DOI: 10.5061/dryad.zcrjdfn8n
title: Screening the Sigma LOPAC®1280 library of compounds for protective effects against cisplatin-induced oto- and nephrotoxicity

description: 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.
description: Ototoxicity (<i>in
 vivo</i>)<i> </i>and Nephrotoxicity (<i>in vitro)
 </i>Drug Screens: The Sigma LOPAC<sup>®</sup>1280 drug
 library (Sigma-Aldrich, St. Louis, MO, USA) was used for both the
 <i>in vitro </i>and <i>in vivo </i>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: <em>Casper (</em>White <em>et al.,
 2008) </em>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®<sup>1280</sup> library at 0.01 mM. Three hours later,
 larvae were treated with vehicle control, or cisplatin (0.02 mM, the
 approximate predetermined EC<sub>50</sub>, 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-1<sup>TM</sup> 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% CO<sub>2</sub> at 37°C. Cells were authenticated and
 confirmed mycoplasma-free by IDEXX BioAnalytics (Columbia, MO, USA). Cells
 were either untreated, treated with the approximate EC<sub>50
 </sub>of cisplatin (0.005 mM) alone, or both the EC<sub>50
 </sub>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
 alamarBlue<sup>TM</sup> (Thermo Fisher Scientific, Waltham,
 MA) assay, according to the manufacturer’s instructions. The negative
 control (culture media with drug and alamarBlue<sup>TM</sup>,
 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.