Kenneth Ramos

An in vitro approach to the study of target organ toxicity of drugs and chemicals

SummaryA major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.

Cardiotoxicity of tricyclic antidepressants in primary cultures of rat myocardial cells.

Primary cultures of myocardial cells were used to evaluate the cardiotoxic potential of various tricyclic antidepressants (TCAs). Lactate dehydrogenase (LDH) leakage, cellular viability, and beating rates were measured to compare the cardiotoxicity of amitriptyline, desipramine, imipramine, and nortriptyline. Tricyclic antidepressants were added to the cultures to give final concentrations of 1 X 10(-5), 1 X 10(-4), and 1 X 10(-3) M. Treatments lasted 1 and 4 h. All TCAs tested caused significant release of LDH and decreased cellular viability when added at 1 X 10(-3) M for 1 and 4 h. Amitriptyline was the only compound that caused significant LDH release 4 h after exposure to lower doses. Decreased viability was observed 4 h after exposure to all TCAs at a concentration of 1 X 10(-4) and 1 X 10(-3) M. Arrhythmias were observed 1 h after exposure to 1 X 10(-5) and 1 X 10(-4) M amitriptyline. All doses of amitriptyline inhibited beating 4 h after exposure. Imipramine, desipramine, and nortriptyline at a concentration of 1 X 10(-5) M decreased the beating rates of cultured myocytes 1 and 4 h after exposure. Arrhythmias and/or total inhibition of beating were observed when the cultures were exposed to higher concentrations of these compounds. Based on these data, the rank order of cardiotoxicity was amitriptyline greater than imipramine = desipramine greater than nortriptyline.

Cocaine-induced cardiotoxicity in vitro.

A growing number of reports have related cocaine use with the onset of myocardial infarction in young otherwise healthy individuals. Although the cardiac effects of cocaine have traditionally been attributed to sympathomimetic stimulation, several studies have suggested that cocaine may be directly cardiotoxic. The purpose of this study was to evaluate the cardiotoxic effects of cocaine in an in vitro preparation devoid of sympathetic innervation. Primary cultures of rat cardiac muscle and non-muscle cells were prepared from hearts excised from 3-5-day-old Sprague-Dawley rats. Cultures were exposed to various cocaine concentrations (1 x 10(-7)-1 x 10(-3)m) for 1-24 hr. Beating activity, morphological status and lactate dehydrogenase (LDH) leakage were evaluated following cocaine exposure. A decrease in the beating activity of cultured muscle cells was observed 1 hr after exposure to the highest cocaine concentrations (1 x 10(-5)-1 x 10(-3)m) tested. Similar results were obtained 24 hr after exposure. Morphological alterations in muscle cells were evident only after exposure to the highest concentration (1 x 10(-3)m). Vacuoles appeared 1 hr after cocaine exposure and were replaced by dark granules within 24 hr. LDH release was significantly elevated in the muscle cell cultures exposed to 1 x 10(-3)m cocaine for 24 hr. The pattern of cocaine-induced morphological alterations and enzyme leakage was similar in non-muscle cells. These data suggest that cocaine induces direct toxic effects on both cardiac muscle and non-muscle cells maintained in an environment free of neuronal and hormonal influences.

Cytotoxicity of isoproterenol to cultured heart cells: Effects of antioxidants on modifying membrane damage

Primary cultures of rat myocytes were used to study the cardiac damage induced by toxic doses of L-isoproterenol (ISO). Cultures were exposed to varying concentrations of ISO (2.4 X 10(-5), 1 X 10(-4), and 5 X 10(-4) M) for 0.5, 1.5, 4, and 12 hr. Mitochondrial membrane fragility, myocyte potassium content (as an index of sarcolemmal damage), and cellular glutathione content were used to evaluate cellular injury. A significant increase in mitochondrial fragility was observed 0.5 hr after treatment with 5 X 10(-4) M ISO. Lower doses caused an increase in mitochondrial fragility 1.5 hr after exposure. Longer durations of ISO exposure (4 and 12 hr) were necessary to decrease cellular potassium content. Glutathione levels were minimally affected by ISO. L-Ascorbic acid (5 X 10(-3) M) or sodium bisulfite (9.6 X 10(-4) M) were added to the cultures to determine if antioxidants prevent the toxicity caused by ISO. The presence of L-ascorbic acid or sodium bisulfite in ISO-treated myocyte cultures prevented the toxic changes in mitochondrial fragility and myocyte potassium content. The data indicate that antioxidants may be useful in reducing injury induced by toxic doses of ISO. Furthermore, mitochondrial injury may be involved significantly with the development of ISO-induced cardiotoxicity.

Role of calcium in isoproterenol cytotoxicity to cultured myocardial cells

Primary cultures of rat myocardial cells were used to evaluate the cellular dynamics of calcium accumulation after exposure to isoproterenol (ISO). Non-toxic concentrations of ISO (2.4 X 10(-7) M) caused a gradual increase in myocyte calcium uptake. These effects peaked 3 min after exposure and returned to control levels within 2 min. Toxic concentrations of ISO caused a biphasic increase in calcium uptake. The initial phase peaked 1 min after exposure and returned to control levels by 3 min. A second phase was characterized by a progressive increase in calcium uptake that plateaued 10 min after exposure. Ascorbic acid (AA, 5 X 10(-3) M) and sodium bisulfite (SB, 9.6 X 10(-4) M) did not modify the calcium uptake of the initial phase, whereas propranolol (1 X 10(-6) M) and verapamil (1 X 10(-5) M) prevented the initial rise in calcium uptake. In contrast, the antioxidants prevented the the second phase of ISO-induced calcium uptake, whereas verapamil and propranolol did not. The toxic accumulation of calcium induced by ISO may be due to oxidative damage of the sarcolemma. Antioxidants may prevent the formation of oxidative metabolites from ISO and the subsequent calcium overload. Our results show that agents which modify slow calcium-channel transport do not prevent ISO-induced calcium overload in our cell culture system.

Preventory by L(−) ascorbic acid of isoproterenol-induced cardiotoxicity in primary cultures of rat myocytes

Primary cultures of rat myocytes were exposed to various doses of L-isoproterenol (ISO) for 4, 12 and 24 h. L-ascorbic acid (AA) was added to some cultures immediately after exposure to ISO to determine if antioxidants reduce the toxicity caused by ISO. Leakage of lactate dehydrogenase (LDH) and cell viability were used as indices of cell injury. A significant increase in LDH release was found 24 h after exposure to 1 X 10(-4) M ISO alone. Higher doses (5 X 10(-4) and 1 X 10(-3) M) caused significant enzyme release 4, 12 and 24 h after exposure. The viability of cultures exposed to toxic doses of ISO for 4 h was not affected. A dose-dependent decrease in cell viability was observed 12 h and 24 h after exposure to ISO. L-ascorbic acid (5 X 10(-3) and 1.5 X 10(-2) M) significantly reduced the LDH release caused by ISO. The enzyme release from cultures exposed to 5 X 10(-4) and 5 X 10(-3) M AA alone was similar to that of control cultures. However, 1.5 X 10(-2) and 3 X 10(-2) M AA caused marked LDH release. The viability of cultures subjected to ISO in the presence of 5 X 10(-3) M AA was similar to that of controls. Our results show that AA protects against the LDH release and decreased cellular viability caused by toxic doses of ISO.

Cell injury of cultured rat myocardial cells after reoxygenation of hypoxic cultures in the presence and absence of calcium.

SummaryPrimary cultures of rat myocytes were deprived of oxygen (approximately 5 mm Hg, pO2) for 2 h in the presence or absence of calcium and were subsequently incubated for different time periods under normoxic (approximately 120 mm Hg, pO2) conditions. Myocyte calcium content was determined at the end of the oxygen-free period and after repletion of oxygen. Lactate dehydrogenase (LDH) release from the cells into the medium was used as an index of cell injury. Cultures deprived of oxygen in the absence of calcium showed a significant decrease in myocyte calcium content at the end of the oxygen-free period. However, the calcium content of myocyte cultures deprived of oxygen in the presence of calcium did not change significantly. The enzyme release of both groups of oxygendeprived cultures was similar to that of controls. A progressive increase in myocyte calcium content was observed 5, 10, 15, and 30 min after repletion of oxygen in both groups. Reoxygenation of cultures subjected to hypoxia in the presence of calcium caused minimal LDH leakage. However, major enzyme release was observed 30 min after oxygen repletion in cultures previously subjected to hypoxia in a calcium-free environment. The present study shows that reoxygenation injury is more severe in myocyte cultures previously deprived of both oxygen and calcium than in cultures deprived of oxygen in the presence of calcium.

Accumulation of45calcium as an index of cell injury and cytotoxicity in cultured cells

Measurement of45calcium uptake in cultured cells can be used as a sensitive indicator of chemicalinduced alterations in plasma membrane permeability to calcium. Accumulation of large amounts of calcium may alter the structural and functional integrity of several cellular compartments and result in cell injury.

Cellular injury of primary cultures of rat myocytes incubated in calcium-free medium followed by recovery in calcium.

SummaryPrimary monolayer cultures of rat myocardial cells were used to study the cellular injury that occurs when calcium is reintroduced after a period of calcium depletion. Cultures were treated with a calcium-free balanced salt solution (BSS) for 2 h and were then incubated for different time periods in the presence of normocalcemic BSS (2.5 mM CaCl2). Myocyte calcium content was determined after the calcium-free period and after incubation in the presence of calcium. Leakage of cytoplasmic lactate dehydrogenase (LDH) into the medium was used as an index of cell injury. At the end of the calcium-free period there was a significant decrease in the total cellular content of calcium, and LDH release was minimal. After incubation in normocalcemic BSS, the myoctte calcium content increased progressively with time and cellular injury was manifested by significant leakage of LDH. The calcium content of treated cells reached control levels 10 min after calcium repletion. Maximal keakage of LDH was observed 60 min after the restoration of calcium. The calcium content of treated cultures was higher than that of control cultures 120 min after incubation in normocalcemic BSS.

Attenuation by antioxidants of Na+/K+ ATPase inhibition by toxic concentrations of isoproterenol in cultured rat myocardial cells.

We have reported previously that toxic concentrations of isoproterenol caused severe alterations in the structural integrity of the sarcolemma and mitochondria found in primary cultures of rat myocardial cells [8, 9]. Mitochondrial injury was observed 1.5 h after exposure to isoproterenol, whereas leakage of intracellular ions and enzymes was observed only after prolonged exposures (greater than 4 h). Ascorbic acid and sodium bisulfite prevented the cytotoxic effects of isoproterenol in our cell culture system. Takeo et al. [13] suggested that adrenochrome (an oxidative metabolite of epinephrine) specifically inhibits the activity of the sodium/potassium ATPase. Other investigators have shown that an indole metabolite of epinephrine inhibited actomyosin ATPase [1, 4]. These inhibitory actions may result from an interaction between the oxidative metabolites and sulfhydryl groups present in the enzyme [13]. Inhibition of the sodium/potassium ATPase is associated with an increase in the intracellular concentration of Na+ and Ca2+ and a decrease in intracellular K+. Changes in the intracellular concentration of these ions are commonly seen in heart cell damage and contractile failure [2]. The present study was designed to determine if isoproterenol, a synthetic catecholamine, inhibits the sodium/potassium ATPase activity in a primary culture system of rat myocardial cells.