Cytoprotective Activities of Water-Soluble Fullerenes in Zebrafish Models - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 257

(Hamamatsu Photonics, Hamamatsu City, Japan). Images were analyzed with the
Adobe Photoshop 6.0 computer program (Adobe, San Jose, CA).

19.2.8 Vertebrate Animal Care and Safety

The Office of Laboratory Animal Welfare (OLAW), National Institutes of Health
(NIH), has approved Phylonix' Animal Welfare Assurance #A4191-01, effective
July 14, 2003, for a 5-year approval period. We euthanize zebrafish of all ages
by overexposure to tricaine methanesulfonate; adult fish are then rapidly frozen.
These procedures are consistent with the American VeterinaryMedical Association's
(AVMA) Panel on Euthanasia. Approximately 1500 animals were used in this study.

19.3 RESULTS

19.3.1 General Remarks and In Vitro Properties

Three different classes of water-soluble fullerenes, dendrofullerenes (2-7), C3-like
fullerenes (1 and 8-10), and oxo-aminofullerenes (11 and 12) (Fig. 19.1), were
tested in five different zebrafish assays to assess both overall toxicity (measured by
LC 50 ) and efficacy in protecting against three different tissue-specific chemical
cytotoxins: cisplatinum, gentamicin, and 6-hydroxydopamine. Cisplatinum is a
widely used and highly effective anticancer drug and also causes apoptosis in
noncancerous hair cells of auditory system and renal tubular cells in mammals. A
similar pattern of cytotoxicity was observed in lateral-line neuromast hair cells of
zebrafish embryos after exposure to cisplatinum. Gentamicin and related amino-
glycoside antibiotics induce apoptosis and cell loss in auditory hair cells in
mammals and dorsal mechanoreceptor hair cells in zebrafish and represent a major
cause of acquired deafness in children (Yoshikawa, 1980; Stavroulaki et al., 1999).
6-Hydroxydopamine is taken up byCNS neurons, particularly by tyrosine hydroxylase-
containing dopaminergic neurons in the central nervous system, and causes
oxidative injury and apoptotic death (Nagatsu et al., 2000). The preferential
destruction of dopaminergic neurons in mammals has led to widespread use of
6-hydroxydopamine as a model system for induction and study of Parkinson's-like
syndromes in experimental animals. The data in Table 19.1 indicate that both
dendrofullerenes 2-7 and C3-like anionic fullerenes 8 and 9 show substantial
antioxidant activity against superoxide, whereas the activity of the cationic deri-
vatives 10-12 is considerably lower (Witte et al., 2007). The results in Table 19.1
were obtained using xanthine/xanthine oxidase-generated superoxide. Similar
results have been obtained with potassium superoxide and direct spectroscopic
detection of superoxide (Ivanovic et al., unpublished data). The fact that anionic
fullerenes interact much stronger with the positively charged cytochrome c than
cationic derivatives demonstrates that electrostatic interactions are the major
driving force for this binding process (Witte et al., 2007).