Miloslav Dobrota

Induction of apoptosis by a novel copper-based anticancer compound, casiopeina II, in L1210 murine leukaemia and CH1 human ovarian carcinoma cells.

The activity of casiopeina II [Cu(1,4-dimethyl-1, 10-phenanthroline)(glycine)NO(3)], a novel anticancer agent, was tested in two cell lines, L1210 murine leukaemia, CH1 human ovarian carcinoma, cisplatin-resistant and sensitive. Exposure of the cells to a range of concentrations of casiopeina II indicates that this copper complex kills cells by apoptosis and necrosis. Condensed chromatin and nuclear fragmentation were observed after exposure to casiopeina II. The caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) almost completely inhibited apoptosis induced by cisplatin; however, casiopeina II-induced apoptosis was inhibited only by 50-70%. These data are consistent with caspase activation (measured by Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; Z-DEVD-AFC) by casiopeina II and cisplatin and confirm that caspases are activated in the apoptotic cell death induced by casiopeina II. DNA fragmentation was observed in L1210 cells, but not in CH1 cells. No difference in susceptibility to induction of apoptosis by casiopeina II was found between sensitive and cisplatin resistant cells. In this work we show that the novel copper-based antineoplastic agent casiopeina II is highly active against murine and human cancer cell lines, including cell lines resistant to cisplatin.

Sources of the proteins of rat bile.

The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component. The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an alpha2-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.

Endocytic vesicles in liver carry polymeric IgA from serum to bile

The distributions both of endogenous IgA and of injected 125I-labelled IgA were determined amongst the components of a liver homogenate. Rate zonal sedimentation, under conditions where separation was principally determined by particle size, showed that IgA was tightly bound to material which sedimented in the size range of the larger endoplasmic reticulum fragments. Further fractionation of the components within this size range according to their densities, by isopycnic centrifugation, showed that the IgA was associated with small vesicles with a density range of 1.12--1.17 g/ml, quite distinct from endoplasmic reticulum fragments. We therefore conclude that the IgA is present in liver cells in a distinct class of vesicles, which are, presumably, responsible for the transport of IgA from blood to bile.

Kinetics and mechanism of uptake of platinum-based pharmaceuticals by the rat small intestine

The absorption of two platinum-based pharmaceuticals, cisplatin and carboplatin, was studied using in vitro and in situ models. By utilizing everted rat small intestine, it was found that absorption of both drugs was linear with time up to 60 min and was not saturable up to a concentration of 1.0 mM. Moreover, uptake against a concentration gradient could not be demonstrated and absorption was not reduced by metabolic inhibition or anoxic conditions. These results indicate the lack of involvement of an active transport mechanism for cisplatin and carboplatin and imply that absorption across the gastrointestinal tract is by passive diffusion. Cisplatin was absorbed more readily than carboplatin, both in vitro and in situ. In situ both drugs were found to disappear from the intestinal lumen following first-order kinetics. The results of in situ studies indicate that a decrease in pH of the perfusion medium leads to an increase in absorption of carboplatin into the systemic blood. This report establishes the fact that both cisplatin and carboplatin are absorbed across the gastro-intestinal tract and indicates that preclinical trials involving oral administration of platinum-based pharmaceuticals could be justified.

Comparative responses to mode of oral administration and dose of ochratoxin A or nephrotoxic extract of Penicillium polonicum in rats

Administration of Penicillium polonicum extract to male Sprague-Dawley rats (200 g), either mixed in feed or given daily by gavage, for 5 days, had no clinical effects. However, at necropsy on day 6 marked histopathological changes occurred in renal tubule epithelia, including mitotic figures, karyomegalic nuclei, and frequent apoptosis identified specifically by TUNEL methodology and confocal microscopy. Ochratoxin A given similarly to rats (daily, 1 mg or 0.2 mg) was also clinically asymptomatic except for the 1 mg dose given by gavage; rats in this group lost weight. Marked renal tubular necrosis, though even without any significant accompanying apoptosis, was evident only at this higher dose by gavage; it was associated also with the highest incidence of renal DNA adducts and a disproportionately high concentration of ochratoxin A in plasma on day 6. Significantly fewer renal DNA adducts were detected in rats given 1 mg ochratoxin A in feed. The study demonstrates the potential for exaggerated toxicological responses to ochratoxin A administered by gavage through predicted consequential surges in the circulating concentration of the mycotoxin.

Haptoglobin-mediated transfer of haemoglobin from serum into bile

The majority of the proteins present in rat bile are also present in rat serum [I]. These proteins common to serum and bile fall into two distinct groups. In the first group are major serum proteins which are present in bile at -0.1% of their concentration in serum. The relative concentrations of these proteins appear inversely related to their molecular weight (R.H.H., B.M.M. unpublished) and it would seem likely that, as has been suggested in man and dog [2,3], the presence of these proteins in bile is due to diffusion across the tight junctions between hepatocytes. However two serum proteins, identified in [1] as IgA and ‘bile protein 8’ are relatively concentrated in bile. IgA is known to be actively transported from blood to bile [4]. The mechanism of transport involves the binding of IgA to secretory component on the sinusoidal surface of hepatocytes [S ,6] and transport across the hepatocytes in endocytic vesicles [7,8]. These vesicles are not, however, solely concerned with transporting IgA and its receptor secretory component, to bile. Exam~ation of the distribution of a bile protein 8 amongst liver cell fractions showed that it was also associated with vesicles similar in size to the vesicles which contain IgA [9]. Moreover newly made bile protein 8 and newly made secretory component appeared in bile after a similar time lag [lo]. We now show that the role of bile protein 8 is to transfer haemoglobin from serum to bile and that bile protein 8 is haptoglobin partially or totally saturated with haemoglobin.

Metal toxicity in two rodent species and redox potential: Evaluation of quantitative structure-activity relationships

A quantitative structure-activity relationship study of acute toxicity in the mouse and rat is described for the soluble salts of a relatively large number of metals (between 25 and 30 in total). Electrode potential is the major determinant of acute metal toxicity (R = 0.85 and 0.86) for an intraperitoneal dose in the mouse, whereas the addition of ionic radius and polarizability enables the inclusion of notable outliers in the original expression, such as beryllium and barium, thus giving a good correlation (R = 0.87) with toxicity for 27 metal compounds. These findings are rationalized on the basis of relative ease of ionization, electron affinity, and transport factors of the metals and their ions, thus being consistent with the hard and soft acids and bases properties of metals and their biological reactivities.

Binding of lanthanides to cell membranes in the presence of ligands

The effect of a series of ligands on the binding of the lanthanide, europium (Eu), to rabbit intestinal cell membranes was investigated in vitro. When tested as Eu-ligand complexes (ratio of Eu:ligand, 1:2) of intermediate stability (log stability constant, log K1, for the reaction Eu + L = EuL, of about 7-12) such as Eu-citrate and Eu-nitrilotriacetate (NTA), Eu was available for uptake in a soluble form by intestinal brush-border membrane vesicles (BBMV) in phosphate- and bicarbonate-free solutions at pH 7.2. Ligands with lower log K1 did not maintain Eu in solution whilst those of higher affinity did not donate it to membranes. Generally, there was a clear relationship between log K1 of the Eu-ligand complex and the binding of Eu to BBMV. This relationship identifies ligands that can effectively donate Eu to vesicles under these conditions. BBMV uptake of Eu was due to binding at two sites. Binding to the diethylenetriaminepentaacetate (DTPA)-sensitive site predominated at 20 degrees C and uptake by the DTPA-insensitive site was enhanced at 37 degrees C. Only trace amounts of the bound Eu appeared to be internalized within the vesicles. In the presence of physiological concentrations of phosphate and bicarbonate in cell culture medium, Eu was precipitated from most complexes (at 1:2 and 1:5 Eu:ligand ratio) except DTPA and albumin. Eu precipitation could be prevented by increasing the ligand:Eu ratio. When isolated hepatocytes in cell culture medium were incubated with EuCl3, about 60% of Eu was bound to the cells; Eu-albumin was not bound by hepatocytes.

Distribution and excretion of lanthanides: comparison between europium salts and complexes

Europium (152,154Eu) was intravenously injected into rats as: (i) the chloride salt at pH 7.4, (ii) the chloride salt at pH 3, (iii) the albumin complex and (iv) the DTPA complex, and tissue uptake was determined 24 h later. For the chlorides, the target organ for uptake was liver (about 60% of dose) whilst europium complexes were rapidly excreted in urine and were predominantly taken up into the kidney (about 0.5% of dose) and bone. Liver uptake of EuCl3, pH 7.4, corresponded to that of a colloidal material with most 152Eu present in the non-hepatocyte population; however, EuCl3, pH 3, was handled in a different manner, with significant uptake by hepatocytes. The differing tissue distributions of EuCl3 and Eu-albumin suggest that plasma albumin does not readily bind injected EuCl3. Renal uptake of europium, although a relatively low proportion of the injected dose, was associated with many subcellular fractions, including lysosomes, suggesting significant intracellular uptake and thus possible retention.

A simple gradient maker for use with zonal rotors

Abstract A simple gradient maker based on the addition and removal of liquid from a mixing vessel at independently variable rates has been developed and tested. The mathematical form of the gradients produced has been calculated and a program developed to fit gradients formed by this machine to those required by theoretical considerations.