New 8-hydroxyquinoline and catecholate iron chelators: influence of their partition coefficient on their biological activity.

Four new hexadendate chelators, three hydroxyquinoline-based, Csox, O-Trensox, Cox750, and one catecholate-based CacCam-which have comparable skeletal structures and pFe, but widely different partition coefficients, (Kpart), 0.01, 0.02, 1 and 3.2 respectively, have been tested for their iron chelating efficacy in vitro by two methods. First, by their ability to remove iron from ferritin in solution or second, to remove iron from iron-loaded hepatocytes in vitro. Our objective was to ascertain the importance of Kpart and pFe, on the biological efficiency of the molecule. Previous studies proposed that an ideal value of Kpart of 1 should give maximum biological activity. Mobilization of iron by Csox and CacCAM from ferritin was similar and furthermore more efficient than desferrioxamine B. In the iron-loaded hepatocyte cultures, the three hydroxyquinoline chelators, although showing diversity in terms of lipophilicity, appeared to be very similar in their capacity to chelate iron. CacCAM, the unique catecholate, was the most efficient of the molecules tested, as well as being the least toxic in the cellular model despite having the lowest value of pFe. In conclusion, the use of the partition coefficient and pFe, as tools for predicting biological activity of iron chelators should be not generalized. Further studies are required in order to understand the influence of the structure on the biological activity of the molecule.

Antiproliferative and apoptotic effects of O-Trensox, a new synthetic iron chelator, on differentiated human hepatoma cell lines.

We investigated the effects of a new iron chelator, O-Trensox (TRX), compared with desferrioxamine (DFO), on proliferation and apoptosis in cultures of the human hepatoblastoma HepG2 and hepatocarcinoma HBG cell lines. Our results show that TRX decreased DNA synthesis in a time- and dose-dependent manner and with a higher efficiency than DFO. Mitotic index was also strongly decreased by TRX and, unexpectedly, DFO inhibited mitotic activity to the same extent as TRX, thus there is a discrepancy between the slight reduction in DNA synthesis and a large decrease in mitotic index after DFO treatment. In addition, we found that TRX induced accumulation of cells in the G(1) and G(2) phases of the cell cycle whereas DFO arrested cells in G(1) and during progression through S phase. These data suggest that the partial inhibition of DNA replication observed after exposure to DFO may be due to a lower efficiency of metal chelation and/or that it does not inhibit the G(1)/S transition but arrests cells in late S phase. The effects of both TRX and DFO on DNA synthesis and mitotic index were reversible after removing the chelators from the culture medium. An apoptotic effect of TRX was strongly suggested by analysis of DNA content by flow cytometry, nuclear fragmentation and DNA degradation in oligonucleosomes and confirmed by the induction of a high level of caspase 3-like activity. TRX induced apoptosis in a dose- and time-dependent manner in proliferating HepG2 cells. In HBG cells, TRX induced apoptosis in proliferating and confluent cells arrested in the G(1) phase of the cell cycle, demonstrating that inhibition of proliferation and induction of apoptosis occurred independently. DFO induced DNA alterations only at concentrations >100 microM and without induction of caspase 3-like activity, indicating that DFO is not a strong inducer of apoptosis. Addition of Fe or Zn to the culture medium during TRX treatment led to a complete restoration of proliferation rate and inhibition of apoptosis, demonstrating that Fe/Zn-saturated TRX was not toxic in the absence of metal depletion. These data show that TRX, at concentrations of 20-50 microM, strongly inhibits cell proliferation and induces apoptosis in proliferating and non-proliferating HepG2 and HBG cells, respectively.

Irniine, a pyrrolidine alkaloid, isolated from Arisarum vulgare can induce apoptosis and/or necrosis in rat hepatocyte cultures.

The effects of irniine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, on rat hepatocyte primary cultures and rat liver epithelial cell line (RLEC) were studied. Cytotoxicity was first evaluated by LDH release, MTT and NR tests and MDA production, while cellular alterations were visualized by electron microscopy and DNA gel-electrophoresis. In hepatocyte and RLEC cultures, a major toxicity appeared at 40 microM of irniine and was demonstrated by an increase in LDH release and decreases in MTT reduction and NR uptake while concentrations lower than 40 microM did not induce significant changes in these parameters. However, we observed an increase in MDA production at 30 microM. Important alterations of the nuclei and mitochondria were also visualized by electron microscopy in cells treated with 50 microM. Using DNA gel-electrophoresis, we demonstrated that irniine at 40 and 50 microM induced DNA damage. All together these results demonstrate that: (1) Irniine induces a significant hepatotoxicity. (2) Irniine toxicity is not mediated by a metabolic derivative since RLEC, which do not contain a monooxygenase system, were also affected by this compound. (3) Irniine induces a significant DNA damage and oxidative stress which leads to cell death by necrosis and/or by apoptosis. Moreover, our data suggest that the alkaloid irniine contained in A. vulgare may be involved in the toxic symptoms observed after medicinal use or consumption of the plant tubers as food both by humans and animals.

Cytoprotection and iron mobilization in rat hepatocyte cultures by a new synthetic dihydroxamate chelator.

The cytoprotection and iron mobilization effect of a new dihydroxamate chelator 1,1 bis [(11-N-hydroxy)-2,5,11-triaza-1,6,10-trioxo dodecanyl] ethane or KD was studied in primary rat hepatocyte cultures exposed to iron-citrate. Lactate dehydrogenase (LDH) release and malondialdehyde (MDA) production were measured as indexes of cytotoxicity. Cell viability was evaluated using the [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] (MTT) reduction test. To demonstrate that this chelator was able to decrease iron uptake or increase iron release from the hepatocytes, labelled cells were obtained by maintaining the cultures in the presence of 0.02 microM 55Fe-citrate. The efficacy of KD was compared to desferrioxamine B (DFO) at stoechiometry concentrations. After 24 h of exposure to 50 microM of iron-citrate, a significant release of LDH and MDA was observed. Cell viability was also significantly decreased. When 100 microM of KD were added at the same time as iron, LDH and MDA release was decreased and cell viability was improved. In the presence of the same chelator concentration, a net decrease of iron uptake by the cells was observed as attested by the low intracellular 55Fe level. Moreover, in the 55Fe loaded hepatocytes, the chelator increased the iron extracellular level indicating its iron release effect from the cells. In all tested experimental conditions, the efficacy of 100 microM of the dihydroxamate chelator KD was close to that of 50 microM of the trihydroxamate chelator DFO. In conclusion, KD is effective at a level comparable to DFO in protecting rat hepatocytes against the toxic effect of iron-citrate by decreasing the uptake of the metal and increasing its release from the cells. This synthetic compound appears to have some potential therapeutical interest and the results obtained encourage the synthesis of new hydroxamate ligands.

Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures.

Toxicity of bgugaine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, was studied in three different liver cell culture models: (1) the rat hepatocyte primary culture; (2) a liver epithelial cell line; and (3) the human hepatoblastoma cell line HepG2. Cytotoxicity was evaluated by LDH release, MTT reduction and MDA production. DNA fragmentation was analysed by flow cytometry or DNA gel-electrophoresis. In hepatocyte and epithelial cell cultures, drug toxicity appeared at 30 microM and was evaluated by an increase in LDH release, a decrease in MTT reduction and a higher level of MDA production. Bgugaine concentrations lower than 30 microM did not induce changes in these parameters. In HepG2 cells, bgugaine treatment also induced LDH release at concentrations of 40 and 50 microM. DNA fragmentation, analysed in the HepG2 cell line by flow cytometry, was observed in cultures exposed to 50 microM bgugaine. However, using DNA gel-electrophoresis, we demonstrated that lower bgugaine concentrations (10, 20 and 30 microM) also induced DNA damage. Our results show that: (1) bgugaine induces an important hepatotoxicity; (2) bgugaine toxicity is not mediated by a metabolic derivative; and (3) bgugaine induces a significant DNA damage. Therefore, our data suggest that the alkaloid bgugaine contained in Arisarum vulgarae may be involved in the toxicologic symptoms observed after consumption of this plant tubers by humans and animals.

Iron mobilisation and cellular protection by a new synthetic chelator O-Trensox.

We tested a new synthetic, 8-hydroxyquinoline-based, hexadentate iron chelator, O-Trensox and compared it with desferrioxamine B (DFO). Iron mobilisation was evaluated: (i) in vitro by using ferritin and haemosiderin; DFO mobilised iron much more rapidly from ferritin at pH 7.4 than did O-Trensox, whereas at pH 4, ferritin and haemosiderin iron mobilisation was very similar with both chelators; (ii) in vitro by using cultured rat hepatocytes which had been loaded with 55Fe-ferritin; here DFO was slightly more effective after 100 hr than O-Trensox; (iii) in vivo administration i.p. to rats which had been iron-loaded with iron dextran; O-Trensox mobilised 51.5% of hepatic iron over two weeks compared to 48.8% for DFO. We also demonstrated the effect of O-Trensox in decreasing the entry of 55Fe citrate into hepatocyte cultures. The protective effect of O-Trensox against iron toxicity induced in hepatocyte cultures by ferric citrate was shown by decreased release of the enzymes lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotranferase (ALT) from the cultures and, using electron paramagnetic resonance (EPR) measurements, decreased production of lipid radicals. O-Trensox was more effective than DFO in quenching hydroxyl radicals in an acellular system.