DOI: 10.1002/jbt.21964
Evaluation in zebrafish model of the toxicity of rhodamine
B-conjugated crotamine, a peptide potentially useful for
diagnostics and therapeutics
Judy Yuet-Wa Chan
1Hefeng Zhou
1Yiu Wa Kwan
2Shun Wan Chan
3Gandhi Radis-Baptista
4Simon Ming-Yuen Lee
11State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China
3State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
Uni-versity, Kowloon, Hong Kong, China
4Laboratory of Biochemistry and Biotechnology, Institute for Marine Sciences, Federal University of Ceara, Fortaleza, Brazil
Correspondence Simon Ming Yuen Lee Gandhi Rádis-Baptista (Emails: simonlee@umac.mo; gandhi.radis@ufc.br; g1rad2baptist3@gmail.com)
Contract Grant Sponsor: Macau Science and Technology Development Fund
Contract Grant Numbers: 069/2015/A2, 134/2014/A3.
Contract Grant Sponsor: Research Committee, University of Macau.
Contract Grant Numbers: MYRG2016-00129-ICMS-QRCM, MYRG2015-00214-MYRG2016-00129-ICMS-QRCM, and MYRG2016-00133-ICMS-QRCM. Contract Grant Sponsor: Program on Toxinology (Issue 2010).
Contract Grant Sponsor: CNPq. Judy Yuet-Wa Chan and Hefeng Zhou con-tributed equally to this work.
Abstract
Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4�M, which effectively kill zebrafish larvae in less than 10 min. With non-lethal
con-centrations (<1�M), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the car-diac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translo-cated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.
K E Y W O R D S
acute toxicity, rhodamine B-crotamine, screening of pharmaceuticals, venom-derived peptides, zebrafish model
1
I N T RO D U C T I O N
Peptides, diverse in structure and function, are present in all living organisms and play a key role in a number of biological activities and cellular processes. In venomous animals like snakes and spiders, pep-tides form part of lethal toxin cocktails that are injected into victims or prey.[1]Peptides expressed in the venom of such animals can
inter-fere with and modulate neurotransmission, blood coagulation, blood pressure, tissue integrity, cell signaling, and even immune response.[2,3]
Despite comprising “toxic weapons,” venom-derived peptides display target selectivity and specificity, which make them useful in a wide range of applications in medicine and biotechnology.[4,5]
Crotamine is a 42 amino acid long defensin-like cationic peptide, purified from the venom of the South American rattlesnake,Crotalus durissus terrificus. Soon after its discovery, crotamine was demon-strated to induce muscle contractions by increasing Na+permeability of skeletal muscle membranes, producing spasmodic seizure and tonic convulsion in animals.[6–8] Further studies have shown that
crotamine exerts its pharmacological action not only through the modulation of the Na+-channel of the junctional region of muscle
fibers of vertebrates,[9,10] but also by interacting with subtypes of
the K+-channel, as evidenced with lower eukaryote models[11]and
with electrophysiological models of cloned channels expressed in Xenopus laevis oocytes.[12] At the millimolar concentration,
2 of 7 CHAN ET AL.
crotamine is a toxic peptide to eukaryotic muscle cells and tis-sues, whereas in the low micromolar-to-submicromolar range, it displays cell-penetrating, antitumor, antimicrobial, antifungal, and antiparasite properties.[7,8,13–16] Interestingly, crotamine can form
complex with nucleic acids and carry DNA into cells, serving as an intracellular delivery agent.[17–19] Detailed work showed that
cro-tamine is a peptide that preferentially and selectively targets actively proliferating eukaryotic cells,[17]thereby being capable of
discrimi-nating and inducing death to a number of tumors.[7,8]Indeed, when
cyanine 3-conjugated crotamine is injected i.p. in nude mice engrafted with melanoma, it was able to travel through the bloodstream, reach the tumor site, accumulate in tumor mass, and induce cell death of cancer cells.[20]
Zebrafish (Danio rerio) are small tropical fish that have unique advantages for experimental research in biochemistry, genetics, and pharmacology, making them ideal for toxicological studies. Small size, short generation time, easy accessibility and manipulation, trans-parent body, and the availability of thousands of mutants represent distinct characteristics and advantages for the evaluation of sub-stances that can cause acute toxicities and interference with organism development.[21]Another advantage is that the toxicity of numerous
molecules can be assessed in zebrafish, even in minute quantities (i.e., less than 1 mg of pure sample), by observing the phenotypic changes and abnormal functions of various physiological systems (e.g., cardiovascular and neurological systems). Additionally, transgenic lines of zebrafish have been developed that respond to exposure to toxicants. These genetic modifications make zebrafish genotypes sen-sitive to toxic components contained in industrial pollutants and other kinds of residual waste.[22,23] Thus, zebrafish comprises a reliable
vertebrate model for the evaluation of the toxicity of compounds and the biosafety of pharmaceuticals.[24]Recently, we have successfully
mapped the minimal toxic/non-toxic regions of a rattlesnake venom cathelicidin (vipericidin), by assessing the toxicity in zebrafish of peptide fragments and fluorescent-labeled derivatives.[25]
In the present study, we examined thein vivotoxicity of rhodamine B-conjugated crotamine (RhoB-crotamine), a peptide potentially use-ful for diagnosis and experimental anti-proliferative therapies, on the development of zebrafish embryos, as well as on the cardiovascular and neurobehavioral systems of zebrafish larvae.
2
M AT E R I A L S A N D M E T H O D S
2.1
Zebrafish and RhoB-crotamine
Wild-type zebrafish were purchased from a local fish supplier. Trans-genic fish lines Tg (cmlc2: GFP) and Tg (fli-1: EGFP) were purchased from Zebrafish International Resource Center (ZIRC, Eugene, OR). Crotamine conjugated to RhoB at the N-terminal (RhoB-crotamine) was synthesized by Cellmano Biotech Limited (Hefei, China) and was used throughout the whole study. The purity of RhoB-labeled cro-tamine (molecular weight: 5350.87) was>98%. The lyophilized
pep-tide was resuspended in DMSO to make a stock solution of 1 mM and kept in aliquots at−20°C until use.
2.2
Zebrafish husbandry
The procedures for zebrafish culture, breeding, embryo collection, embryonic and larval culture, as well as observation of fluorescent sig-nal in the zebrafish tissues, were performed according to the standard protocols described in the Zebrafish Handbook (4thEdition).[26]The
collection of embryos and the peptide exposure experiments were all carried out at 28°C in embryo medium (13.7 mM NaCl, 540 mM KCl, pH 7.4, 25 mM Na2HPO4, 44 mM KH2PO4, 300 mM CaCl2, 100 mM MgSO4, 420 mM NaHCO3, pH 7.4). Ethical approval for animal exper-imentation was granted by the Animal Research Ethics Committee of the University of Macau, Macau, China.
2.3
Assessment of RhoB-crotamine interference in
a zebrafish survival test
Wild-type zebrafish larvae at 3 days post-fertilization (3 dpf) were ran-domly distributed into a 24-well microplate (10–12 fish per well) and exposed to 1 mL solutions of crotamine in zebrafish embryo medium at increasing concentrations (0, 0.5, 1, 2, 4, 8, and 16�M) and different
time points. Mortality was then measured by observing the heart beats of the zebrafish.
2.4
Assessment of RhoB-crotamine interference in
zebrafish embryonic development
Wild-type zebrafish embryos (1 dpf) were exposed to various concen-trations of RhoB-crotamine (0.25–1�M) in 24-well plates at 28°C for 48 h, in a dark environment in order to avoid degradation of the flu-orescent crotamine. The exposure media was replaced every 24 h. At least 10 embryos were grouped in each well. The occurrence of defor-mities, absence of tail detachment, absence of somite, light pigmen-tation, and coiled body were observed and reported daily for 48 h by means of a stereomicroscope. The experiment was repeated three times.
2.5
Assessment of RhoB-crotamine interference
in zebrafish cardiac functions
Tg(cmlc2:GFP) zebrafish embryos were used for the cardiotoxicity assay. All embryos were cultivated in embryo medium containing 0.003% of 1-phenyl-2-thiourea to block pigmentation from 1 dpf. Meanwhile, 2 dpf zebrafish embryos were dechorionated, as men-tioned above, and then randomly distributed into a 24-well microplate (12–14 fish per well), treated with 1 mL solutions of RhoB-crotamine in zebrafish embryo medium at various concentrations (0, 0.25, 0.5, 1, �M). After 2 days of incubation, zebrafish were immersed into
F I G U R E 1 Survival rate of zebrafish larvae upon treatment with crotamine. Zebrafish larvae (3 dpf) were treated with 0–16�M of
RhoB-crotamine for 10 min, 30 min, 3 h, and 24 h. Survival of the larvae was then determined by observing the heart beating. Survival rate is expressed as mean percentage±S.E.M. with 8–10 larvae in each group. ****P<0.0001 versus 0�M crotamine
F I G U R E 2 Abnormal development of zebrafish embryos induced by RhoB-crotamine. Hatching rate, pigmentation, and body shape were eval-uated after 48 h treatment with crotamine (0–1�M) (A–C). Representative images are shown for normal and malformed zebrafish embryos (D).
Data are expressed as mean±S.E.M. with 8–10 embryos in each group
functions were evaluated according to various parameters, which were measured as described previously.[27]Images from the videos were
used to measure the longitudinal axis length (a) and lateral axis length (b) between the myocardial borders of ventricles at end-diastole (EDV) and end-systole (ESV), respectively. In order to measure the HR, the
number of heartbeats in each 15 s segment was counted. The ventricu-lar volume at EDV and ESV in the ventricu-larvae was calculated from the heart dimensions using the formula for a prolate spheroid:V=4/3�ab2.
4 of 7 CHAN ET AL.
F I G U R E 3 Cardiac dysfunction induced by RhoB-crotamine in zebrafish larvae. Heart functions were evaluated by measuring the heart rate (A), stroke volume (B), cardiac output (C), and fractional shortening (D) after 48 h incubation with or without crotamine. Data are expressed as mean±S.E.M. with 8–10 larvae in each group. *P<0.05 versus 0�M RhoB-crotamine
F I G U R E 4 Attenuation of locomotion in zebrafish by RhoB-crotamine. After incubation with crotamine (0–1�M) for 15 min, the
swimming distance of zebrafish larvae was recorded by the Zebrabox System. Each treatment included 8–10 zebrafish. Data are expressed as mean±S.E.M. **P<0.01 versus 0�M crotamine
CO=SV×HR, %FS=(Diastolic diameter−Systolic diameter)/(Systolic diameter)×100%.
2.6
Measurement of locomotion after exposure of
zebrafish to RhoB-crotamine
Behavioral assay was performed at concentrations of crotamine that did not induce death in the zebrafish embryo. Wild-type zebrafish larvae (6 dpf) were transferred into the wells of a 96-well plate (1 larvae/well). The fish were acclimatized for 30 min and increasing
concentrations of RhoB-crotamine (0, 0.25, 0.5, 1�M) were added to
each well (8–10 fish in each group). After a pre-incubation period (i.e., 15 min incubation with the peptide), the swimming behavior of the lar-vae was recorded for 10 min. The total swimming distance reached by each individual zebrafish was then analyzed using the Zebrabox system (View Point, Champagne-au-Mont-d’Or, France).
2.7
In vivo
distribution of RhoB-crotamine
Tg (fli-1: EGFP) zebrafish larvae were exposed to 1 �M
RhoB-conjugated crotamine for 24 h and mounted on microscope glass slides. The distribution of RhoB-crotamine in zebrafish body and tissues was visualized using an IX81 motorized inverted fluorescence microscope (Olympus Company, Tokyo, Japan).
3
R E S U LT S
3.1
RhoB-crotamine induced the death of zebrafish
larvae
The mortality of zebrafish induced by various concentrations of fluo-rescently labeled crotamine was assessed at 10 min (A), 30 min (B), 3 h (C), and 24 h (D). Results in Figure 1 showed that RhoB-crotamine at concentrations higher than 4�M caused death of zebrafish larvae, in
F I G U R E 5 Distribution of crotamine in zebrafish larvae. Tg (fli-1: EGFP) zebrafish larvae (green color) were exposed to 1�M
rhodamine-conjugated crotamine for 24 h. The distribution of RhoB-crotamine (red color) was visualized using an inverted fluorescence microscope under GFP excitation (A) and bright field (B), respectively. The experiment was repeated three times and similar results were obtained each time
3.2
RhoB-crotamine caused embryonic
malformations in zebrafish
Abnormal formation of zebrafish embryos was observed at all tested concentrations of RhoB-crotamine (0.25–1.0�M), after 48 h
incuba-tion. Nearly 100% of embryos were unable to hatch. After breaking the yolk sac, light pigmentation (8–26%) and coiled body (20–58%) were observed in zebrafish exposed to the peptide (Figure 2).
3.3
RhoB-crotamine induced cardiac dysfunction in
zebrafish larvae
The cardiac functions of zebrafish larvae were evaluated by measur-ing the HR, SV, CO, and FS. From the results shown in Figure 3, cardiac dysfunction with a significant decrease in heartbeat, SV, and CO was observed with 1�M of RhoB-crotamine treatment.
3.4
RhoB-crotamine suppressed the locomotion
of zebrafish larvae
The locomotion of individual zebrafish larvae was measured by track-ing the swimmtrack-ing route in each well of a 96-well plate. Total swimmtrack-ing distance in 10 min was analyzed and is shown in Figure 4. After 15 min incubation with various concentrations of RhoB-crotamine, the swim-ming distance upon treatment with 0.5 and 1�M RhoB-crotamine was
significantly attenuated (P<0.01).
3.5
Distribution of RhoB-crotamine in zebrafish
tissues
The tissue distribution of RhoB-crotamine in zebrafish was observed under excitation in a fluorescence microscope. At the same time, the vascular system of Tg (fli-1: EGFP) zebrafish could be observed by expression of enhanced green fluorescent protein. From Figure 5, it can
be seen that RhoB-crotamine was mainly localized in the yolk sac and intestine.
4
D I S C U S S I O N
A handful of peptide toxins has been isolated from animal venom that modulate specific molecular targets and some of them converted into therapeutics and diagnostics.[28–30]Despite the high potential for drug
discovery and development, toxicity is a concern. The toxicity tests of bioactive peptide toxins are often carried outin vitro, as exempli-fied by cell viability tests[31]andin vivoin mouse model.[32]Herein,
we have evaluated several bioanalytical parameters in two develop-mental stages of zebrafish (embryo and larvae) in response to the potential toxicities caused by RhoB-labeled crotamine—a peptide use-ful for diagnosis and adjuvant therapies against cancer and eukaryotic pathogens.
RhoB-crotamine was lethal to zebrafish embryos and disrupted the cardiac and locomotive system of zebrafish larvae. The LD50of RhoB-crotamine to zebrafish was equivalent to 4�M (Figure 1). The
con-trastive difference between the non-toxic/non-lethal concentration of 2�M and the LD50of 4�M of RhoB-crotamine could be explained by the ratio of peptide to target that exerts a given pharmacological or toxicological effect; in this case, responding suggestively accord-ing to an all-or-none mechanism, as observed for cecropin A—a 37-residue, amphipathic�-helical antimicrobial peptide with high cationic
character.[33]Based on this all-or-none kinetic model, at relatively low
concentration (2�M), RhoB-crotamine would interact to its target, but
without causing any observable toxic effect; by doubling the peptide concentration (i.e., 4�M), the number and mass of interacting
pep-tide molecules on-target (peppep-tide/target ratio) consequently increase and the toxicity is effectively reached. In non-lethal concentrations (<1�M), crotamine caused malformation in zebrafish embryos. In
6 of 7 CHAN ET AL.
exposure to RhoB-crotamine hatching was delayed or abolished (Fig-ure 2A). Thus, RhoB-crotamine interfered with and arrest proliferative (embryonic) cells of zebrafish. By breaking the yolk sac and observ-ing the morphology of zebrafish, coiled body and lighter pigmenta-tion were evidenced (Figures 2B and 2C), showing that non-lethal con-centration of RhoB-crotamine adversely affected the development of zebrafish embryo. As observed for other anticancer peptides,[34]
cro-tamine that was cytotoxic to distinct tumor cells was similarly toxic to zebrafish embryos. Moreover, the fact that RhoB-crotamine heav-ily accumulates in the yolk sac is of note, since the permeability of vitelline membrane and yolk syncytial layer to hydrophilic substances is very low.[35]Importantly, the retention of RhoB-crotamine in yolk
sac indicated it could potentially cause liver toxicity in mammals. Sev-eral compounds in clinical use that are known hepatotoxic, for exam-ple, acetaminophen, aspirin, tetracycline, cyclophosphamide, and ery-thromycin, induce liver degeneration, reduce liver size, and accumulate to yolk-sac.[36]
Complete cardiac functions in zebrafish can be established as early as 2 dpf. Crotamine decreased the cardiac functions under chronic exposition (Figure 3). Such cardiac toxicity could be explained by the interference of crotamine with ion channels. In mammals, crotamine disturbs Ca2+ion flux in the sarcoplasmic reticulum by opening the
ryanodine receptor.[37]Moreover, the crotamine cytotoxicity toward
tumor cells in culture was dependent of intracellular calcium increase that triggers calcium-mediated cell death.[20]
The RhoB-crotamine toxicity on zebrafish behavior was also addressed. Rho-B crotamine attenuated the zebrafish swimming dis-tance, within a short time interval (Figure 4). The interference with neural and muscle tissues of zebrafish might be inferred by the fact that crotamine selectively inhibits KV1.1, KV1.2, and KV1.3 channels with a low IC50(∼300 nM).[12]Studies reported that crotamine can
pene-trate into tumor cells and induce death at micromolar concentrations (<1�M, i.e., 1.0 nmol/mL), but it is harmless to healthy and
quies-cent cells at conquies-centrations 10-fold higher.[17]Consequently, one can
expect that malignant or healthy proliferating stages of cells could be sensitive to crotamine and derivatives. In fact, zebrafish embryos were very sensitive to RhoB-crotamine, in concentrations that are harmless to most healthy mammalian cells. Taken together, our zebrafish model proved to be a suitablein vivosystem to investigate the absorption, biodistribution and bioavailability of peptide drug leads; it was sensi-tive enough to assess the acute dysfunction in target zebrafish tissues and the toxicity in whole organism, caused by theranostic peptide can-didates from venom sources.
C O N F L I C T O F I N T E R E S T
The authors declare no conflicts of interests in association with this work.
R E F E R E N C E S
[1] J. White,Ther. Drug Monit.2000,22(1), 65–68. [2] R. J. McCleary, R. M. Kini,Toxicon2013,62, 56–74.
[3] Y. S. Chan, R. C. Cheung, L. Xia, J. H. Wong, T. B. Ng, W. Y. Chan,Appl. Microbiol. Biotechnol.2016,100(14), 6165–6181.
[4] Y. N. Utkin,World J. Biol. Chem.2015,6(2), 28–33.
[5] R. J. Lewis, M. L. Garcia,Nat. Rev. Drug Discov.2003,2(10), 790– 802.
[6] M. A. Hayashi, F. D. Nascimento, A. Kerkis, V. Oliveira, E. B. Oliveira, A. Pereira, G. Radis-Baptista, H. B. Nader, T. Yamane, I. Kerkis, I. L. Tersar-iol.Toxicon2008,52(3), 508–517.
[7] G. Rádis-Baptista, I. Kerkis,Curr. Pharm. Des.2011,17(38), 4351– 4361.
[8] I. Kerkis, M. A. Hayashi, A. R. Prieto da Silva, A. Pereira, P. L. De Sa Junior, A. J. Zaharenko, G. Rádis-Baptista, A. Kerkis, T. Yamane,Biomed. Res. Int.2014, 675985.
[9] C. C. Chang, K. H. Tseng,Br. J. Pharmacol.1978,63(3), 551–559. [10] C. T. Rizzi, J. L. Carvalho-de-Souza, E. Schiavon, A. C. Cassola, E. Wanke,
L. R. Troncone,Toxicon2007,50(4), 553–562.
[11] N. Y. Yount, D. Kupferwasser, A. Spisni, S. M. Dutz, Z. H. Ramjan, S. Sharma, A. J. Waring, M. R. Yeaman,Proc. Natl. Acad. Sci. U.S.A.2009,
106(35), 14972–14977.
[12] S. Peigneur, D. J. Orts, A. R. Prieto da Silva, N. Oguiura, M. Boni-Mitake, E. B. Oliveira, A. J. Zaharenko, J. C. Freitas, J. Tytgat,Mol. Pharmacol.
2012,82(1), 90–96.
[13] A. Pereira, A. Kerkis, M. A. Hayashi, A. S. Pereira, F. S. Silva, E. B. Oliveira, A. R. Prieto da Silva, T. Yamane, G. Rádis-Baptista, I. Kerkis,
Expert Opin. Investig. Drugs2011,20(9),1189–1200.
[14] N. Oguiura, M. Boni-Mitake, R. Affonso, G. Zhang,J. Antibiot. (Tokyo) 2011,64(4), 327–331.
[15] E. S. Yamane, F. C. Bizerra, E. B. Oliveira, J. T. Moreira, M. Rajabi, G. L. Nunes, A. O. de Souza, I. D. da Silva, T. Yamane, R. L. Karpel, P. I. Silva Jr., M. A. Hayashi,Biochimie2013,95(2), 231–240.
[16] S. El Chamy Maluf, C Dal Mas, E. B. Oliveira, P. M. Melo, A. K. Carmona, M. L. Gazarini, M. A. Hayashi,Peptides2016,78, 11–16.
[17] A. Kerkis, I. Kerkis, G. Rádis-Baptista, E. B. Oliveira, A. M. Vianna-Morgante, L. V. Pereira, T. Yamane, FASEB J.2004,18(12), 1407-1409.
[18] P. C. Chen, M. A. Hayashi, E. B. Oliveira, R. L. Karpel,PLoS One2012,
7(11), e48913.
[19] F. D. Nascimento, M. A. Hayashi, A. Kerkis, V. Oliveira, E. B. Oliveira, G. Rádis-Baptista, H. B. Nader, T. Yamane, I. L. Tersariol, I. Kerkis.J. Biol. Chem.2007,282(29), 21349–21360.
[20] F. D. Nascimento, L. Sancey, A. Pereira, C. Rome, V. Oliveira, E. B. Oliveira, H. B. Nader, T. Yamane, I. Kerkis, I. L. Tersariol, J. L. Coll, M. A. Hayashi,Mol. Pharm.2012,9(2), 211–221.
[21] Toxicology NRCUCoD. Scientific Frontiers in Developmental Toxi-cology and Risk Assessment, 4th ed. Washington (DC): National Academies Press (US);2000.
[22] M. Vaughan, R. van Egmond,Atla-Altern. Lab. Anim.2010,38(3), 231– 238.
[23] N. S. Sipes, S. Padilla, T. B. Knudsen,Birth Defects Res. C2011,93(3), 256–267.
[24] C. W. Liu, F. Xiong, H. Z. Jia, X. L. Wang, H. Cheng, Y. H. Sun, X. Z. Zhang, R. X. Zhuo, J. Feng,Biomacromolecules2013,14(2), 358–366. [25] L. Wang, J. Y. W. Chan, J. V. Rego, C. M. Chong, N. Ai, C. B. Falcao,
S. M. Y. L. Rádis-Baptista,BBA-Gen. Subjects2015,1850(6), 1253-1260.
[26] M. Westerfield, The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed. Eugene: University of Oregon Press;
[27] K. S. Wilson, J. Baily, C. S. Tucker, G. Matrone, S. Vass, C. Moran, K. E. Chapman, J. J. Mullins, C. Kenyon, P. W. Hadoke, M. A. Denvir,Mol. Cell. Endocrinol.2015,414, 120–131.
[28] Y. Zhang,Dongwuxue Yanjiu2015,36(4), 183–222.
[29] J. W. Fox, S. M. Serrano,Curr. Pharm. Des.2007,13(28), 2927–2934. [30] A. L. Harvey,Toxicon2014,92, 193–200.
[31] G. Fotakis, J. A. Timbrell,Toxicol. Lett.2006,160(2), 171–177. [32] S. Pacor, A. Giangaspero, M. Bacac, G. Sava, A. Tossi,J. Antimicrob.
Chemother.2002,50(3), 339–348.
[33] S. M. Gregory, A. Cavenaugh, V. Journigan, A. Pokorny, P. F. Almeida,
Biophys. J.2008,94(5), 1667–1680.
[34] M. G. Morash, S. E. Douglas, A. Robotham, C. M. Ridley, J. W. Gallant, K. H. Soanes,Dis. Model Mech.2011,4(5), 622–633.
[35] J. Kopeika, T. Zhang, D. Rawson,Cryo Lett.2006,27(5), 319–328. [36] A. D. Vliegenthart, C. S. Tucker, J. Del Pozo, J. W. Dear,Br. J. Clin.
Phar-macol.2014,78(6), 1217–1227.
[37] J. E. Fletcher, M. Hubert, S. J. Wieland, Q. H. Gong, M. S. Jiang,Toxicon 1996,34(11–12), 1301–1311.
How to cite this article: Chan JY, Zhou H, Kwan YW, et. al. Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnos-tics and therapeudiagnos-tics.J Biochem Mol Toxicol. 2017;00:e21964.
