In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells. Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae. By applying PEPITA to characterize ototoxic drug interaction outcomes, we have discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Treading Water: Tire Wear Particle Leachate Recreates an Urban Runoff Mortality Syndrome in Coho but Not Chum Salmon.

Tire tread wear particles (TWP) are increasingly recognized as a global pollutant of surface waters, but their impact on biota in receiving waters is rarely addressed. In the developed U.S. Pacific Northwest, acute mortality of adult coho salmon (Oncorhynchus kisutch) follows rain events and is correlated with roadway density. Roadway runoff experimentally triggers behavioral symptoms and associated changes in blood indicative of cardiorespiratory distress prior to death. Closely related chum salmon (O. keta) lack an equivalent response. Acute mortality of juvenile coho was recently experimentally linked to a transformation product of a tire-derived chemical. We evaluated whether TWP leachate is sufficient to trigger the acute mortality syndrome in adult coho salmon. We characterized the acute response of adult coho and chum salmon to TWP leachate (survival, behavior, blood physiology) and compared it with that caused by roadway runoff. TWP leachate was acutely lethal to coho at concentrations similar to roadway runoff, with the same behaviors and blood parameters impacted. As with runoff, chum salmon appeared insensitive to TWP leachate at concentrations lethal to coho. Our results confirm that environmentally relevant TWP exposures cause acute mortalities of a keystone aquatic species.

A ubiquitous tire rubber-derived chemical induces acute mortality in coho salmon.

In U.S. Pacific Northwest coho salmon (Oncorhynchus kisutch), stormwater exposure annually causes unexplained acute mortality when adult salmon migrate to urban creeks to reproduce. By investigating this phenomenon, we identified a highly toxic quinone transformation product of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), a globally ubiquitous tire rubber antioxidant. Retrospective analysis of representative roadway runoff and stormwater-affected creeks of the U.S. West Coast indicated widespread occurrence of 6PPD-quinone (<0.3 to 19 micrograms per liter) at toxic concentrations (median lethal concentration of 0.8 ± 0.16 micrograms per liter). These results reveal unanticipated risks of 6PPD antioxidants to an aquatic species and imply toxicological relevance for dissipated tire rubber residues.

Effects of salinity on olfactory toxicity and behavioral responses of juvenile salmonids from copper.

Dissolved copper is one of the more pervasive and toxic constituents of stormwater runoff and is commonly found in stream, estuary, and coastal marine habitats of juvenile salmon. While stormwater runoff does not usually carry copper concentrations high enough to result in acute lethality, they are of concern because sublethal concentrations of copper exposure have been shown to both impair olfactory function and alter behavior in various species in freshwater. To compare these results to other environments that salmon are likely to encounter, experiments were conducted to evaluate the effects of salinity on the impairment of olfactory function and avoidance of copper. Copper concentrations well within the range of those found in urban watersheds, have been shown to diminish or eliminate the olfactory response to the amino acid, l-serine in freshwater using electro-olfactogram (EOG) techniques. The olfactory responses of both freshwater-phase and seawater-phase coho and seawater-phase Chinook salmon, were tested in freshwater or seawater, depending on phase, and freshwater-phase coho at an intermediate salinity of 10‰. Both 10‰ salinity and full strength seawater protected against the effects of 50µg copper/L. In addition to impairing olfactory response, copper has also been shown to alter salmon behavior by causing an avoidance response. To determine whether copper will cause avoidance behavior at different salinities, experiments were conducted using a multi-chambered experimental tank. The circular tank was divided into six segments by water currents so that copper could be contained within one segment yet fish could move freely between them. The presence of individual fish in each of the segments was counted before and after introduction of dissolved copper (<20µg/L) to one of the segments in both freshwater and seawater. To address whether use of preferred habitat is altered by the presence of copper, experiments were also conducted with a submerged structural element. The presence of sub-lethal levels of dissolved copper altered the behavior of juvenile Chinook salmon by inducing an avoidance response in both freshwater and seawater. While increased salinity is protective against loss of olfactory function from dissolved copper, avoidance could potentially affect behaviors beneficial to growth, survival and reproductive success.

Confirmation of Stormwater Bioretention Treatment Effectiveness Using Molecular Indicators of Cardiovascular Toxicity in Developing Fish.

Urban stormwater runoff is a globally significant threat to the ecological integrity of aquatic habitats. Green stormwater infrastructure methods such as bioretention are increasingly used to improve water quality by filtering chemical contaminants that may be harmful to fish and other species. Ubiquitous examples of toxics in runoff from highways and other impervious surfaces include polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause functional and structural defects in developing fish hearts. Therefore, abnormal heart development in fish can be a sensitive measure of clean water technology effectiveness. Here we use the zebrafish experimental model to assess the effects of untreated runoff on the expression of genes that are classically responsive to contaminant exposures, as well as heart-related genes that may underpin the familiar cardiotoxicity phenotype. Further, we assess the effectiveness of soil bioretention for treating runoff, as measured by prevention of both visible cardiac toxicity and corresponding gene regulation. We find that contaminants in the dissolved phase of runoff (e.g., PAHs) are cardiotoxic and that soil bioretention protects against these harmful effects. Molecular markers were more sensitive than visible toxicity indicators, and several cardiac-related genes show promise as novel tools for evaluating the effectiveness of evolving stormwater mitigation strategies.