Epoxyscillirosidine Induced Cytotoxicity and Ultrastructural Changes in a Rat Embryonic Cardiomyocyte (H9c2) Cell Line.

Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside-induced intoxication in ruminants in South Africa. The toxic principle, 1α, 2α-epoxyscillirosidine, is a bufadienolide. To replace the use of sentient animals in toxicity testing, the aim of this study was to evaluate the cytotoxic effects of epoxyscillirosidine on rat embryonic cardiomyocytes (H9c2 cell line). This in vitro cell model can then be used in future toxin neutralization or toxico-therapy studies. Cell viability, evaluated with the methyl blue thiazol tetrazolium (MTT) assay, indicated that a hormetic dose/concentration response is characterized by a biphasic low dose stimulation and high dose inhibition. Increased cell membrane permeability and leakage, as expected with necrotic cells, were demonstrated with the lactate dehydrogenase (LDH) assay. The LC50 was 382.68, 132.28 and 289.23 µM for 24, 48, and 72 h respectively. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed at higher doses. Ultra-structural changes suggested that the cause of H9c2 cell death, subsequent to epoxyscillirosidine exposure, is necrosis, which is consistent with myocardial necrosis observed at necropsy. Based on the toxicity observed, and supported by ultra-structural findings, the H9c2 cell line could be a suitable in vitro model to evaluate epoxyscillirosidine neutralization or other therapeutic interventions in the future.

Novel and efficient synthesis of 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-one analogues.

The efficient synthesis of some 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-ones was investigated. 22-Iodocyclo-18,21-dinorcholenones were prepared from cyclo-18,21-dinorcholenones using I(2)/DMAP/pyridine system firstly. The cross coupling reaction of 22-iodocyclo-18,21-dinorcholenones and 1-alkynes was carried out efficiently catalyzed by tetrakis(triphenylphosphine) palladium/cuprous iodide in the presence of base diisopropylethylamine. This strategy offered a very straightforward and efficient method for access to conjugated alkynyl cyclo-18,21-dinorcholenones from the cyclo-18,21-dinorcholenones and 1-alkynes in excellent overall yields. Evaluation of the synthesized compounds for cytotoxicity against KB, HeLa, MKN-28 and MCF-7 cell lines showed that the 22-alkynylcyclodinorchoenones possessing hydroxylethyl and hydroxylmethyl mono-substituted side chain at the end of alkynyl group have significantly inhibition activity.

Synthesis and mutagenicity of a ring-A-aromatized bile acid, 3-hydroxy-19-nor-1,3,5(10)-cholatrien-24-oic acid.

It has been presumed that ring-A-aromatized bile acids are produced from biliary bile acids by intestinal flora and the acids thus formed participate in the large bowel carcinogenesis. One of these acids is probably 3-hydroxy-19-nor-1,3,5(10)-cholatrien-24-oic acid, judged from the literatures. Consequently, this acid was synthesized from previously prepared 3-methoxy-19-nor-1,3,5(10)-cholatrien-24-ol. The phenolic ether was successively oxidized with pyridinium chlorochromate and wet silver oxide to give 3-methoxy-19-nor-1,3,5(10)-cholatrien-24-oic acid in high yield, which, after successive treatments with methanol containing a catalytic amount of p-toluenesulfonic acid, a combination of aluminum chloride and ethanethiol, and alkali, gave the desired compound in satisfactory yield. The compound was not mutagenic in Salmonella tester strains TA 98 and TA 100, but it increased the mutagenicity of 2-aminoanthracene when both were applied to plates together. When compared with cholic, deoxycholic, and lithocholic acids, the investigated compound exhibited about two to threefold increase of mutagenicity in the latter assay.