular Biology,Developmental Toxicology : 15-24.
","pubmedId":"","doi":"10.1007/978-1-61779-867-2_3"},{"reference":"Lai CH, Chou CY, Ch'ang LY, Liu CS, Lin Wc. 2000. Identification of Novel Human Genes Evolutionarily Conserved inCaenorhabditis elegansby Comparative Proteomics. Genome Research 10: 703-713.
","pubmedId":"","doi":"10.1101/gr.10.5.703"},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"},{"reference":"Hobert O. 2010. Neurogenesis in the nematode Caenorhabditis elegans. WormBook : 10.1895/wormbook.1.12.2.
","pubmedId":"","doi":"10.1895/wormbook.1.12.2"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Giunti Sn, Andersen N, Rayes D, De Rosa MaJ. 2021. Drug discovery: Insights from the invertebrate Caenorhabditis elegans. Pharmacology Research & Perspectives 9: 10.1002/prp2.721.
","pubmedId":"","doi":"10.1002/prp2.721"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Byrd R. 1994. Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits. Fundamental and Applied Toxicology 22: 511-518.
","pubmedId":"","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"S�nchez C, B�ges� K, Ebert B, Reines E, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology 174: 10.1007/s00213-004-1865-z.
","pubmedId":"","doi":"10.1007/s00213-004-1865-z"},{"reference":"Bymaster F. 2001. Comparative Affinity of Duloxetine and Venlafaxine for Serotonin and Norepinephrine Transporters in vitro and in vivo, Human Serotonin Receptor Subtypes, and Other Neuronal Receptors. Neuropsychopharmacology 25: 871-880.
","pubmedId":"","doi":"10.1016/S0893-133X(01)00298-6\\"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"06094722-7a77-4d83-83fe-5a7ce6c85401","decision":"revise","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater activity than citalopram, duloxetine was more potent than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/a99ce23a55303f166a362234726594b1.png"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs initially dissolved in different solvents. A) Responsiveness to SSRIs with drug stocks originally dissolved in water (n=4). B) Responsiveness to citalopram or escitalopram when their drug stocks are dissolved in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with drug stocks originally dissolved in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine when their drug stocks are dissolved in DMSO (n=4).
","imageTitle":"Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were either dissolved in water, DMSO, or ethanol, starting at between 20-50 mM and then diluted down to 4 mM in water to start serial dilutions in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/([number of responsive+number of nonresponsive])). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"
Caenorhabditis (C.) elegans is an invertebrate model organism that has been used in research to explore areas as diverse as development(Pincus and Slack 2010) to stress responses (Rodriguez, Snoek et al. 2013). This organism is advantageous in these studies for a number of reasons, most notably its relatively short maturation cycle (from egg to fertile adult in about 72 hours), its relative ease of cultivation, and its short lifespans (Tejeda-Benitez and Olivero-Verbel 2016) Furthermore, they can also be cultivated in liquid or on agar plates (Boyd, Smith et al. 2012), which allows for a higher degree of flexibility in their use. These nematodes also have proteins that are homologous to their mammalian counterparts (Lai, Chou et al. 2000). For example, they also have the serotonin transporter mod-4, which is analogous to that found in mammals (Yu, Vogt et al. 2023). Although C. elegans lacks a norepinephrine transporter or the neurotransmitter norepinephrine itself, they utilize the neurotransmitter octopamine instead (Chase and Koelle 2007).
One underexploited avenue that involves C. elegans is that of drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt 2017), these animals have rarely been part of the normal drug development procedures (Letizia, Cornaglia et al. 2018), even though these medications have targets within C. elegans (Hobert 2013). This is most likely because C. elegans does not have some key systems that mammalian model organisms have, such as a circulatory system and conscious behavioral characteristics and abilities (Giunti, Andersen et al. 2021). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale 2015), potentially the nonmammalian C. elegans can offer a cheaper alternative that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
In this study, we evaluated, in C. elegans, the toxicity of reuptake inhibitors, selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), in drug solutions within a 96-well plate exposure format. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang, Han et al. 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham 1994). None have been tested in C. elegans, even though these drugs may target homologous reuptake transporters in C. elegans. Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs with their original stock solution dissolved in water (FIG 1A). We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+ 0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe 1998). For citalopram and escitalopram, we also tried to dissolve their original stock solution in DMSO or ethanol (FIG 1B). When dissolved in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram in DMSO, but not citalopram, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez, Bøgesø et al. 2004). This effect was like that observed when the original stock of citalopram and escitalopram was dissolved in ethanol (escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were dissolved in water (FIG 1C). We observed toxicity for only duloxetine (duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50 are observed for all SNRIs. This may be due to the absence of a norepinephrine transporter (Bymaster, Dreshfield-Ahmad et al. 2001). However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For example, in mammals, duloxetine has some additional affinity toward its dopamine transporters (Bymaster, Dreshfield-Ahmad et al. 2001). For milnacipran and levomilnacipran, their lack of toxicity may be because of their more selective affinity to the norepinephrine transporter compared to the dopamine transporter (Bymaster, Dreshfield-Ahmad et al. 2001). For venlafaxine, we dissolved its original drug stock in DMSO, and similar to that observed in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors. Nevertheless, it does provide some indication of what is actually observed in mammalian models, such as the fact that milnacipran did not demonstrate toxicity in C. elegans since this organism lacks its main target. Other indicators of C. elegans output may be better gauges of drug toxicology such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the initial solvent used to dissolve the drug. This indicates that the method of stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans cannot replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Pincus Z, Slack FJ. 2010. Developmental biomarkers of aging in Caenorhabditis elegans. Developmental Dynamics 239: 1306-1314.
","pubmedId":"","doi":"10.1002/dvdy.22224"},{"reference":"Rodriguez M, Snoek LB, De Bono M, Kammenga JE. 2013. Worms under stress: C. elegans stress response and its relevance to complex human disease and aging. Trends in Genetics 29: 367-374.
","pubmedId":"","doi":"10.1016/j.tig.2013.01.010"},{"reference":"Tejeda-Benitez L, Olivero-Verbel J. 2016. Caenorhabditis elegans, a Biological Model for Research in Toxicology. Reviews of Environmental Contamination and Toxicology,Reviews of Environmental Contamination and Toxicology Volume 237 : 1-35.
","pubmedId":"","doi":"10.1007/978-3-319-23573-8_1"},{"reference":"Boyd WA, Smith MV, Freedman JH. 2012. Caenorhabditis elegans as a Model in Developmental Toxicology. Methods in Molecular Biology,Developmental Toxicology : 15-24.
","pubmedId":"","doi":"10.1007/978-1-61779-867-2_3"},{"reference":"Lai CH, Chou CY, Ch'ang LY, Liu CS, Lin Wc. 2000. Identification of Novel Human Genes Evolutionarily Conserved inCaenorhabditis elegansby Comparative Proteomics. Genome Research 10: 703-713.
","pubmedId":"","doi":"10.1101/gr.10.5.703"},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"},{"reference":"Hobert O. 2010. Neurogenesis in the nematode Caenorhabditis elegans. WormBook : 10.1895/wormbook.1.12.2.
","pubmedId":"","doi":"10.1895/wormbook.1.12.2"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Giunti Sn, Andersen N, Rayes D, De Rosa MaJ. 2021. Drug discovery: Insights from the invertebrate Caenorhabditis elegans. Pharmacology Research & Perspectives 9: 10.1002/prp2.721.
","pubmedId":"","doi":"10.1002/prp2.721"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Byrd R. 1994. Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits. Fundamental and Applied Toxicology 22: 511-518.
","pubmedId":"","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"S�nchez C, B�ges� K, Ebert B, Reines E, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology 174: 10.1007/s00213-004-1865-z.
","pubmedId":"","doi":"10.1007/s00213-004-1865-z"},{"reference":"Bymaster F. 2001. Comparative Affinity of Duloxetine and Venlafaxine for Serotonin and Norepinephrine Transporters in vitro and in vivo, Human Serotonin Receptor Subtypes, and Other Neuronal Receptors. Neuropsychopharmacology 25: 871-880.
","pubmedId":"","doi":"10.1016/S0893-133X(01)00298-6\\"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"bb63cc50-b631-47de-8eb2-92dd090dc804","decision":"revise","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater activity than citalopram, duloxetine was more potent than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/a99ce23a55303f166a362234726594b1.png"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs with the original stock solutions initially prepared in different solvents. A) Responsiveness to SSRIs with the original stock solutions initially prepared in water (n=4). B) Responsiveness to citalopram or escitalopram with stock solutions initially prepared in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with stock solutions initially prepared in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine with the original stock solutions prepared in DMSO (n=4).
","imageTitle":"
Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were prepared first by either dissolving in water, DMSO, or ethanol, establishing an original stock concentration between 20 and 50 mM. From these original stock concentrations, the drugs were then diluted to 4 mM in water. Thereafter, serial dilutions were performed in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/total). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"One underexploited avenue for Caenorhabditis (C.) elegans is drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt 2017), these animals have rarely been part of the normal drug development procedures (Letizia et al., 2018), even though medications have targets within C. elegans (Hobert 2013). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale 2015), potentially, the nonmammalian C. elegans can offer a cheaper alternative that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
In this study, we evaluated, in C. elegans, the toxicity of reuptake inhibitors, selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs), within a 96-well plate exposure format. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang et al., 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham 1994). Limited studies have evaluated the toxicological effects of these medications in C. elegans, even though these drugs target the serotonin transporter mod-5, which is analogous to that found in mammals (Ranganathan et al., 2001; Yu et al., 2023). Furthermore, these animals contain a serotonergic pathway that is responsive to antidepressants (Weinshenker et al., 1995). Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs by first preparing their original stock solution in water, followed by serial dilutions in water (FIG 1A). We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe 1998). For citalopram and escitalopram, we also repeated this experiment by initially preparing their original stock solution in DMSO or ethanol, followed by serial dilutions in water (FIG 1B). When prepared first in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram, but not citalopram, when first prepared in DMSO, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez et al., 2004). This effect was observed when the original stock of citalopram and escitalopram was first prepared in ethanol (escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were initially prepared in water, followed by serial dilutions in water (FIG 1C). We observed toxicity for only duloxetine (duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For venlafaxine, we also prepared its original drug stock in DMSO followed by serial dilutions in water. Like that observed when prepared in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors. Other indicators of C. elegans output may be better gauges of drug toxicology, such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the solvent used to initially dissolve the drug. This indicates that the method of original stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans may not replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Byrd R. 1994. Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits. Fundamental and Applied Toxicology 22: 511-518.
","pubmedId":"","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Ranganathan R, Sawin ER, Trent C, Horvitz HR. 2001. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21(16): 5871-84.
","pubmedId":"11487610","doi":""},{"reference":"S�nchez C, B�ges� K, Ebert B, Reines E, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology 174: 10.1007/s00213-004-1865-z.
","pubmedId":"","doi":"10.1007/s00213-004-1865-z"},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Weinshenker D, Garriga G, Thomas JH. 1995. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci 15(10): 6975-85.
","pubmedId":"7472454","doi":""},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[{"reviewer":{"displayName":"Michael Koelle"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"98b9ffb1-cf72-4169-ab3f-269fd48a1c2c","decision":"edit","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater activity than citalopram, duloxetine was more potent than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/263ed8c5c34be5c5a2e4cbba2dd615b4.jpg"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs with the original stock solutions initially prepared in different solvents. A) Responsiveness to SSRIs with the original stock solutions initially prepared in water (n=4). B) Responsiveness to citalopram or escitalopram with stock solutions initially prepared in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with stock solutions initially prepared in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine with the original stock solutions prepared in DMSO (n=4).
","imageTitle":"
Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were prepared first by either dissolving in water, DMSO, or ethanol, establishing an original stock concentration between 20 and 50 mM. From these original stock concentrations, the drugs were then diluted to 4 mM in water. This was performed to minimize the issue of solubility of these drugs even in diluted concentrations. Thereafter, serial dilutions were performed in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. The volume was 100 μl of water per well. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/total). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"One underexploited avenue for Caenorhabditis elegans is drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt, 2017), these animals have rarely been part of the normal drug development procedures (Letizia et al., 2018), even though medications have targets within C. elegans (Hobert, 2013). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale, 2015), potentially the nonmammalian models can offer a cheaper alternative (Khabib et al., 2022) that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
This study evaluates the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) in C. elegans. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson, 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang et al., 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham, 1994). Limited studies have evaluated the toxicological effects of these medications in C. elegans, even though these drugs target the serotonin transporter mod-5, which is analogous to that found in mammals (Ranganathan et al., 2001; Yu et al., 2023). Furthermore, these animals contain a serotonergic pathway that is responsive to antidepressants (Weinshenker et al., 1995). Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs by first preparing their original stock solution in water, followed by serial dilutions in water (FIG 1A). We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe, 1998). For citalopram and escitalopram, we also repeated this experiment by initially preparing their original stock solution in DMSO or ethanol, followed by serial dilutions in water (FIG 1B). When prepared first in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram, but not citalopram, when first prepared in DMSO, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez et al., 2004). This effect was observed when the original stock of citalopram and escitalopram was first prepared in ethanol (Escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were initially prepared in water, followed by serial dilutions in water (FIG 1C). We observed toxicity for only duloxetine (Duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For venlafaxine, we also prepared its original drug stock in DMSO followed by serial dilutions in water. Like that observed when prepared in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (Desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors, as observed with the high concentrations needed to render the animals unresponsive. What may also contribute to this is that the route of absorption of the C. elegans of these medications are different from mammals, with the cuticle forming a barrier to limit drug absorption (Xiong et al., 2017). Other indicators of C. elegans output may be better gauges of drug toxicology, such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the solvent used to initially dissolve the drug. This indicates that the method of original stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans may not replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Byrd R. 1994. Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits. Fundamental and Applied Toxicology 22: 511-518.
","pubmedId":"","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Khabib MNH, Sivasanku Y, Lee HB, Kumar S, Kue CS. 2022. Alternative animal models in predictive toxicology. Toxicology 465: 153053.
","pubmedId":"34838596","doi":""},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Ranganathan R, Sawin ER, Trent C, Horvitz HR. 2001. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21(16): 5871-84.
","pubmedId":"11487610","doi":""},{"reference":"S�nchez C, B�ges� K, Ebert B, Reines E, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology 174: 10.1007/s00213-004-1865-z.
","pubmedId":"","doi":"10.1007/s00213-004-1865-z"},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Weinshenker D, Garriga G, Thomas JH. 1995. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci 15(10): 6975-85.
","pubmedId":"7472454","doi":""},{"reference":"Xiong H, Pears C, Woollard A. 2017. An enhanced C. elegans based platform for toxicity assessment. Sci Rep 7(1): 9839.
","pubmedId":"28852193","doi":""},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"d334bc50-d7c2-43a1-9d56-d02ea369a1e9","decision":"revise","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater activity than citalopram, duloxetine was more potent than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/263ed8c5c34be5c5a2e4cbba2dd615b4.jpg"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs with the original stock solutions initially prepared in different solvents. A) Responsiveness to SSRIs with the original stock solutions initially prepared in water (n=4). B) Responsiveness to citalopram or escitalopram with stock solutions initially prepared in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with stock solutions initially prepared in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine with the original stock solutions prepared in DMSO (n=4).
","imageTitle":"
Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were prepared first by either dissolving in water, DMSO, or ethanol, establishing an original stock concentration between 20 and 50 mM. From these original stock concentrations, the drugs were then diluted to 4 mM in water. This was performed to minimize the issue of solubility of these drugs even in diluted concentrations. Thereafter, serial dilutions were performed in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. The volume was 100 μl of water per well. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/total). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"One underexploited avenue for Caenorhabditis elegans is drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt, 2017), these animals have rarely been part of the normal drug development procedures (Letizia et al., 2018), even though medications have targets within C. elegans (Hobert, 2013). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale, 2015), potentially the nonmammalian models can offer a cheaper alternative (Khabib et al., 2022) that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
This study evaluates the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) in C. elegans. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson, 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang et al., 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham, 1994). Limited studies have evaluated the toxicological effects of these medications in C. elegans, even though these drugs target the serotonin transporter mod-5, which is analogous to that found in mammals (Ranganathan et al., 2001; Yu et al., 2023). Furthermore, these animals contain a serotonergic pathway that is responsive to antidepressants (Weinshenker et al., 1995). Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs by first preparing their original stock solution in water, followed by serial dilutions in water (FIG 1A). We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe, 1998). For citalopram and escitalopram, we also repeated this experiment by initially preparing their original stock solution in DMSO or ethanol, followed by serial dilutions in water (FIG 1B). When prepared first in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram, but not citalopram, when first prepared in DMSO, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez et al., 2004). This effect was observed when the original stock of citalopram and escitalopram was first prepared in ethanol (Escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were initially prepared in water, followed by serial dilutions in water (FIG 1C). We observed toxicity for only duloxetine (Duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For venlafaxine, we also prepared its original drug stock in DMSO followed by serial dilutions in water. Like that observed when prepared in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (Desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors, as observed with the high concentrations needed to render the animals unresponsive. What may also contribute to this is that the route of absorption of the C. elegans of these medications are different from mammals, with the cuticle forming a barrier to limit drug absorption (Xiong et al., 2017). Other indicators of C. elegans output may be better gauges of drug toxicology, such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the solvent used to initially dissolve the drug. This indicates that the method of original stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans may not replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Byrd R. 1994. Developmental Toxicology Studies of Fluoxetine Hydrochloride Administered Orally to Rats and Rabbits. Fundamental and Applied Toxicology 22: 511-518.
","pubmedId":"","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Khabib MNH, Sivasanku Y, Lee HB, Kumar S, Kue CS. 2022. Alternative animal models in predictive toxicology. Toxicology 465: 153053.
","pubmedId":"34838596","doi":""},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Ranganathan R, Sawin ER, Trent C, Horvitz HR. 2001. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21(16): 5871-84.
","pubmedId":"11487610","doi":""},{"reference":"Sánchez C, Bøgesø KP, Ebert B, Reines EH, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology (Berl) 174(2): 163-76.
","pubmedId":"15160261","doi":""},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Weinshenker D, Garriga G, Thomas JH. 1995. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci 15(10): 6975-85.
","pubmedId":"7472454","doi":""},{"reference":"Xiong H, Pears C, Woollard A. 2017. An enhanced C. elegans based platform for toxicity assessment. Sci Rep 7(1): 9839.
","pubmedId":"28852193","doi":""},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"41bc8ade-716a-47f2-9788-591ae853ae03","decision":"accept","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater toxicity than citalopram, duloxetine was more toxic than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/263ed8c5c34be5c5a2e4cbba2dd615b4.jpg"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs with the original stock solutions initially prepared in different solvents. A) Responsiveness to SSRIs with the original stock solutions initially prepared in water (n=4). B) Responsiveness to citalopram or escitalopram with stock solutions initially prepared in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with stock solutions initially prepared in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine with the original stock solutions prepared in DMSO (n=4).
","imageTitle":"
Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were prepared first by either dissolving in water, DMSO, or ethanol, establishing an original stock concentration between 20 and 50 mM. From these original stock concentrations, the drugs were then diluted to 4 mM in water. This was performed to minimize the issue of solubility of these drugs even in diluted concentrations. Thereafter, serial dilutions were performed in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. The volume was 100 μl of water per well. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/total). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"One underexploited avenue for Caenorhabditis elegans is drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt, 2017), these animals have rarely been part of the normal drug development procedures (Letizia et al., 2018), even though medications have targets within C. elegans (Hobert, 2013). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale, 2015), potentially the nonmammalian models can offer a cheaper alternative (Khabib et al., 2022) that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
This study evaluates the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) in C. elegans. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson, 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang et al., 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham, 1994). Limited studies have evaluated the toxicological effects of these medications in C. elegans, even though these drugs target the serotonin transporter mod-5, which is analogous to that found in mammals (Ranganathan et al., 2001; Yu et al., 2023). Furthermore, these animals contain a serotonergic pathway that is responsive to antidepressants (Weinshenker et al., 1995). Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs by first preparing their original stock solution in water, followed by serial dilutions in water (FIG 1A). We note that C. elegans drug exposures are typically performed in isotonic saline to avoid subjecting animals to osmotic stress, and that our resulting data are likely influenced by these hypotonic conditions. We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe, 1998). For citalopram and escitalopram, we also repeated this experiment by initially preparing their original stock solution in DMSO or ethanol, followed by serial dilutions in water (FIG 1B). When prepared first in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram, but not citalopram, when first prepared in DMSO, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez et al., 2004). This effect was observed when the original stock of citalopram and escitalopram was first prepared in ethanol (Escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were initially prepared in water, followed by serial dilutions in water (FIG 1C). We observed toxicity for only duloxetine (Duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For venlafaxine, we also prepared its original drug stock in DMSO followed by serial dilutions in water. Like that observed when prepared in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (Desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors, as observed with the high concentrations needed to render the animals unresponsive. What may also contribute to this is that the route of absorption of the C. elegans of these medications is different from mammals, with the cuticle forming a barrier to limit drug absorption (Xiong et al., 2017). Other indicators of C. elegans output may be better gauges of drug toxicology, such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the solvent used to initially dissolve the drug. This indicates that the method of original stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans may not replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Byrd RA, Markham JK. 1994. Developmental toxicology studies of fluoxetine hydrochloride administered orally to rats and rabbits. Fundam Appl Toxicol 22(4): 511-8.
","pubmedId":"8056199","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Khabib MNH, Sivasanku Y, Lee HB, Kumar S, Kue CS. 2022. Alternative animal models in predictive toxicology. Toxicology 465: 153053.
","pubmedId":"34838596","doi":""},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Ranganathan R, Sawin ER, Trent C, Horvitz HR. 2001. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21(16): 5871-84.
","pubmedId":"11487610","doi":""},{"reference":"Sánchez C, Bøgesø KP, Ebert B, Reines EH, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology (Berl) 174(2): 163-76.
","pubmedId":"15160261","doi":""},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Weinshenker D, Garriga G, Thomas JH. 1995. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci 15(10): 6975-85.
","pubmedId":"7472454","doi":""},{"reference":"Xiong H, Pears C, Woollard A. 2017. An enhanced C. elegans based platform for toxicity assessment. Sci Rep 7(1): 9839.
","pubmedId":"28852193","doi":""},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1782783290015"}]},{"id":"8fbfb38f-95b7-4af8-a997-fd594b7b891c","decision":"publish","abstract":"Drug toxicity assessment is important for drug development. Here, we evaluated whether the invertebrate model Caenorhabditis elegans can be used to assess the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs). Drug-induced non-responsiveness served as a functional measure of toxicity. Overall, responses did not consistently parallel those of mammals, although select trends were conserved. Escitalopram showed greater toxicity than citalopram, duloxetine was more toxic than milnacipran, and desvenlafaxine, but not venlafaxine, produced toxicity. These results suggest that C. elegans cannot replace mammalian testing, but may serve as a rapid and low-cost prescreening model.
","acknowledgements":"We would like to thank Lillian Fleisher, Kyle Ryan Macaraag, Vincent Newland, Timothy Cho and Danielle Valls for their critical review of the manuscript.
","authors":[{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis"],"email":"Christopherehernandez88@gmail.com","firstName":"Christopher E.","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of the Health Sciences, Henderson, Nevada, USA"],"departments":["College of Graduate Studies"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"avanstone670@student.roseman.edu","firstName":"Alexandra ","lastName":"Van Stone","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Roseman University of Health Sciences, Henderson, Nevada, United States"],"departments":["College of Pharmacy"],"credit":["dataCuration","formalAnalysis","investigation","methodology","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"cso@roseman.edu","firstName":"Christopher","lastName":"So","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-1393-0333"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This research received no external funding.
","image":{"url":"https://portal.micropublication.org/uploads/263ed8c5c34be5c5a2e4cbba2dd615b4.jpg"},"imageCaption":"Figure 1: Response of C. elegans to increasing concentrations (100 nM-4 mM) of select SSRIs and SNRIs with the original stock solutions initially prepared in different solvents. A) Responsiveness to SSRIs with the original stock solutions initially prepared in water (n=4). B) Responsiveness to citalopram or escitalopram with stock solutions initially prepared in DMSO (n=4) or ethanol (n=4). C) Responsiveness to SNRIs with stock solutions initially prepared in water (n=4). D) Responsiveness to venlafaxine and desvenlafaxine with the original stock solutions prepared in DMSO (n=4).
","imageTitle":"
Concentration–Response Assessment of SSRI and SNRI Toxicity in Caenorhabditis elegans
","methods":"The original drug stocks were prepared first by either dissolving in water, DMSO, or ethanol, establishing an original stock concentration between 20 and 50 mM. From these original stock concentrations, the drugs were then diluted to 4 mM in water. This was performed to minimize the issue of solubility of these drugs even in diluted concentrations. Thereafter, serial dilutions were performed in water on a 96-well plate. All experimental groups, including vehicle controls, were subjected to identical exposure conditions and durations. The volume was 100 μl of water per well. For C. elegans maintenance, animals were maintained at 20 °C. The C. elegans N2 strain was synchronized by bleaching, then allowed to grow on NGM plates with OP50 for 48 hours, reaching the L4 stage. Thereafter, animals were collected by aspiration in water, and 50-80 animals were transferred into a well within a 96-well plate with concentrations of drugs or vehicles. The C. elegans were then incubated in select concentrations for 24 hours at 20 °C. The next day, each well with various concentrations of drugs or vehicles was prodded with a platinum rod, and the number of responsive and nonresponsive animals was scored. These data were then expressed as % responsive (number of responsive/total). These data were then analyzed by Graphpad Prism, and LC50s were expressed as LC50 + standard error of the mean.
","reagents":"Fluoxetine, duloxetine, and venlafaxine were purchased from TCI Chemicals Inc (Portland, OR). Citalopram and escitalopram were purchased from Thermo Fisher (Waltham, MA). Desvenlafaxine, milnacipran, and levomilnacipran were purchased from Selleckchem (Houston, TX). Paroxetine and sertraline were purchased from Matrix Scientific (Elgin, SC).
","patternDescription":"One underexploited avenue for Caenorhabditis elegans is drug testing and toxicity assessment. Although some studies have used C. elegans for testing drug toxicity after the fact (Hunt, 2017), these animals have rarely been part of the normal drug development procedures (Letizia et al., 2018), even though medications have targets within C. elegans (Hobert, 2013). However, with the cost of drug testing in mammalian models very high (Doke and Dhawale, 2015), potentially the nonmammalian models can offer a cheaper alternative (Khabib et al., 2022) that will not replace the required animal tests prior to drug approval, but may give guidance as to the drug design and development process.
This study evaluates the toxicity of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) in C. elegans. These drugs were selected because they are prescribed for mental health disorders (Machado and Einarson, 2010) and are generally considered safe, although some side effects are more noticeable than others (Wang et al., 2018). Furthermore, their toxicities, prior to drug approval for clinical use, were tested in rabbits, mice, and rats (Byrd and Markham, 1994). Limited studies have evaluated the toxicological effects of these medications in C. elegans, even though these drugs target the serotonin transporter mod-5, which is analogous to that found in mammals (Ranganathan et al., 2001; Yu et al., 2023). Furthermore, these animals contain a serotonergic pathway that is responsive to antidepressants (Weinshenker et al., 1995). Therefore, it would be of interest to assess their drug-induced toxicities in nematodes and explore if the toxic effects found within this organism would have been predictive of the toxicity found in the mammalian model organisms.
First, we assessed the toxicity of SSRIs by first preparing their original stock solution in water, followed by serial dilutions in water (FIG 1A). We note that C. elegans drug exposures are typically performed in isotonic saline to avoid subjecting animals to osmotic stress, and that our resulting data are likely influenced by these hypotonic conditions. We observed a loss of responsiveness, which may stem from drug toxicity, for fluoxetine, paroxetine, and sertraline (Fluoxetine LC50: 0.51+0.15 mM; paroxetine LC50: 2.1+0.9 mM; and sertraline LC50: 0.28+0.0001 mM). This, however, was not observed for citalopram and escitalopram. This rank order of LC50 does not correlate with either reported mouse or rat LC50, where sertraline is least toxic (Davies and Kluwe, 1998). For citalopram and escitalopram, we also repeated this experiment by initially preparing their original stock solution in DMSO or ethanol, followed by serial dilutions in water (FIG 1B). When prepared first in DMSO, citalopram demonstrated no toxic effects, but escitalopram showed toxicity at the highest concentration (escitalopram LC50: 708+29 mM). This observed effect with escitalopram, but not citalopram, when first prepared in DMSO, is consistent with the greater pharmacological activity of escitalopram reported in mammalian models (Sánchez et al., 2004). This effect was observed when the original stock of citalopram and escitalopram was first prepared in ethanol (Escitalopram LC50: 11.17+0.48 mM) (FIG 1B).
Next, we assessed the toxicity of SNRIs when their original chemical stocks were initially prepared in water, followed by serial dilutions in water (FIG 1C). We observed toxicity for only duloxetine (Duloxetine LC50: 0.88+0.17 mM). This observation differs from that observed in rats, where LC50s are observed for all SNRIs. However, the fact that duloxetine does demonstrate an LC50 in our studies suggests duloxetine may contribute to this through an alternate target in C. elegans. For venlafaxine, we also prepared its original drug stock in DMSO followed by serial dilutions in water. Like that observed when prepared in water, no toxic effects were observed (FIG 1D). On the other hand, when its active metabolite desvenlafaxine was used in this experiment, we observed toxicity (Desvenlafaxine LC50: 1.24+0.15 M). This suggests that C. elegans may have limited metabolic capacity to convert venlafaxine to its active metabolite.
These findings highlight the limitations in using C. elegans viability and non-responsiveness alone to predict mammalian toxicity of reuptake inhibitors, as observed with the high concentrations needed to render the animals unresponsive. What may also contribute to this is that the route of absorption of the C. elegans of these medications is different from mammals, with the cuticle forming a barrier to limit drug absorption (Xiong et al., 2017). Other indicators of C. elegans output may be better gauges of drug toxicology, such as worm growth, development, feeding rate, and motility. These endpoints may provide more informative measures of drug-induced dysfunction collectively alongside viability than observing this variable alone. Notably, drug potency varies depending on the solvent used to initially dissolve the drug. This indicates that the method of original stock preparation can influence apparent toxicity in C. elegans assays. Altogether, these results suggest that while C. elegans may not replace mammalian toxicology models, it may serve as a rapid, low-cost prescreening tool to prioritize compounds before vertebrate testing.
","references":[{"reference":"Byrd RA, Markham JK. 1994. Developmental toxicology studies of fluoxetine hydrochloride administered orally to rats and rabbits. Fundam Appl Toxicol 22(4): 511-8.
","pubmedId":"8056199","doi":"10.1006/faat.1994.1058 "},{"reference":"Davies TS, Kluwe WM. 1998. Preclinical Toxicological Evaluation of Sertraline Hydrochloride. Drug and Chemical Toxicology 21: 521-537.
","pubmedId":"","doi":"10.3109/01480549809002220"},{"reference":"Doke SK, Dhawale SC. 2015. Alternatives to animal testing: A review. Saudi Pharmaceutical Journal 23: 223-229.
","pubmedId":"","doi":"10.1016/j.jsps.2013.11.002"},{"reference":"Hobert O. 2013. The neuronal genome of Caenorhabditis elegans. WormBook : 1-106.
","pubmedId":"","doi":"10.1895/wormbook.1.161.1"},{"reference":"Hunt PR. 2016. The C. elegans model in toxicity testing. Journal of Applied Toxicology 37: 50-59.
","pubmedId":"","doi":"10.1002/jat.3357 "},{"reference":"Khabib MNH, Sivasanku Y, Lee HB, Kumar S, Kue CS. 2022. Alternative animal models in predictive toxicology. Toxicology 465: 153053.
","pubmedId":"34838596","doi":""},{"reference":"Letizia MC, Cornaglia M, Tranchida G, Trouillon R, Gijs MAM. 2018. A design of experiment approach for efficient multi-parametric drug testing using aCaenorhabditis elegansmodel. Integrative Biology 10: 48-56.
","pubmedId":"","doi":"10.1039/c7ib00184c"},{"reference":"Machado M, Einarson TR. 2009. Comparison of SSRIs and SNRIs in major depressive disorder: a meta-analysis of head-to-head randomized clinical trials. Journal of Clinical Pharmacy and Therapeutics 35: 177-188.
","pubmedId":"","doi":"doi:10.1111/j.1365-2710.2009.01050.x"},{"reference":"Ranganathan R, Sawin ER, Trent C, Horvitz HR. 2001. Mutations in the Caenorhabditis elegans serotonin reuptake transporter MOD-5 reveal serotonin-dependent and -independent activities of fluoxetine. J Neurosci 21(16): 5871-84.
","pubmedId":"11487610","doi":""},{"reference":"Sánchez C, Bøgesø KP, Ebert B, Reines EH, Braestrup C. 2004. Escitalopram versus citalopram: the surprising role of the R-enantiomer. Psychopharmacology (Berl) 174(2): 163-76.
","pubmedId":"15160261","doi":""},{"reference":"Wang SM, Han C, Bahk WM, Lee SJ, Patkar AA, Masand PS, Pae CU. 2018. Addressing the Side Effects of Contemporary Antidepressant Drugs: A Comprehensive Review. Chonnam Medical Journal 54: 101.
","pubmedId":"","doi":"10.4068/cmj.2018.54.2.101"},{"reference":"Weinshenker D, Garriga G, Thomas JH. 1995. Genetic and pharmacological analysis of neurotransmitters controlling egg laying in C. elegans. J Neurosci 15(10): 6975-85.
","pubmedId":"7472454","doi":""},{"reference":"Xiong H, Pears C, Woollard A. 2017. An enhanced C. elegans based platform for toxicity assessment. Sci Rep 7(1): 9839.
","pubmedId":"28852193","doi":""},{"reference":"Yu J, Vogt MC, Fox BW, Wrobel CJJ, Fajardo Palomino D, Curtis BJ, et al., Schroeder. 2022. Parallel pathways for serotonin biosynthesis and metabolism in C. elegans. Nature Chemical Biology 19: 141-150.
","pubmedId":"","doi":"10.1038/s41589-022-01148-7"}],"title":"Evaluating Caenorhabditis elegans as a Toxicity Model for Reuptake Inhibitors
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