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The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and alpha-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of alpha-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.

Isolated hepatocytes have been employed to investigate the uptake of the heavy metal cadmium ( 109 Cd). A rapid initial phase of uptake was followed by a second slower phase, both of which exhibited a linear relationship between velocity of uptake and substrate concentration. The presence of potassium cyanide (1 m m ) and carbonyl cyanide- m -chlorophenyl hydrazone (2 μ m ) did not cause any change in the uptake of Cd (200 μ m ). This suggests that the uptake of Cd into isolated hepatocytes is not an active process. Incubation of 10 μ m Cd in the presence of zinc (50 and 130 μ m ) produced a decrease in the velocity of uptake of only the first phase. Pretreatment of rats with zinc, which elevates hepatic metallothionein, resulted in isolated hepatocytes which showed a greater uptake rate for the second phase when compared to hepatocytes isolated from control rats. The results therefore demonstrate that uptake of Cd by isolated rat hepatocytes is a biphasic response in which at least part of the first phase appears to be carrier mediated with no indication of involvement of a carrier in the second phase of uptake. This second phase may be related to binding of Cd to intracellular components, since elevation of hepatic metallothionein was found to increase the uptake velocity of only this phase.

The biochemical mechanism involved in cadmium-induced cellular injury remains to be elucidated. Various theories have been proposed to account for this action, one of which is an interaction with reduced sulfhydryl (SH) groups of membranes. The ability of a known SH group reducing agent, dithiothreitol (DTT), to interact with cadmium-induced damage to isolated hepatocytes was investigated. Cadmium chloride produced a cytotoxic response similar to that previously demonstrated. When added just prior to cadmium chloride, DTT was found to protect against the metal-induced injury. If the cells were first exposed to cadmium for 15 min before DTT was added, it was found that the cells could be rescued from the ensuing expression of toxicity due to cadmium. This result was regardless of the fact that by 15 min the cells had accumulated a quantity of cadmium that was associated with cell injury. Furthermore, DTT did not exert its action by decreasing the amount of cadmium accumulation by the cells. Under some experimental conditions, an increased cadmium uptake in the presence of DTT was observed. While it is not clear as to whether the chelating or thiol reductive properties of DTT are of prime importance in the demonstrated protection, the data show that toxicity is not due to a simple, single irreversible interaction between cadmium and membrane SH groups.

Recent evidence has renewed interest in the hypothesis that Ca plays a central role in cell death. It was previously found that Cd and CuCl2 cause loss of viability of isolated hepatocytes. It was therefore of interest to determine whether Ca was intimately involved with the toxic effect of these metals. Some of the chemicals that were previously shown to be toxic through a mechanism involving Ca (amphotericin B, lysolecithin, and Ca ionophore A23187) were also included in the study. Hepatocytes were incubated with one of these chemicals and samples taken at various time points up to 120 min for estimation of cell viability (intracellular K+ and leakage of aspartate aminotransferase) and lipid peroxidation. The toxic effects due to Cd or Cu were not ameliorated on omission of Ca from the incubation medium. Furthermore, of the other three chemicals investigated, only the toxic properties of the Ca ionophore were effectively blocked by incubation in a Ca-free medium. The results of this study do not support the hypothesis that Ca plays a ubiquitous role in the death of liver cells.

Chlorpromazine has been shown to have no effect on the uptake of the endogenous bile salt substrate, taurocholate, by isolated rat hepatocytes. It has been shown, however, to inhibit directly release of taurocholate from pre-loaded cells over extended incubation. However, there was no inhibition of the efflux process per se as shown by similar initial rates of taurocholate efflux in the presence or absence of chlorpromazine. Pretreatment of rats with chlorpromazine (100 mumoles/kg) resulted in no change in the ability to transport (that is, accumulate or secrete) taurocholate by hepatocytes isolated 2, 24, 36, 48, or 60 hr later. The data indicate that, if a direct effect on bile acid transport is important in chlorpromazine induced biliary dysfunction, then it involves release rather than uptake at the cell membrane. However, as efflux itself is not inhibited chlorpromazine may interfere with release of taurocholate from intracellular sites.

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.

Several phenothiazines have been shown to inhibit NK cell-mediated cytotoxicity (NKC) and antibody-dependent cell-mediated cytotoxicity (ADCC) effected by human peripheral blood lymphocytes. Of those tested, chlorpromazine was found to inhibit at the lowest concentration (5 microM) with a 90% inhibition at 10 microM for NKC and 70% inhibition for ADCC. Pre-incubation of either effector or target cells with chlorpromazine did not provide any evidence of inhibition in subsequent cytotoxicity assays. The data for chlorpromazine in particular may be of clinical significance. In vivo inhibition would presumably require the presence of the drug since preincubation did not produce inhibition of NK or K cell function.

Ethinyl estradiol is a well documented, predictable cholestatic agent. The direct effects of this drug on hepatocellular uptake and efflux have been investigated. Uptake of taurocholate and ouabain, which are both actively transported, was inhibited, while uptake of cadmium, which occurs by a combination of facilitated and simple diffusion, was unaffected. The Vmax for taurocholate uptake was not altered by the presence of ethinyl estradiol, whereas the Km was increased, suggesting a reduced affinity of receptor for taurocholate. Efflux of taurocholate from pre-loaded cells remained unchanged in the presence of ethinyl estradiol. The data are not entirely consistent with an interference of Na+,K+-ATP ase by ethinyl estradiol being an important site of action. The observations may help to explain the mechanism of biliary dysfunction induced by ethinyl estradiol.

Interactions between Cd and other metal ions are important from both nutritional and toxicological aspects. As Cd is toxic to isolated hepatocytes, these cells can be used to investigate the effects of other metals on Cd-induced cellular injury. Isolated hepatocytes were incubated at 37 degrees C with vehicle (saline); Cd (200 or 400 microM); or Cd plus Cr, Mn, Zn, Ni, Pb, Se, or Fe (200-1000 microM). Evidence of cellular injury was assessed by loss of intracellular K+ and aspartate aminotransferase from the hepatocytes. Effects on lipid peroxidation, as measured by concentration of the thiobarbituric acid reactants, were assessed. Uptake of 109Cd and interaction of the other metal ions with this accumulation were also quantitated. Cell injury due to Cd was consistently reduced by Cr, Mn, Zn, Pb, and Fe. Lipid peroxidation due to Cd was inhibited by Cr, Mn, and Zn. All the metals except Ni produced an increase in the amount of Cd accumulated by hepatocytes. There was no consistent relation between reduction of cellular toxicity and either inhibition of lipid peroxidation or uptake of Cd. These experiments show that (1) protective properties of some metals seen in vivo can be demonstrated at the cellular level and (2) protective effects of metals in general on Cd-induced cellular toxicity are not due to a decrease in either Cd uptake or lipid peroxidation.

It has been proposed that the peroxidation of lipids may account for the toxicity of the heavy metal, mercury. Recent evidence has discounted a similar theory for another heavy metal, cadmium, when examined using isolated rat hepatocytes. Such a system provides the opportunity to examine several parameters and treatments simultaneously on aliquots from the same parent population of hepatocytes. Thus, isolated hepatocytes were incubated with mercuric chloride at concentrations ranging from 10 to 200 μ m for 20, 40, or 60 min of incubation. Lipid peroxidation was found to increase with the concentration of HgCl 2 and incubation time, as assessed by increases in thiobarbituric acid-reacting substances and in ethane in the gas phase of the flasks. Cell viability was reduced by Hg 2+ as determined by the release of cytoplasmic lactate dehydrogenase from hepatocytes. Reduced glutathione also declined in the presence of the higher concentrations of Hg 2+ , suggesting this decrease to be a manifestation of the toxic response. The increases in thiobarbituric acid reactant concentrations were closely related to loss of cytoplasmic lactate dehydrogenase, suggesting a possible causative role for lipid peroxidation in the toxic response. However, addition of the antioxidant N,N′ -diphenyl- p -phenylenediamine or (+)-cyanidanol-3 to the cell suspensions inhibited lipid peroxidation, but only (+)-cyanidanol-3 could partially reduce the Hg 2+ -induced cytotoxicity. This finding indicates that the lipid peroxidation associated with loss of viability of isolated rat hepatocytes is not directly responsible for this cell injury.

The utility of isolated rat hepatocytes as a model system for screening potential chelators in treatment of Cd intoxication was studied. The ability of the chelators diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 2,3-dimercaptosuccinic acid (DMSA), diethyldithiocarbamic acid (DDC), d,1-penicillamine (PEN), nitrilotriacetic acid (NTA), and 2,3-dimercaptopropanol (BAL) to decrease the cellular concentration of Cd was correlated with the previously reported effectiveness of these agents in the treatment of Cd intoxication in vivo. The results of cellular studies with either control or metallothionein-induced hepatocytes were compared to the in vivo effect of the chelators administered before or after the induction of metallothionein, respectively. The effects of DTPA, EDTA, DDC, and BAL in the hepatocyte model screening system correlated well with their reported in vivo effects. The results with NTA, PEN, and DMSA were not correlated as well but were explained by the relative differences between in vivo doses versus in vitro concentrations of the respective chelators. Therefore, the proposed model for screening potential chelators for use in cadmium intoxication appears to be a system which may prove to be an economical and rapid method to facilitate the search for efficacious chelators.

Hepatocytes isolated from Sprague-Dawley rats were exposed to carbon tetrachloride together with various concentrations of trichloroethylene over a 40-fold range. A potentiation of carbon tetrachloride-induced lipid peroxidation by trichloroethylene and an enhanced toxicity on combined exposure were clearly demonstrated. Additionally, rats were treated 2.5 hr before isolation of hepatocytes, which were then exposed to carbon tetrachloride. Lipid peroxidation and potassium ion leakage were increased in these cells. Some incubations included the antioxidant N,N′ -diphenyl- p -phenylenediamine (DPPD) while others contained dithiothreitol (DTT), a thiol reducing compound. DPPD inhibited lipid peroxidation while DTT did not. Neither, however, was able to inhibit the toxicity. Assays to estimate total and nonprotein bound sulfhydryl groups were also performed. There was no indication of a causative role for cellular sulfhydryl groups in the enhanced toxicity. Therefore, our data show that lipid peroxidation is not responsible for the trichloroethylene-induced enhancement of toxicity in hepatocytes due to carbon tetrachloride. Furthermore, there is no evidence to indicate a role for sulfhydryl groups in this response.

The effects of low-level cadmium (Cd) administration to rats on animal health, liver and kidney thiols, metallothionein, and glutathione reductase (GSSG reductase) and their modulation by cysteine (as a possible protector) and diethyl maleate (as a possible potentiator) have been investigated. Male Sprague-Dawley rats were treated with sodium or Cd acetate (25 micrograms Cd/kg) orally five times a week for 6 weeks. A second group of animals received cysteine (500 mg/kg; po) before each gavage while a third group received diethyl maleate (DEM) (0.85 mg/kg; ip) in addition to sodium or Cd acetate. When rats were treated with cadmium alone neither weight gain nor serum parameters indicative of hepato- or nephro-toxicity were affected. However, acid-soluble thiols, primarily glutathione, were decreased by about 25% in liver only. A tendency to a decrease in hepatic protein thiols was also noted. No changes were observed for hepatic or renal metallothionein in response to this low level of cadmium administration alone or in combination with the other treatments. Animals receiving cysteine, either alone or with cadmium, showed decreased body weight gain, but no change in serum parameters. Acid-soluble thiols in liver were lower in cysteine-treated rats (24%) and cysteine + Cd (33%) while kidney thiols were unaffected. Administration of DEM alone or with Cd did not cause any alteration in body weight gain. When given DEM + Cd, however, an increase in serum bilirubin was observed, which suggests interference with hepatobiliary function. Acid-soluble thiols were decreased by DEM alone (45%) and DEM + Cd (51%) in liver while renal thiols showed no change. Our data indicate that low-level Cd gavage decreases hepatic cellular thiols but not those of kidney. Cysteine gavage does not protect from the Cd-related effect. Indeed, cysteine itself was found to reduce acid-soluble thiols under the experimental conditions. This was observed only in liver, as was the decrease in thiols due to DEM treatment. DEM administration together with Cd resulted in signs of liver toxicity. There is no indication that inhibition of GSSG reductase by Cd might be involved in the thiol-decreasing effect of short-term repeated low-level gavage of Cd to rats.

Ofurace, oxadixyl, and alachlor were studied for their effect on hepatic xenobiotic biotransformation in male rats by dosing i.p. with 1 or 100 mg/kg of chemical for 7 days. Ofurace decreased ethoxyresorufin-O-deethylase and aniline p-hydroxylase activities but induced ethoxycoumarin-O-deethylase activity. Glutathione S-transferase and aminopyrine N-demethylase activities responded in a non dose-dependent manner, while cytochrome P-450 content and aldrin epoxidase activities were unchanged. Oxadixyl induced P-450 content, and ethoxycoumarin-O-deethylase and aminopyrine N-demethylase activities. It left ethoxyresorufin-O-deethylase, aldrin epoxidase and epoxide hydrolase activities unchanged and decreased aniline p-hydroxylase activities. Alachlor induced all activities excepting aldrin epoxidase (no change) and aniline p-hydroxylase (decreased). The data indicate that each of these acylanilide pesticides interacts with the monooxygenase system but with differing patterns.

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.

Fast atom bombardment mass spectrometric methodologies for carbohydrate structure determination are intimately linked to classical procedures such as derivatization and hydrolysis. This paper reviews the current status of carbohydrate fast atom bombardment mass spectrometry and reports on two new protocols which we are using to optimize sensitivity and fragmentation.

The toxicities of various combinations of trichloroethylene (TRI), tetrachloroethylene (TET) and 1,1,1-trichloroethane (TCE) were examined in suspensions of rat hepatocytes and in vivo. For each pair and for the three solvents together, an interactive toxicity was demonstrated in vitro, as determined by release of potassium ion and cytoplasmic enzymes. A similar pattern of response was found after administration to the intact rat for increases in plasma alanine aminotransferase and sorbitol dehydrogenase, both indices of hepatotoxicity. Plasma urea levels were significantly elevated on exposure to the three chemicals together. Thus a remarkably similar pattern of toxicity was found in vitro and in vivo, which supports the possible use of hepatocyte suspensions as a screening procedure for toxicity of mixtures.

The thermal effects of continuous wave carbon dioxide laser irradiation on human teeth were investigated. Internal temperature changes were monitored by means of electrical thermistors implanted within the pulp chambers of 20 extracted, unerupted human molar teeth. One-hundred test exposures at various powers and durations were obtained. Linear regression/correlation analysis of the data suggests a direct relationship between the independent variable, exposure energy (joules), and the dependent variable, internal temperature, under the conditions of this study.

Various metals, in particular, cadmium, have been found to interfere with a variety of aspects of immune function. Recently, spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) have been widely investigated due to their purported role in immune surveillance. Thus, the effects of cadmium on these two processes were examined. The effects of zinc and the combination of these two metals were also investigated. Both cadmium and zinc were found to inhibit SCMC and ADCC in a dose-dependent manner. In combination, the effects of the metal were generally additive.

The in vivo effect of cadmium (Cd) with or without prior administration of L -cysteine (Cys) or diethylmaleate (DEM) on hepatic and renal cytochrome c oxidase (Cyt- c -Ox), essential metals and mitochondrial thiols was investigated. Male Sprague—Dawley rats were given 25 μg Cd/kg (as Cd acetate) orally 5 times a week for 6 weeks. Different groups of animals additionally received either Cys (500 mg/kg per day, p.o.) or DEM (0.85 mg/kg, i.p.) by multiple administration. Parameters were determined 1 day after the last gavage. Cadmium decreased the activity of Cyt- c -Ox in mitochondria of livers but not in those of kidneys. Copper in both tissue and mitochondria were unaffected whereas hepatic tissue iron decreased by 50% upon Cd gavage. Cysteine pretreatment increased hepatic and especially renal mitochondrial Cd, but diminished the Cd effect on Cyt- c -Ox and increased hepatic tissue iron. Both DEM and DEM/Cd treatment decreased Cyt- c -Ox by 50% in liver but not in kidneys. Metallothionein was not significantly altered by either treatment. Considering data from all the experimental groups Cyt- c -Ox activity seems to be related rather to the amount of protein thiols than to either copper or iron in hepatic mitochondria. The data demonstrate the high susceptibility of hepatic vs. renal mitochondria and suggest the involvement of thiols in Cyt- c -Ox activity.