Hematotoxicity response in rats by the novel copper-based anticancer agent: casiopeina II.

The in vivo toxicity of the novel copper-based anticancer agent, casiopeina II (Cu(4,7-dimethyl-1,10-phenanthroline)(glycine)NO3) (CII), was investigated. Casiopeinas are a family of copper-coordinated complexes that have shown promising anticancer activity. The major toxic effect attributed to a single i.v. administration of CII (5 mg/kg dose) in the rat was an hemolytic anemia (reduced hemoglobin concentration (HB), red blood cell (RBC) count and packed cell volume (PCV) accompanied by a marked neutrophilic leukocytosis) 12 h and 5 days after administration, attributed to a direct erythrocyte damage. Increased reticulocyte levels and presence of normoblasts in peripheral blood 5 days post-administration indicated an effective erythropoietic response with recovery at 15 days. Increase in spleen weight and the morphological evidence of congestion of the red pulp (RP) with erythrocytes (E) resulting in a higher ratio of red to white pulp (WP) was consistent with increased uptake of damaged erythrocytes by the reticuloendothelial system observed by histopathology and electron microscopy. Extramedullary hemopoiesis was markedly increased at 5 days giving further evidence of a regenerative erythropoietic response that had an effective recovery by 15 days. Morphological changes in spleen cellularity were consistent with hematotoxicity, mainly a reduction of the red pulp/white pulp ratio, increase in erythrocyte content at 12 h, and an infiltration of nucleated cells in the red pulp at 5 days, with a tendency towards recovery 15 days after administration. The erythrocyte damage is attributed to generation of free radicals and oxidative damage on the membrane and within cells resulting from the reduction of Cu(II) and the probable dissociation of the CII complex.

Effects on male rats of di-(2-ethylhexyl) phthalate and di-n-hexylphthalate administered alone or in combination.

The effects of phthalate esters of branched chain alcohols, typified by di-(2-ethylhexyl)phthalate (DEHP) differ from those of esters of straight chain alcohols typified by di-n-hexyl phthalate (DnHP). The former induce liver enlargement and proliferation of hepatic peroxisomes, while the latter cause no peroxisome proliferation but cause fat accumulation in the liver. Both classes of phthalate esters are hypolipidaemic and cause thyroid changes associated with an increased rate of thyroglobulin turnover. As phthalate esters are used as mixtures, we have examined the effect of mixtures of the compounds. Groups of five male Wistar albino rats were administered either control diet or diets containing either 10000 ppm of DEHP, 10000 ppm of DnHP or 10000 ppm DEHP plus 10000 ppm DnHP for 14 days. Rats receiving diets containing DEHP showed the expected increase in relative liver weight, in "peroxisomal" fatty acid oxidation and in CYP4A1. Serum triglyceride and serum cholesterol were also reduced, and the thyroid showed the histological changes mentioned above. Rats consuming diets containing DnHP showed no increase in relative liver weight and no induction of peroxisomal fatty acid oxidation or CYP4A1. However, there was a marked accumulation of fat in the liver. The fall in serum cholesterol was similar to that in rats treated with DEHP, but the fall of serum triglyceride was more pronounced. Thyroidal changes were again observed. In general, changes in rats treated with a mixture of DEHP and DnHP were very similar to those found with rats treated with DEHP alone. The liver was enlarged, and peroxisomal fatty acid oxidation and CYP4A1 were both induced. The amount of fat in the liver was much less than in rats receiving DnHP alone. Thyroid changes were similar to those in rats receiving the individual compounds. The effect on serum cholesterol seemed additive, but the levels of serum triglyceride were intermediate between the groups receiving the single compounds.

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.

Proliferation of hepatic peroxisomes in rats following the intake of green or black tea.

Rats maintained on green, black or decaffeinated black tea (2.5%, w/v) as their sole drinking fluid displayed higher hepatic CN- insensitive palmitoyl CoA oxidase activity than controls; the extent of increase was similar with the three types of tea. Morphological examination of the liver using electron microscopy revealed an increase in the number of peroxisomes in the tea-treated animals. The same treatment of the animals with green and black tea resulted in a similar rise in hepatic microsomal lauric acid hydroxylation. Analysis by HPLC of the aqueous tea extracts employed in the current study showed that the total flavanol content of the green variety was much higher than the black varieties, and confirmed the absence of caffeine in the decaffeinated black tea. It may be concluded from the present studies that neither caffeine nor flavanoids are likely to be responsible for the proliferation of peroxisomes observed in rats treated with tea.

Structural and functional integrity of precision-cut liver slices in xenobiotic metabolism: a comparison of the dynamic organ and multiwell plate culture procedures.

1. Objectives were two-fold: (1) to compare the viability of precision-cut liver slices in two culture systems, namely the dynamic organ and the multiwell plate; and (2) to evaluate whether increasing the number of slices per incubation results in a proportional increase in the extent of metabolism. 2. With both culturing systems, the major products of 7-ethoxycoumarin metabolism were the sulphate and glucuronide conjugates of 7-hydroxycoumarin with very low levels of the free compound. When the multiwell plate procedure was used, metabolism increased linearly for at least 10 h, whereas it tended to plateau after 6 h in the dynamic organ culture system. At preincubations > 10 h, significantly more metabolism of 7-ethoxycoumarin was seen in the slices cultured using the multiwell system compared with the dynamic organ system. 3. Morphological evaluation employing light and electron microscopy revealed that liver slices incubated using the multiwell system were structurally better preserved compared with those incubated using the dynamic organ system. 4. Using the multiwell system, increasing the number of slices per incubation from one to two resulted in only a modest increase in the metabolism of 7-ethoxycoumarin. The rate of metabolism of this substrate was much higher with one liver slice when expressed per mg homogenate protein. 5. It is concluded that (1) the multiwell plate culture system for culturing slices is superior to the dynamic organ system in studying the metabolism of xenobiotics following long-term incubations, (2) increasing the number of slices per incubation does not result in a corresponding increase in the rate of metabolism, and (3) in both culture systems optimal viability appears to be within 24 h of incubation.

Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases.

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.

The LEC rat possesses reduced hepatic selenium, contributing to the severity of spontaneous hepatitis and sensitivity to carcinogenesis.

The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC rats, which develop a spontaneous form of hepatitis at 3-4 months of age, and compared to trace metal concentrations in the LEA rat, its asymptomatic congenic strain. Consistent with results found by other groups, copper was found to accumulate within the liver of LEC rats to levels more than 50 times those measured in LEA rats. In addition, liver selenium concentration in LEC rats was found to be around 50% of that in LEA rats. The enzyme activity, and RNA for the selenium dependent enzyme, glutathione peroxidase, was also found to be reduced in LEC rat liver. These results indicate that hepatic selenium in the LEC rat is depleted and that, as a result of this, the capacity to protect cells from copper-induced free-radical damage is reduced.

The effects of platinum chemotherapy on essential trace elements.

The effects of cisplatin chemotherapy on the metabolism of essential trace elements were investigated in 12 patients before and after treatment with cisplatin. In serum, the mean post-treatment concentrations of Cu 913.91 mumol 1-1), Zn (9.57 mumol 1-1) and Mg (0.54 mumol 1-1) were significantly reduced compared with the pre-treatment levels 919.35, 11.86 and 0.67) while Se, caeruloplasmin and C-reactive protein concentrations were unaltered. Urinary excretion of Cu, Mg and Zn were enhanced. The urinary N-acetyl-beta-D-glucosaminidase activity (a marker of proximal renal tubular dysfunction) was also increased and suggests that the mechanism for decrease of certain trace elements in serum during treatment could be increased urinary excretion caused by impaired cellular metabolism. It is not clear whether the loss of trace elements via the urine has any implication for the clinical status of cancer patients treated with cisplatin.

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.

Biochemical and morphological effects of contrast media on the kidney.

The intravenous use of roentgen contrast media (CM) is associated with a low incidence of renal impairment. This paper considers the intravascular handling and retention of CM in relation to effects on renal function - specifically the ability of the kidney to reabsorb and catabolise low molecular weight proteins. Renal morphology following experimental administration of a high dose of an isotonic dimeric CM (iodixanol at 3 g I/kg) in rats showed numerous, large, protein-containing vacuoles or droplets in the cells of the proximal convoluted tubule. These were fully formed within 3.5 hours. The process of vacuole-formation involving the uptake of CM appears to be analogous to dextran uptake that occurs via fluid phase endocytosis. These vacuoles or CM droplets are abundant for 7 days but then slowly decline over several weeks. The quantitative recovery of (14)C iodixanol (3g I/kg) from the kidneys between 3.5 hours to 7 days after administration was about 1% of the dose, with some 0.2% of the original dose still present at 28 days. Subcellular analysis to determine the site of the radiolabel showed that the (14)C was associated with lysosomal marker enzymes. The CM-induced vacuoles/droplets are most probably giant lysosomes, which contain the intracellularly retained CM. Co-administration of tracer doses of (125)I-labelled cytochrome C with iodixanol showed some impairment of low molecular weight protein reabsorption, but remarkably this process was not effected when the vacuoles were fully formed. The conspicuous morphology of the vacuoles, the CM retention and the transient proteinuria and enzymuria cannot presently be associated with any functionally significant impairment of tubular or cellular processes.

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.

Effect of oral aluminium citrate on short-term tissue distribution of aluminium.

Aluminium (Al) concentrations in the plasma, bone, lung, liver, kidney, spleen, duodenum and brain of rats were measured 2, 4 and 24 hr after a single oral dose of 0.46 mmol as Al citrate (1:5 molar ratio). Compared with control animals, very high concentrations were found at 2 hr post-administration in plasma (539 micrograms/litre) and in all tissues except the brain where Al did not change throughout the 24-hr period. The increased levels in the liver (161 ng/g) and lung (89.7 ng/g) at 2 hr were maintained until 4 hr and then decreased. At 24 hr the plasma value decreased to 24.6 micrograms/litre as compared with the peak value of 539 micrograms/litre. In a typical soft tissue such as the kidney the peak at 2 hr of 682 ng/g decreased to 241 ng/g, which was still more than 10-fold greater than the control level. Uniquely, in the case of bone Al increased throughout the period of the experiment. Our results indicate that Al in the citrate form is readily absorbed and that it appears to equilibrate rapidly between plasma and the intracellular compartments of most soft tissues but does not readily permeate the blood-brain barrier. In a group of rats previously given silicic acid in the drinking water and co-administered with the Al dose, the tissue Al distribution pattern at 4 hr post-administration was modified in comparison with the test animals not loaded with silicic acid. Al concentrations in plasma and soft tissues were significantly reduced except for the spleen, in which Al increased, and there was complete inhibition of the very high Al uptake/deposition in bone.

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.

Effect of fasting on lysosomes in kidney cortex of glomerulonephritic rats.

The effect of food restriction (FR) on the kidney cortex lysosomes prepared by rate and isopycnic zonal centrifugation was studied in rats with passive Heymann glomerulonephritis (PHN). FR reduced the renal mass by 41%, but the capacity for handling of labelled endocytosed proteins by the lysosomes was not different from fed PHN rats. While PHN with heavy proteinuria increased the recovery of lysosomal enzymes in the large lysosomes located in the proximal tubule, no changes were observed in FR-PHN rats in spite of significant proteinuria. The density of the small lysosomes was significantly shifted/reduced (from 1,200 and 1,235 g/ml to 1,185 and 1,225 g/ml, respectively) in both fed and FR-PHN rats, suggesting that the handling of extra loads of protein may enhance the absorptive function of small lysosomes found in the lower part of the nephron. FR reduced the mechanical fragility of lysosomes in the kidney cortex of PHN-rats. The highly increased urinary excretion of lysosomal enzymes in fed PHN rats was not observed in FR-PHN rats. As a conclusion, FR reduces both the fragility of lysosomes and the proportion of digestive enzymes in fragile lysosomes. These lysosomal enzymes may be of pathogenic importance in PHN causing cell damage when liberated from disrupted lysosomes.

Lysosomes of the renal cortex: heterogeneity and role in protein handling.

Rate sedimentation of the kidney cortical mitochondrial/lysosomal (ML) fraction yields two distinct classes of lysosomes: the large lysosomes or protein droplets and a heterogeneous broad band of smaller lysosomes. The protein droplets which are recovered as a well defined zone of high purity also sediment as a homogeneous band after equilibrium banding at a density of 1.235 g/ml in sucrose. The small lysosomes co-sediment with other subcellular organelles as a broad band, indicated by the distribution of various acid hydrolases, which exhibit subtle heterogeneity among these small lysosomes. The distribution of renin containing granules indicates that in size they represent a distinct subpopulation of small lysosomes. Further fractionation of small lysosomes by equilibrium banding separates two distinct populations at densities 1.20 (small light) and 1.235 g/ml (small dense). Comparison of lysosomal populations fractionated in these studies with the distribution of lysosomal acid hydrolases along the different segments of the nephron suggests that large and small dense lysosomes probably originate from the proximal tubule while the small light lysosomes may contain lysosomes from the distal tubule. Very small, lysosome-like organelles subfractionated from the 'microsomes' may constitute a mixture of small light lysosomes, lysosomal fragments and endocytic vesicles from a variety of cell types. Time course studies with 3H labelled Cd-thionein, following intravenous administration, suggests that uptake in the kidney cortex is very rapid and that catabolism takes place in two distinct phases: rapid breakdown starting in the endosome compartment and slower breakdown in lysosomes. From the association of labelled lysozyme (125I) and Cd-thionein (109Cd) it appears that all the different lysosomal populations identified are at some stage involved with uptake and catabolism of these two proteins.

Latency of acid hydrolases in rat kidney cortex.

1. Some lysosomal populations in the rat kidney cortex appear to be mechanically weak and are readily disrupted by gentle homogenization, while other populations remain intact even after repeated homogenization. 2. Lysosomes in the rat kidney cortex appear to be resistant to hypertonic media but are readily disrupted under hypotonic conditions. 3. Lysosomes in rat kidney cortex are readily disrupted when incubated in isotonic sucrose at 37 degrees C. 4. Measurement of total and free activity of three acid hydrolases: N-acetyl-beta-D-glucosaminidase (NAG), acid beta-galactosidase and acid beta-glycerophosphatase, indicates that the latency of these enzymes is relatively low in the homogenate (10-29%) and the ML-fraction (14-42%), but high (60-95%) in the purified large lysosomes (protein droplets). 5. The latency of purified small lysosomes is relatively lower (30-60%) than that of large lysosomes, suggesting that small lysosome populations are relatively permeable to the acid hydrolase substrates. 6. Latency variations of acid hydrolases amongst subcellular fractions appear to reflect the heterogeneity of lysosomal populations present in the kidney cortical homogenate.

Changes in lysosome populations in the rat kidney cortex induced by passive Heymann glomerulonephritis.

Acute passive Heymann glomerulonephritis in rats induced heavy proteinuria and highly increased urinary activity of N-acetyl-beta-D-glucosaminidase, acid beta-galactosidase and acid phosphatase. The cortical activity of these acid hydrolases was increased essentially in the large lysosomes as demonstrated by subfractionation of the lysosome-rich mitochondrial-lysosomal fraction, by rate zonal centrifugation. Banding density of small lysosomes shifted or reduced to slightly lower value (1.225 g/ml), which is between the banding densities of small 'light' (1.20 g/ml) and small 'dense' lysosomes (1.235 g/ml) in normal rat kidney cortex. Labelled protein reabsorbed in the proximal tubule is recovered in these populations of small lysosomes as well as in the large lysosomes or 'protein droplets'. Glomerulonephritis also induced a new population of small 'light' lysosomes (density 1.185-1.195 g/ml) enriched in cathepsin D. The previously demonstrated morphological, biochemical, and physiological heterogeneity of renal lysosomes was confirmed and emphasized in the kidney cortex of glomerulonephritic rats. The main changes in the lysosomal populations appear to reflect the increased protein reabsorption as confirmed by the proteinuria.

The lysosomal distribution of cathepsin B in the rat kidney cortex.

Subcellular distribution of cathepsin B following subfractionation of the kidney cortex mitochondrial/lysosomal fraction by rate sedimentation indicates that this enzyme is mainly associated with the large, fast sedimenting lysosomes (protein droplets). A small proportion of cathepsin B is also present in the small lysosomes which cosediment with mitochondria, peroxisomes, and brush border and other large membrane vesicles. Amongst this broad spectrum of small lysosomes the distribution of cathepsin B, together with other acid hydrolases is associated with the more rapidly sedimenting lysosomes whilst cathepsin D differs in being associated with the slowest sedimenting lysosomes. Equilibrium banding in sucrose gradients shows the large lysosomes band at a density of 1.235 g/ml and that the small lysosomes have two distinct populations at densities 1.20 and 1.235 g/ml. Cathepsin B (and also cathepsin D and acid ribonuclease) appears to be associated only with lysosomes of high density. The various other acid hydrolases assayed are found in all the lysosomal populations. Small and large lysosomes of high density are very rich in a number of proteinases and therefore most probably represent lysosomal populations involved in the catabolism of proteins taken up from the glomerular filtrate.