Upon exploration of the chemistry of the combination
of ruthenium/arene
with anthraquinone alizarin (L), three new complexes with the general
formulas [Ru(L)Cl(η6-p-cymene)]
(C1), [Ru(L)(η6-p-cymene)(PPh3)]PF6 (C2), and [Ru(L)(η6-p-cymene)(PEt3)]PF6 (C3) were synthesized and characterized using spectroscopic
techniques (mass, IR, and 1D and 2D NMR), molar conductivity, elemental
analysis, and X-ray diffraction. Complex C1 exhibited
fluorescence, such as free alizarin, while in C2 and C3, the emission was probably quenched by monophosphines and
the crystallographic data showed that hydrophobic interactions are
predominant in intermolecular contacts. The cytotoxicity of the complexes
was evaluated in the MDA-MB-231 (triple-negative breast cancer), MCF-7
(breast cancer), and A549 (lung) tumor cell lines and MCF-10A (breast)
and MRC-5 (lung) nontumor cell lines. Complexes C1 and C2 were more selective to the breast tumor cell lines, and C2 was the most cytotoxic (IC50 = 6.5 μM
for MDA-MB-231). In addition, compound C1 performs a
covalent interaction with DNA, while C2 and C3 present only weak interactions; however, internalization studies
by flow cytometry and confocal microscopy showed that complex C1 does not accumulate in viable MDA-MB-231 cells and is detected
in the cytoplasm only after cell permeabilization. Investigations
of the mechanism of action of the complexes indicate that C2 promotes cell cycle arrest in the Sub-G1 phase in MDA-MB-231,
inhibits its colony formation, and has a possible antimetastatic action,
impeding cell migration in the wound-healing experiment (13% of wound
healing in 24 h). The in vivo toxicological experiments
with zebrafish indicate that C1 and C3 exhibit
the most zebrafish embryo developmental toxicity (inhibition of spontaneous
movements and heartbeats), while C2, the most promising
anticancer drug in the in vitro preclinical tests,
revealed the lowest toxicity in in vivo preclinical
screening.
Zebrafish (Danio rerio) is a tropical fish species widely used in research, worldwide. The development of genetically modified animals and the increasing number of zebrafish breeding facilities due to their emerging use in several research fields, opened room for new ethical challenges for research carried out with this species. It is necessary to raise the scientific community’s awareness of the ethical standards and laws in force, on animal research. Thus, the aim of the current study is to describe 10 Rs ethical principles by using zebrafish as model system in research. The classical 3 Rs concerning animal welfare, namely replacement, reduction and refinement; and the added 7 Rs related to scientific (registration, reporting, robustness, reproducibility and relevance) and conduct principles (responsibility, and respect) in zebrafish research are herein presented and critically discussed. The use of these 10 Rs by researchers, institutions and the Animal Ethics Committee is recommended to support regulations, decision-making about and the promotion of zebrafish health and welfare in research.
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