Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 253

single well of a 96-well plate), and transparent up to 10 days post fertilization (dpf),
the toxic and cytoprotective effects of small molecules can be studied in situ during
this period. Finally, since the morphological structure of zebrafish embryos has been
mapped to the single-cell level, the toxic, apoptotic, and cytoprotective effects on
various organs can be tracked to approximately the single-cell level in situ (Rodriguez
and Driever, 1997; Cole and Ross, 2001; Langheinrich et al., 2002; Parng et al., 2002).
Our results indicate that many of our anionic water-soluble fullerenes generally
exhibit little overall toxicity at concentrations of 400 m M or greater, while cationic

fullerenes exhibit significant toxicity at 100 Mor less. We also observed that anionic

fullerenes provide significant protection against apoptosis induced by three known
chemical cytotoxins, cisplatinum, gentamicin, and 6-hydroxydopamine, which pro-
duce similar patterns of cell injury and death in zebrafish and humans.

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19.2 MATERIALS AND METHODS

19.2.1 Compounds

Water-soluble fullerenes 1-12 were prepared according to our previously reported
synthesis protocols (Witte et al., 2007, and references cited therein). In order to carry
out the biological tests, 25-100mM stock solutions in 10% DMSO and 0.1 N HCl
of 1-12 were prepared. All stocks were stored at 20 C and diluted before use.

19.2.2 Standard Procedures for Embryo Collection

Embryos were generated by natural pairwise mating, as described in The Zebrafish
Book (Westerfield, 1993). Four to five pairs of adult zebrafish were set up for each
mating, and, on average, 100-150 embryos per pair were generated. Embryos were
maintained at 28 C in fish water (200mg Instant Ocean Salt per liter of deionized
water; pH 6.6-7.0 maintained with 2.5mg/L Jungle pH Stabilizer (Jungle Labora-
tories Corporation, Cibolo, TX); conductivity 670-760 S). Embryos were cleaned

(dead embryos removed) and sorted by developmental stage (Kimmel et al., 1995) at 6
and 24 h post fertilization (hpf). Because the embryo receives nourishment from an
attached yolk sac, no feeding was required for 7dpf.

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19.2.3 Determination of LC 50 and Organ Toxicity

19.2.3.1 Treatment of Embryoswith 1-12 Zebrafish embryos at 24hpf were
distributed into 96-well cell culture plates, one embryo per well in 100 Lfishwater

containing the compound. Embryos were exposed from day 1 to day 5 post fertilization.
Typically, the fish water contains PBS at a final concentration of 10%. Treated embryos
were compared with untreated and 0.1% DMSO-treated controls.

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19.2.3.2 Measurement of LC 50 and Lethality Curves Mortality was
recorded every 24 h. At 120hpf, total mortality was used to generate the