Utilization of methionine as a sulfhydryl source for metallothionein synthesis in rat primary hepatocyte cultures

Metallothioneins (MT) contain a high concentration of cysteine which bind heavy metals. Exposure of liver cells to metals induces the synthesis of MT and thus causes the cells to draw upon their sulfhydryl (SH) pools. The utilization of methionine as compared with that of cysteine as a source of SH for the synthesis of MT has not been shown. Therefore, studies were designed to determine whether methionine, in addition to cysteine, serves as an SH donor for Zn-induced synthesis of MT in rat hepatocyte cultures. Hepatocytes were able to synthesize only low levels of MT when the concentration of both amino acids was extremely low; however, when either of the amino acids was present at a high concentration, production of MT was independent of the other amino acid concentration. Subsequently, induction of MT was compared in four media: complete (0.5 mM methionine, 0.5 mM cysteine), Met (0.5 mM methionine), Cys (0.5 mM cysteine), and SH free (-SH). Somewhat higher concentrations of MT were produced by the hepatocytes in the Met than in the Cys media and no differences were observed between the Met and the complete media. By contrast, GSH synthesis was much more dependent on methionine than on cysteine for its synthesis. Incorporation studies with 35S-labeled cysteine and methionine indicated that lower concentrations of MT found in hepatocytes in the Cys media may be due to less accumulation of cysteine by the hepatocytes. Cellular accumulation of cysteine was initially rapid and then reached a plateau, whereas the rate for methionine accumulation was more constant and eventually obtained higher cellular levels. To provide additional evidence for the role of methionine in MT production, a known inhibitor of the cystathionine pathway, DL-propargylglycine (PPG), was added to each of the four media. Reductions in MT levels were not observed in the cells cultured in the complete and Cys media; however, a 95% reduction was observed in the cells cultured in the Met media. In summary, the present results suggest that both cysteine and methionine can serve as a SH source for MT synthesis, and that the availability of SH in most culture mediums would not limit the synthesis of MT. Whereas methionine is a much better SH source than cysteine for GSH synthesis in hepatocyte cultures, it is only slightly better for MT synthesis.     

Induction of metallothionein by steroids in rat primary hepatocyte cultures

The purpose of this study was to characterize the induction of metallothionein (MT) by steroids in rat primary hepatocyte cultures. Comparison of the characteristics of MT induction by a steroid (dexamethasone) to that by metals (Zn and Cd), examination of the involvement of the glucocorticoid receptor in the steroid induction of MT, and determination of the potency and effectiveness of a number of steroids were studied. In general, the patterns of MT induction by metals and steroids were quite different. For metals, the maximal MT induction (12- to 39-fold) was limited by toxicity whereas for steroids, a plateau in MT induction (fivefold) occurred at noncytotoxic concentrations. Steroids elicited an increase in MT at concentrations that were one-hundredth to one-thousandth less than that of metals. A combination of metal and steroid increased the induction of MT to a level higher than achieved by metal or steroid alone. The effectiveness of steroids at inducing MT was related to their ability to induce a specific glucocorticoid effect, induction of tyrosine aminotransferase. For specific classes of steroids, synthetic glucocorticoids were more potent than the metals in inducing MT, but endogenous glucocorticoids, mineralocorticoids, androgens, and estrogens were less potent than the metals. The concentration of corticosterone, the major endogenous glucocorticoid of rats, required to induce MT in hepatocytes was 100 times higher than concentrations achievable in the plasma of rats. In conclusion, in rat hepatocytes dexamethasone was a more potent but less effective inducer of MT than Zn or Cd; synthetic glucocorticoids were more potent but endogenous adrenalcorticoids (i.e., glucocorticoids, mineralocorticoids, androgens, and estrogens) were both less potent and less effective inducers of MT than were metals, suggesting that glucocorticoids may not be the mediator for stress-induced MT induction; and induction of MT by steroids correlated well with the induction of tyrosine aminotransferase, supporting the involvement of a hormone-receptor complex in the induction of MT by steroids.     

Alteration in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite

Patterns of protein synthesis in primary cultures of rat kidney proximal epithelial tubule cells were examined following exposure to gallium (Ga) chloride, indium (In) chloride, and sodium arsenite. After incubation with these chemicals for 20 hr, newly synthesized proteins were labeled with [35S]methionine. 35S-labeled proteins in the cells were separated by SDS/polyacrylamide gel and two-dimensional gel electrophoresis and detected by fluorography. A protein with molecular weight (Mr) of 30,000 was markedly induced by exposure to 300 microM Ga or 10 microM arsenite, and synthesis of proteins with Mr of 85,000, 71,000, 65,000, 51,000, 38,000, and 28,000 was also increased by Ga or arsenite. Arsenite exposure increased synthesis of eight different proteins, which were not induced by Ga. No significant changes in protein synthesis were observed with 300 microM In exposure. Release of lactate dehydrogenase from the cells was not significantly increased by exposure to concentrations of 300 microM Ga and 3 microM arsenite or less. In the absence of overt cell injury, the induction of these proteins may be useful as an early indicator for assessing exposure to Ga.     

Formation of lead-induced inclusion bodies in primary rat kidney epithelial cell cultures: Effect of actinomycin D and cycloheximide

Cells were isolated from adult rat kidney by mild trypsin digestion and maintained in Eagles MEM-d-valine medium for 3 days to form a monolayer primary culture of epithelial cells. When these cells in monolayer were exposed to a medium containing lead nitrate (4.5 × 10^?4m) complexed with glutamic acid (5 × 10^?4m), inclusion bodies with characteristic ultrastructure were detected morphologically in the cytoplasm within 4 hr. At 24 hr after lead exposure the inclusion bodies were localized mainly in the nucleus. Addition of actinomycin D (0.05 and 5 μg/ml) or cycloheximide (5 and 10 μg/ml) directly to cultures prior to lead exposure resulted in the absence of inclusion bodies at high concentrations and in fewer inclusions at low concentrations. These inhibitors did not affect the uptake of lead by the cell; but they markedly inhibited incorporation of [³H]leucine into cellular proteins. The initial appearance of inclusion bodies in cytoplasm after lead exposure, its subsequent relocalization in the nucleus and the effect of inhibitors in these processes were also confirmed by quantitation of inclusion bodies ultrastructurally in randomly selected cells in each experiment. These results indicate that active protein synthesis is required for the formation of lead-induced inclusion bodies.     

Erythromycin toxicity in primary cultures of rat hepatocytes

1. Cultured rat hepatocytes were used to study the toxicity of erythromycin base (EB), erythromycin estolate (EE) and a new fluorinated derivative, (8S)-8-fluoroerythromycin A (EF).

2. EF was not cytotoxic after 18 h incubation at concentrations up to 8 × 10^?4 M and EE was much more toxic than EB at all concentrations studied.

3. EE toxicity was greater in a serum-free medium and was not increased by induction of cytochrome P-450 with phenobarbitone.

4. In hepatocytes co-cultured with rat-liver epithelial cells EE, but not EF, raised the cytochrome P-450 content and formed stable cytochrome P-450 complexes with about 40% of the haemoprotein.

5. The lack of correlation between cytochrome P-450 content and cytotoxicity suggests that some of the parent erythromycin drugs and not their metabolites are the toxic entities.     

In vitro evidence of cellular adaptation to ozone toxicity in the rat trachea

Adaptation to prolonged ozone (O3) exposure occurs in the tracheal epithelium of rats and is marked by the presence of ciliated cells with uniform short cilia but is not accompanied by shifts in cell populations, altered characteristics of epithelial secretory cell products, increased cell turnover, or elevated tracheal superoxide dismutase activity. The purpose of this study was to test the hypothesis that adaptation is a result of alterations intrinsic to epithelial cells or to the cells and their matrix, and not due to systemic or neural influences. Rats were preexposed to either filtered air (FA) or 0.96 ppm O3 for 8 hr/night for 60 days, and then their tracheae were removed and exposed to 3 ppm O3 in an explant culture system where behavioral, nasal, exudative, and secretory product influences can be eliminated. After exposure to 3 ppm O3 in vitro, quantitative electron microscopic evaluation demonstrated that the epithelia from the FA preexposure group had significantly more necrotic cells and sloughed cells and fewer ciliated cells than the epithelia from the O3 preexposure group. Thus previous exposure to ozone in vivo induces a change in tracheal epithelium which confers resistance to ozone-induced injury in the explant exposure system.     

Ethanol toxicity in primary cultures of rat myocardial cells

The potential cardiotoxicity of ethanol (EtOH) was evaluated in primary cultures of rat myocardial cells. EtOH cardiotoxicity was assessed in the cells on the basis of cell morphology, lactate dehydrogenase (LDH) leakage, succinate dehydrogenase (SDH) activity, and beating rates. Cells were treated with EtOH at concentrations of 600, 800, and 1000 mg% for duration of 1, 4, and 24 h and then evaluated for cardiotoxicity. Vacuole formation occurred 1 h after exposure to EtOH at 800 and 1000 mg%; by 4 h, cytosolic granular material appeared in these cells. Exposure for 24 h to all concentrations of EtOH resulted in vacuole, granule, and pseudopod formation and loss of cross-striations. Significant LDH leakage occurred at 1 h and 4 h with 800 and 1000 mg% EtOH. LDH release was significantly increased after 24 h with all concentrations. SDH activity was significantly depressed after 24 h with all concentrations of EtOH. Beating rates were altered as early as 1 h after exposure to 800 and 1000 mg% EtOH. After 24 h, those cells exposed to the highest concentrations of EtOH were not beating at all. These data suggest that primary myocardial cell cultures may be used to assess the in vitro cardiotoxicity of EtOH to the myocardial cell.     

Unscheduled DNA synthesis in tracheal epithelial cell cultures

An in vitro method has been established for the isolation and culture of tracheal epithelial cells for the evaluation of chemically induced genotoxicity using an unscheduled DNA synthesis assay. Cell cultures were derived from the Wistar albino rat and the golden Syrian hamster. Epithelial cells were isolated with protease type XIV for 16 hr and allowed to attach for 24 hr on collagen-coated coverslips in multi-well plates. Cells were exposed to the experimental carcinogens benzo[a]pyrene (metabolism-dependent) and methylmethanesulphonate (direct acting) for 24 hr. Benzo[a]pyrene and methylmethanesulphonate induced DNA repair in cultures isolated from hamsters, whereas only methylmethanesulphonate induced unscheduled DNA synthesis in rat tracheal epithelial cells, thus indicating the lack of metabolic activation in the rat cultures. These results could indicate the suitability of this culture system for the evaluation of airborne carcinogens.     

Hepatic toxicity of paraquat in primary cultures of rat hepatocytes

The aim of this work was to evaluate the hepatic effects of toxic and subtoxic doses of paraquat, using a primary culture of adult rat hepatocytes as an experimental model. Concentrations in culture ranged from 10⁻³ to 10⁻⁹ -paraquat. When added to cultures at the time of plating 10⁻³ -paraquat impaired hepatocyte spreading on the substratum and caused cell death which was preceded by a leakage of cytosolic enzymes (lactate dehydrogenase, glutamic-oxaloacetic transaminase). When added to 24-hr cultures, effects on the metabolism of the hepatocytes were observed at paraquat concentrations of 10⁻³ and 10⁻⁴ but not at lower concentrations. In comparison with controls, when paraquat was present at 10⁻³ , gluconeogenesis from fructose and lactate was inhibited by 40 and 58%, respectively, ureogenesis was inhibited by 16% and the albumin synthesis rate was reduced by 62%. The intracellular content of the reduced form of glutathione decreased by 33% and was parallelled by an increase in the oxidized form of glutathione. However, neither cytotoxic nor metabolic effects were observed in cells exposed to 10⁻⁵ , the concentration found in blood after acute intoxication with paraquat.     

The protective effect of metallothionein on the toxicity of various metals in rat primary hepatocyte culture

Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein, has been implicated in the detoxification of Cd. However, whether MT protects against the cellular toxicity of other metals has not been examined thoroughly. This study was therefore designed to determine the effects of Zn-induced MT on the toxicity of seven metals in rat primary hepatocyte cultures. Hepatocytes were grown in monolayer culture for 22 hr and subsequently treated with ZnCl2 (100 microM) for 24 hr which produced a 15-fold increase in MT concentration. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Ag, Co, Cu, Hg, Ni, Pb, or Zn for 24 hr. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all seven metals was significantly less in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 hr) of 110Ag or 203Hg, but markedly altered their subcellular distribution, with metals accumulating more in the cytosol and less in the nuclear, mitochondrial, and microsomal fractions. In the cytosol of control cells, the metals were bound mainly to high-molecular-weight proteins whereas in the Zn-pretreated cells, the metals were mainly associated with MT. In summary, Zn-induced MT in rat primary hepatocyte cultures protects against Ag-, Co-, Cu-, Hg-, Ni-, Pb-, and Zn-induced cytotoxicity. This protection appears to be due to the binding of metals to MT with a concomitant reduction of metal content in critical organelles and proteins.     

Deferoxamine toxicity in hepatoma and primary rat cortical brain cultures.

Deferoxamine is commonly used for treatment of iron intoxication. Because the usual dose is unable to chelate sufficient iron before severe injury occurs, "high-dose" deferoxamine treatment has been proposed. However, several authors have reported severe toxicity after deferoxamine therapy. Although the hemodynamic effects are well described, the cellular toxicity of deferoxamine is unknown. Accordingly, we investigated the cellular toxicity of deferoxamine using in vitro techniques in two cell lines. Brain cells were harvested from fetal rats and cultured for 14-21 days before deferoxamine exposure. Using similar techniques, rat hepatoma cells were grown until confluent. Deferoxamine was added to the cultures to achieve final concentrations of 200-800 microg/ml, corresponding to in vivo infusion rates of 15-60 mg/kg/h. Deferoxamine was removed after 3 or 6 days by changing the medium. Subtoxic FeCl3 (500 mg/dl) was concurrently added to identical cultures to determine if deferoxamine potentiated iron toxicity. Cell viability was measured by a colorimetric assay. The addition of deferoxamine (0.2, 0.4, 0.8 mg/ml) significantly decreased cell viability in both cell groups. The effect of deferoxamine on primary cortical brain cultures was similar for the three concentrations used, and was similar when examined either 72 h or 6 days later. In contrast, hepatoma cell cultures evidenced a dose- dependent cell loss that increased with the length of exposure. The addition ofsubtoxic amounts of FeCl3 (500 microg/dl) in the presence of deferoxamine was protective in all cultures, and abolished deferoxamine-induced cell loss. Interestingly, the addition of serum albumin significantly reduced the amount of iron present in cells, suggesting its potential use to treat iron toxicity. These results suggest that deferoxamine, in the absence of iron, is toxic to cortical brain and hepatoma cells in vitro.     

The chronic toxicity of equine cadmium metallothionein in the rat

The extensive renal tubular necrosis that results in male rats after the intravenous injection of a single, low dose of equine kidney cadmium (Cd), zinc(Zn)-metallothionein (MT) (0.2 mg MT-bound-Cd/kg body wt.) is followed within 72 h by active regeneration. With repeated administration of the same dose at 3-or 4-day intervals, the lesion resolves although, at least initially, the kidney content of Cd increases progressively. At any time during treatment, about 40% of the accumulated Cd is bound as the endogenous (Cd, Cu)MT. The rate of increase in the renal Cd content is dependent on the ratio of CdZn in the injected metalloprotein, and is appreciably less when the constant dose of protein-bound Cd is given as a (2.4 Cd1 Zn)MT, than as a (3.0 Cd1 Zn)MT. On repeated administration of the latter preparation, however, the concentration of Cd in the kidney does not attain a critical concentration, above which persistant tubular damage occurs, but reaches a maximum of about 150–160 g Cd/g wet wt. (after 16 doses) and then declines. After 19 doses of the (2.4 Cd1 Zn)MT under the same conditions, the renal Cd concentration is submaximal and is less (92 g Cd/g wet wt.) than that after either 16 or 27 doses of the (3.0 Cd1 Zn)MT. In animals that are dosed with either of the heterologous MT preparations, the first dose, although not innocuous, seems to protect the kidneys against further damage by subsequent doses. Repeated doses, however, lead to vascular changes, e.g. lymphoid infiltration, periarteriole oedema and dilation of the arcuate veins, and to dilation of the glomerular spaces.     

The modulation by metallothionein of cadmium-induced cytotoxicity in primary hepatocyte cultures

The cytotoxicity of CdCl2 and 2 isoforms of hepatic cadmium-metallothionein (CdMT I and II), was investigated using primary cultures of rat hepatocytes. The cell cultures were exposed to cadmium as CdCl2 or as either isoform of CdMT for a 20-h period at concentrations ranging from 50 to 500 ng Cd X ml-1. Cytotoxicity was assessed by determining the amount of lactic dehydrogenase released from the cells into the incubation medium and the incorporation of [3H] arginine into cell protein. The uptake of Cd by the cells was also measured. Cadmium chloride and both isoforms of CdMT were found to be toxic to hepatocytes although partial protection was afforded by the binding of cadmium to metallothionein (MT). At the higher exposure concentrations and in accordance with the toxicity data, the cells exposed to CdCl2 were found to accumulate more cadmium than those exposed to CdMT. The distribution of cadmium in the culture medium was examined using Sephadex G-75 chromatography. The speciation of cadmium is discussed in relation to its cytotoxicity.     

Induction of metallothionein in rat primary hepatocyte cultures: evidence for direct and indirect induction

The ability of a number of metals and organic chemicals to induce metallothionein (MT) synthesis in primary cultures of rat hepatocytes was tested to determine whether MT induction in vivo results from a direct effect of the agent on the liver or as a result of an indirect, physiologic response to the agent. Hepatocytes were exposed to metals [zinc (Zn), cadmium (Cd), mercury (Hg), manganese (Mn), lead (Pb), cobalt (Co), nickel (Ni), and vanadium (V)] or organic compounds [ethanol, urethane, L-2-oxothiozolidine 4-carboxylate (L-OTCA), or dexamethasone] and were assayed for metallothionein by the Cd/hemoglobin radioassay. Cell viability was monitored by protein synthesis activity and cellular K+ concentration. Increases in MT concentrations were noted for Zn (22-fold), Hg (6.4-fold), Cd (4.8-fold), Co (2.4-fold), Ni (2.2-fold), and dexamethasone (4.5-fold). However, even at maximum tolerated concentrations, Mn, Pb, V, ethanol, urethane, and L-OTCA did not increase MT. The results indicate that Zn, Cd, Hg, Co, Ni and dexamethasone induce MT in vitro and thus are direct inducers of MT synthesis in hepatic tissue. In contrast, Mn, Pb, ethanol, urethane and L-OTCA, which did not increase the MT content of hepatocytes, apparently do so in vivo by an indirect mechanism.     

Interactions of liposome-incorporated amphotericin B with kidney epithelial cell cultures

The polyene antibiotic amphotericin B (AmB) is profoundly cytotoxic to both fungal cells and mammalian cells. We have previously shown that the incorporation of AmB into phospholipid vesicles can markedly reduce the toxicity of the drug for mammalian cells (erythrocytes) without changing its antifungal potency [Mol. Pharmacol. 31:1-11 (1987)]. Because the primary site of in vivo toxicity of AmB is the kidney, here we investigate the effects of free AmB and liposomal AmB (L-AmB) on LLCPK1 cells, a porcine kidney cell line with many characteristics typical of proximal tubule cells. Acute exposure (2 hr) to free AmB inhibits protein synthesis and causes cell detachment and protein loss in LLCPK1 cells, with an IC50 of about 30 micrograms/ml. By contrast, certain formulations of L-AmB have little effect on protein synthesis/protein loss at concentrations of up to 2 mg/ml. The action of liposomes in protecting against acute AmB toxicity extends to effects on sugar transport and on cellular morphology in differentiated cultured kidney cells. Thus, the IC50 for inhibition of sodium-stimulated glucose transport by free AmB is 1.5 micrograms/ml whereas concentrations of L-AmB up to 1 mg/ml do not inhibit this process. However, chronic exposure of cells to L-AmB results in profound toxic effects as manifested by changes in cellular transport functions and cell morphology. Our results suggest that extended periods of proximity between cells and liposomes permit the transfer to toxic amounts of AmB. This may be of importance to the therapeutic use of AmB, for which protracted courses of drug administration are common.     

Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidney

Metallothionein (Mt) is a low molecular weight metalloprotein and its synthesis is induced by various divalent metals (Cd, Zn, Hg and Cu). The Intracellular distribution of Mt in hepatic and renal cells from control and CdCl₂ injected rats was investigated by immunohistochemical methods. Antiserum to purified rat-liver Mt was prepared in rabbits after partial polymerization of the protein. The unlabelled peroxidase-antiperoxidase method using the specific rabbit anti-rat liver Mt provided a sensitive technique to localize Mt in tissue sections. In control rats, Mt or thionein (metal-free protein) was mainly localized in the cytoplasm of hepatocytes, renal collecting duct epithelium and distal convoluted tubular epithelium. In rats, injected intraperitoneally (i.p.) with CdCl₂ (0.6 mg/kg) for 2 weeks, Mt was present mainly in the nuclei which were largely negative in control rats. Repeated injection with CdCl₂ for 4–8 weeks resulted in the appearance of Mt in both the nucleus and cytoplasm. Intraluminal staining was also noted in proximal convoluted tubules along with marked vacuolation in the cytoplasm at 6 and 8 weeks. High intensity staining was observed in proximal convoluted tubules and collecting duct epithelium of the kidneys in CdCl₂ injected rats. The bile duct epithelium in liver samples, renal glomerular mesangial cells, glomerular visceral epithelial cells and vascular smooth muscle cells showed weak to moderately intense staining. No staining was seen in vascular endothelial cells, fibroblasts or leukocytes. The staining for Mt in this technique was abolished when the antibody was absorbed with rat-liver Mt in vitro or by substitution of the antiserum with normal rabbit serum, demonstrating the specificity of the staining reaction for Mt. The results showed the presence of small amounts of Mt predominantly in the cytoplasm of control rat hepatocytes and renal tubular epithelium and its appearance in both the nucleus and cytoplasm after its synthesis induced by CdCl₂ injections.     

Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment

Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10⁵ seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.     

Effects of nitrendipine on cocaine-induced toxicity evaluated in primary myocardial cell cultures

Currently, there is no uniform treatment for abnormal cardiac events precipitated by cocaine use. However, clinical strategies include use of calcium channel antagonists for cardiovascular emergencies. In experimental situations using rats, simultaneous administration of nitrendipine (NIT) with cocaine to the whole animal (1.46 x 10(-3) mg/kg/min of NIT; 2 mg/kg/min of cocaine) and isolated retrograde perfused hearts (Langendorff; 1 x 10(-7) M NIT; 1 x 10(-7) to 1 x 10(-4) M cocaine) normalized cocaine-induced abnormalities in heart rhythm and provided protection from acute cocaine-induced morphological lesions. Using similar concentrations of NIT and cocaine, the purpose of this study was to evaluate the direct cardiac cellular effects of NIT on cocaine-induced alterations in beating activity, morphology, and lactate dehydrogenase (LDH) release in a controlled in vitro system of primary myocardial cell cultures. Cultures were established from hearts of 3-5-day-old Sprague-Dawley rats. After the cells had been maintained in culture for 4 days, evaluation of drug effects were made with exposure to 1 x 10(-3) and 1 x 10(-5) M cocaine alone and combinations of these two concentrations of cocaine with simultaneous exposure to 1 x 10(-7) M NIT for 1 to 24 h. Those cells exposed to 1 x 10(-5) M cocaine alone maintained some beating activity after 1, 4, and 24 h. Beating activity was significantly depressed after treatment with 1 x 10(-3) M cocaine alone and with both combinations of cocaine and NIT. Morphological integrity was maintained in all treatment groups for 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interactions of cis-diamminedichloroplatinum with metallothionein and glutathione in rat liver and kidney

The involvement of metallothionein (MT) in the nephrotoxicity of cis-diamminedichloroplatinum (c-DDP) was investigated in rats using enzyme excretion and histology as indicators of renal damage. In addition, the effects of renal glutathione (GSH) depletion on the nephrotoxicity of c-DDP was assessed by organic anion transport in renal cortical slices. A dose of 6.0 mg c-DDP/kg body wt, i.p. was administered to rats either as a single injection of 6.0 mg/kg or as six daily injections of 1.0 mg/kg. Concentrations of platinum (Pt) after c-DDP injection in both dosing regimens were approximately 12 micrograms/g in kidney and 2 micrograms/g in liver. However, there were no increases in either hepatic or renal concentrations of MT after both series of c-DDP injections. Fractionation of kidney cytosols from c-DDP injected rats on Sephadex G-75 columns revealed that 60-70% of cytosolic Pt was associated with proteins of high molecular weight and 15-20% of the Pt associated with the low molecular weight ligands. No discernable Pt peak was detected in the elution volume of MT. Pretreatment of rats with ZnSO4 increased both hepatic and renal concentrations of MT, but there was no Pt associated with the MT fraction after a subsequent injection of c-DDP. Small increases in the urinary excretion of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase and two brush border enzymes, alkaline phosphatase and gamma-glutamyltranspeptidase were observed 2 and 3 days after a single injection of c-DDP (6.0 mg/kg body wt, i.p.). Urinary creatinine excretion decreased by 50% 1 day after c-DDP injection and continued to decrease for the next 2 days. On the third day after c-DDP treatment, a small but significant decrease in body weight was also observed in the c-DDP injected animals. Pretreatment with Zn did not alter the c-DDP-induced enzymuria or renal tubular damage but slightly attenuated both the decrease in creatinine excretion and the loss in body weight. Uptake of the organic anion, p-aminohippuric acid (PAH) was reduced at 12 and 24 h after c-DDP injection. Reduction of tissue GSH concentrations by pretreatment with buthionine sulfoxime (BSO), resulted in only a slight increase in the c-DDP-induced inhibition of PAH uptake at 24 h after c-DDP injection. These results suggest that, in rats, neither MT nor GSH appear to play major roles in the binding or nephrotoxicity of c-DDP.