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Screening the Sigma LOPAC®1280 library of compounds for protective effects against cisplatin-induced oto- and nephrotoxicity


Wertman, Jaime et al. (2020), Screening the Sigma LOPAC®1280 library of compounds for protective effects against cisplatin-induced oto- and nephrotoxicity, Dryad, Dataset,


Dose-limiting toxicities for cisplatin administration, including ototoxicity and nephrotoxicity, impact the clinical utility of this effective chemotherapy agent and lead to lifelong complications, particularly in pediatric cancer survivors. Using a two-pronged drug screen employing the zebrafish lateral line as an in vivo readout for ototoxicity and kidney cell-based nephrotoxicity assay, we screened 1280 compounds and identified 22 that were both oto- and nephroprotective. Of these, dopamine and L-mimosine, a plant-based amino acid active in the dopamine pathway, were further investigated. Dopamine and L-mimosine protected the hair cells in the zebrafish otic vesicle from cisplatin-induced damage and preserved zebrafish larval glomerular filtration. Importantly, these compounds did not abrogate the cytotoxic effects of cisplatin on human cancer cells. This study provides insights into the mechanisms underlying cisplatin-induced oto- and nephrotoxicity and compelling preclinical evidence for the potential utility of dopamine and L-mimosine in the safer administration of cisplatin.


Ototoxicity (in vivo) and Nephrotoxicity (in vitro) Drug Screens: The Sigma LOPAC®1280 drug library (Sigma-Aldrich, St. Louis, MO, USA) was used for both the in vitro and in vivo drug screens. Each screen was completed one time, with either duplicate wells of HK-2 cells, or 4 zebrafish larvae per well. Cisplatin (Cayman Chemicals, Ann Arbor, MI, USA) was prepared fresh for each experiment as a stock solution of 1.67 mM in 0.9% NaCl.


Ototoxicity Drug Screen: Casper (White et al., 2008) zebrafish embryos were collected and grown in standard E3 medium in a 28°C incubator (Westerfield, 1995). Embryos were cleaned and given new E3 media daily. The use of zebrafish in this study was approved by the Dalhousie University Committee on Laboratory Animals (Protocols #17-131 and #17-055). 

During the drug screen, 72 hours post fertilization zebrafish larvae were incubated with drugs at 35°C, representing a midpoint between the ideal temperature for zebrafish development (28°C) and human cells (37°C). Larvae per well were treated with either the vehicle control or one of the compounds from the Sigma LOPAC®1280 library at 0.01 mM. Three hours later, larvae were treated with vehicle control, or cisplatin (0.02 mM, the approximate predetermined EC50, where neuromast fluorescence was approximately 0.5 fold of control, no drug values), such that larvae were either treated with cisplatin alone, or cisplatin + the library compounds. After a 24 hr incubation with drug(s), larvae were stained with 2 mM YO-PRO-1TM Iodide 491-509 (Thermo Fisher Scientific) according to manufacturer’s instructions, then biosorted to measure peak height (PH) green fluorescence. Larvae were analyzed using a Biosorter, coupled to a Large Particle Handler (Union Biometrica Inc, Holliston, MA, USA). The Biosorter was fitted with a 500 mM fluidics and optics core assembly (FOCA), 1000 mM fluid handling tubing, and a 488 nm laser. PH green fluorescence was recorded. The average fold change in neuromast fluorescence, compared to the no drug control value, of the four larvae treated with each compound is reported in this data set. If the value is reported as zero, this means that at least ¾ larvae died following treatment with the compound + cisplatin, which we considered to be toxic.


Nephrotoxicity Drug Screen: HK-2 human kidney proximal tubule cells (American Type Culture Collection, Manassas, VA, USA) were maintained in standard cell culture conditions, with 5% CO2 at 37°C. Cells were authenticated and confirmed mycoplasma-free by IDEXX BioAnalytics (Columbia, MO, USA). Cells were either untreated, treated with the approximate EC50 of cisplatin (0.005 mM) alone, or both the EC50 of cisplatin + one of the 1280 compounds from the drug library, at a final concentration of 0.01 mM. After a 48 hr incubation with drug(s), cell viability assays were done using an alamarBlueTM (Thermo Fisher Scientific, Waltham, MA) assay, according to the manufacturer’s instructions. The negative control (culture media with drug and alamarBlueTM, but no cells) values were subtracted from experimental values. All values are expressed as fold change in viability, in relation to the no drug control.



Killam Predoctoral Award for Jaime Wertman's Salary

Izaac Walton Killam Doctoral Award for Jaime Wertman's Salary

Killam Predoctoral Award for Jaime Wertman's Salary

Izaac Walton Killam Doctoral Award for Jaime Wertman's Salary