Allison A. Welder

Cardiovascular effects of androgenic-anabolic steroids.

Evidence has accumulated over the pst several years which associates androgenic-anabolic steroid (AAS) use with sudden cardiac death, myocardial infarction, altered serum lipoproteins, and cardiac hypertrophy in humans who habitually use these drugs. Even though some experimental data obtained from animals correlate well with the human findings, the adverse cardiovascular effects of AAS use are poorly understood. The evidence presented in this review suggests that there are at least four hypothetical models of AAS-induced adverse cardiovascular effects: 1) an atherogenic model involving the effects of AAS on lipoprotein concentrations; 2) a thrombosis model involving the effects of AAS on clotting factors and platelets; 3) a vasospasm model involving the effects of AAS on the vascular nitric oxide system; and 4) a direct myocardial injury model involving the effects of AAS on individual myocardial cells. Future studies should be directed at determining the exact mechanisms responsible for AAS-induced adverse cardiovascular effects, at determining the relative contribution of each of these models, and at identifying other possible contributing factors such as metabolism of these steroids and the effects of potential metabolites on various target organs.

The effect of anabolic-androgenic steroids on primary myocardial cell cultures

Although recent case reports suggest that anabolic-androgenic steroids may be directly injurious to the cardiovascular system, the direct myocardial cellular consequences of abuse of these drugs are not known. Therefore, the purpose of this study was to describe the concentration- and time-dependent effects of testosterone cypionate (TC), stanozolol (S), and fluoxymesterone (F) on primary myocardial cell cultures. Evaluation of drug effects were made in 4-d-old primary myocardial cell cultures obtained from 3- to 5-d-old Sprague-Dawley rats. The cultures were exposed to 1 x 10(-4) M, 1 x 10(-6) M, and 1 x 10(-8) M concentrations of TC, S, and F each for 1, 4, and 24 h. Cellular injury was evaluated by alterations in beating activity, induction of morphological alterations, lactate dehydrogenase (LDH) release, neutral red retention, and tetrazolium (MTT) formazan production. Significant alterations in beating activity were observed in the 1 x 10(-4) M TC group in which no beating activity was seen at 1, 4, and 24 h. Morphological integrity was disrupted for the 1 x 10(-4) M TC group at 24 h where destruction of the monolayer was observed. Unlike the cultures treated with the three concentrations of both S and F, significant LDH release was seen at 4 and 24 h with those cultures exposed to 1 x 10(-4) M TC. In the evaluation of neutral red retention, 1 x 10(-4) M TC at 24 h showed a significant decrease in ability to retain the dye.(ABSTRACT TRUNCATED AT 250 WORDS)

A primary culture system of adult rat heart cells for the evaluation of cocaine toxicity

The complex dose-response relationship by which cocaine (Coc) directly precipitates unfavorable cardiac consequences are not known. There appears to be two diametrically opposed cardiovascular actions of Coc. At low doses, the sympathetic nervous system responses dominate, whereas, at high doses, the local anesthetic actions exert the most powerful effects. The purpose of this study was to describe a dose- and time-dependent Coc cardiotoxicity profile in a model of spontaneously contracting adult primary myocardial cell cultures obtained from 60-90-day-old Sprague-Dawley rats. Indices of toxicity determined included contractility, morphology, lactate dehydrogenase release (LDH), mitochondrial tetrazolium formazan (MTT) production and neutral red (NR) formation. After the cells had been grown in culture for 11 days, they were exposed to 1 x 10(-3), 1 x 10(-5), 1 x 10(-7) and 1 x 10(-9) M Coc for 1-24 h. The two lowest doses of Coc (1 x 10(-7) and 1 x 10(-9) M) had little or no effect on the adult heart cell cultures. However, morphological alterations included vacuolization, granulation and pseudopodia formation as early as 1 h after exposure to the highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M). For all time points observed, the two highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M) significantly depressed contractility and induced significant LDH release. MTT formazan production and NR retention were not significantly different from untreated controls for all treatments. By employing an acute Coc exposure paradigm, these data demonstrate that Coc doses greater than or equal to 1 x 10(-5) M induce direct injurious local anesthetic effects on contractility and morphology of spontaneously contracting adult rat myocardial cells in culture.

A primary culture system of adult rat heart cells for the study of toxicologic agents

SummaryTricyclic antidepressants (TCAs) are currently used in the treatment of mental depression and nocturnal enuresis. Clinically, these drugs are useful; however, cardiotoxicity can occur even with therapeutic dosages. For example, TCAs are known to alter myocardial function, induce arrhythmias, and produce heart block in individuals with a normal cardiovascular history. The present study was undertaken to establish a culture system of spontaneously contracting adult primary myocardial cells for toxicologic testing and to examine their contractility, morphology, and lactate dehydrogenase release (LDH) after treatment with one of the most cardiotoxic TCAs, amitriptyline. Primary myocardial cell cultures were obtained from approximately 60- to 90-day-old Sprague-Dawley rats. After the cells had been grown in culture for 11 days, they were treated with amitriptyline (1 × 10−3, 1 × 10−4, and 1 × 10−5M) for 2 to 24 h. The highest concentration of amitriptyline (1 × 10−3M) completely destroyed the cardiac muscle cells. In addition to moderate and severe vacuole, granule, and pseudopodia formation, all contractile activity was inhibited as early as 2 h after exposure to the intermediate concentration of 1 × 10−4M amitriptyline. Significant LDH release did not occur until 8 h after treatment with this intermediate concentration. Even though there was no significant LDH release at all 3 time points tested, there was a 50% decrease in beating activity (154±9 to 77±5 beats/min) and initiation of vacuole formation by 2 h with the lowest concentration of amitriptyline (1 × 10−5M). This study presents a new apparatus for the isolation of adult cardiac myocytes for the establishment of primary cell cultures for toxicologic testing. Furthermore, these data demonstrate that amitriptyline induces a concentration- and time-dependent cardiotoxic profile in a model of spontaneously contracting adult cardiac muscle cells in culture.

A primary culture system of postnatal rat heart cells for the study of cocaine and methamphetamine toxicity

It is now well documented that both cocaine (Coc) and methamphetamine (Meth) are independently capable of inducing injurious effects on the adult and developing myocardium. In addition, when these drugs are used concomitantly such as in polydrug abuse, it has been suggested that they may cause synergistic adverse effects on the myocardium. In this investigation, primary myocardial cell cultures were established from 3-5-day-old Sprague-Dawley rats to describe the adverse effects of Coc and Meth on the myocardium. After the cells were in culture for 4 days, they were exposed to 1 x 10(-5) and 1 x 10(-3) M Coc alone; 1 x 10(-5) and 1 x 10(-3) M Meth alone; and combinations of 1 x 10(-3) M Coc with 1 x 10(-5) M Meth and 1 x 10(-5) M Coc with 1 x 10(-5) M Meth. Lactate dehydrogenase (LDH) release, morphology, and beating activity were evaluated after exposure to the drugs for 1, 4 and 24 h. With all treatment groups for the first 4 h, LDH release was not significantly different from untreated controls. Significant LDH release (P less than 0.001) was exhibited at 24 h with 1 x 10(-3) M Coc alone, 1 x 10(-3) M Meth alone, and 1 x 10(-3) M Coc with 1 x 10(-5) M Meth. For 24 h of treatment, cellular injury (pseudopodia, vacuolization, granulation) induced by 1 x 10(-3) M and 1 x 10(-5) M Coc alone was extensive and minimal, respectively. When 1 x 10(-5) M Meth was added with 1 x 10(-5) M Coc, pseudopodia formation was extensive. No measurable beating activity was observed at 1, 4 and 24 h exposure to 1 x 10(-3) M Coc alone and 1 x 10(-3) M Coc with 1 x 10(-5) M Meth. At 1 h, beating activity after treatment with 1 x 10(-5) M Coc alone and 1 x 10(-5) M Meth alone was not significantly different from untreated controls; however, the percentage of areas exhibiting contractile activity was depressed. Addition of Meth (1 x 10(-5) M) potentiated Coc-induced (1 x 10(-5) M) depression of contractile activity at all 3 time-points. These data suggest that Coc and Meth may interact synergistically at the cellular level to directly potentiate injury to postnatal myocardial cell cultures.

Evaluation of the combined toxic effects of cocaine and ethanol on primary myocardial cell cultures

Since 1978, the most prevalent drug used in conjunction with cocaine in cocaine-associated myocardial deaths is ethanol. Primary myocardial cell cultures were used to evaluate the acute additive cardiotoxic effects of cocaine and ethanol. The cultures were exposed to 1 x 10(-3)m or 1 x 10(-5)m-cocaine or combinations of both concentrations of cocaine with 600 mg ethanol/100 ml. Alterations in beating activity, morphology, and lactate dehydrogenase (LDH) release were evaluated after 1, 4 and 24 hr of treatment. Although cells exposed to 1 x 10(-5)m-cocaine or to 1 x 10(-5)m-cocaine and ethanol were able to retain some beating activity, no beating activity occurred in cells exposed to 1 x 10(-3)m-cocaine with or without ethanol. Morphologically, pseudopodia and disruption of the monolayer were more extensive in cells treated for 4 or 24 hr with the combinations of cocaine and ethanol in comparison with those treated with cocaine alone. After 24 hr, LDH release in cells exposed to 1 x 10(-3)m-cocaine or 1 x 10(-3)m-cocaine with ethanol was elevated over the level in untreated controls. These data suggest that ethanol enhances cocaine-induced beating abnormalities and morphological alterations in primary myocardial cell cultures.

Cocaethylene toxicity in rat primary myocardial cell cultures.

Cocaethylene is a unique cocaine metabolite formed in the presence of ethanol by the liver. Neither acute nor chronic cardiotoxic effects of this metabolite have been investigated. The purpose of this study was to establish a time- and dose-dependent toxicity profile for cocaethylene in primary myocardial cell cultures established from 3-5-day-old Sprague-Dawley rats. Alterations in lactate dehydrogenase (LDH) release, lysosomal neutral red (NR) retention, thiobarbituric acid-reactive substances (TBARS), morphology, and beating activity were evaluated after treatment of cultures with cocaethylene doses ranging from 1.0 x 10(-3) to 1.0 x 10(-9) M from 1 to 24 h. LDH release was significantly elevated after 24 h only with those cultures exposed to the highest dose of cocaethylene (1.0 x 10(-3) M). The highest dose of cocaethylene also significantly depressed NR retention. While all doses of cocaethylene depressed contractile activity and altered cellular morphology by 24 h, there were no TBARS formed up to 15 h. Thus, both low and high doses of cocaethylene are injurious to the cellular integrity and contractility of myocardial cell cultures. Future studies are warranted to determine mechanisms of cocaethylene toxicity in this in vitro model of spontaneously contracting myocardial cells.

Cellular mechanisms of cocaine cardiotoxicity

Although cocaine abuse has been a major drug problem in the United States for over 100 years, it has only been in the last decade that the adverse effects of cocaine on the cardiovascular system have become a serious health issue. The cardiotoxic effects of cocaine are multifactorial and remain a puzzle for investigators to solve. Evidence suggests that cocaine-induced toxic effects on the cardiac and vascular cells include both direct as well as indirect components. In addition, other chemical, physiologic, and environmental factors may further complicate and alter mechanisms and endpoints of cocaine-induced cellular toxicity. In order to fully understand the overall cardiotoxic response to cocaine, the cellular mechanisms and endpoints of toxicity of each of these potentially injurious factors must be identified.

Differential toxicity of cocaine and its isomers, (+)-cocaine and (−)-Ψ-cocaine, is associated with stereoselective hydrolysis by hepatic carboxylesterases in cultured rat hepatocytes

Cocaine induces acute lethal cell injury in rat hepatocytes following N-oxidative metabolic activation by cytochrome P450-dependent and flavin-dependent monooxygenases. Beside this oxidative bioactivation pathway, hepatic carboxylesterases may cleave the carboxymethylester or the benzoylester linkage which leads to molecules found to be non-toxic in vivo. To elucidate the structural requirements of the cocaine molecule for its bioactivation and inactivation, the cytotoxic potential of the natural (-)-cocaine relative to two isomeric forms, (+)-cocaine* (the unnatural enantiomer) and (-)-psi-cocaine (the C2 epimer of the unnatural cocaine) were investigated. Primary short-term cultures of rat hepatocytes obtained from phenobarbital (PB)-pretreated rats were exposed to the drugs for up to 24 h. (-)-Cocaine produced marked time- and concentration-dependent release of lactate dehydrogenase (LDH) into the extracellular medium, whereas the other forms were not cytotoxic (0-1 mM). Furthermore, depletion of cellular glutathione (GSH) with diethylmaleate enhanced LDH release in (-)-cocaine-treated cells and caused marginal cytotoxicity in hepatocytes exposed to the other isomers. To investigate the mechanisms that could be responsible for these isomer-specific effects, the time-dependent metabolic degradation was determined both in cultured hepatocytes and in hepatic microsomes in the presence or absence of the serine carboxylesterase inhibitors, phenylmethylsulfonylfluoride (PMSF) or NaF. All three cocaine analogs were enzymatically degraded, but the rates of ester cleavage greatly varied among the stereoisomers. (-)-Cocaine was primarily N-oxidized via SKF-525A-sensitive pathways, whereas (+)-cocaine was predominantly hydrolyzed by PMSF-sensitive carboxylesterases. In contrast, (-)-psi-cocaine, which is very stable in the absence of cells at 37 degrees C and pH 7.4, was subject to extremely fast enzymatic ester cleavage. In conclusion, these results indicate that the isomer-specific differential cytotoxicity of (-)-cocaine, (+)-cocaine and (-)-psi-cocaine in hepatocytes may be related to stereoselective differences in the rates of hydrolytic inactivation by hepatic carboxylesterases and that the N-oxidative pathway, resulting in hepatocyte injury, may thus be relevant only for (-)-cocaine.

Effects of cocaine and norepinephrine on primary cultures of neonatal rat myocardial cells

Sudden cardiac death associated with cocaine (Coc) abuse in healthy, physically active individuals became a grave concern in the late 1980s. It is well documented that physical activity increases circulating plasma catecholamine levels. Catecholamines as well as Coc are independently capable of inducing toxic cardiac effects. The purpose of this investigation was to evaluate the synergistic or additive toxic effects of norepinephrine (NE) and Coc in primary myocardial cell cultures obtained from 3- to 5-d-old Sprague-Dawley rats. Alterations in lactate dehydrogenase release (LDH), lysosomal neutral red retention (NR), beating activity, and morphology were evaluated after treatment of the cells for 1-24 h with 1 x 10(-3) M Coc alone, 1 x 10(-5) M Coc alone, 1 x 10(-5) M NE alone, 1 x 10(-3) M Coc with 1 x 10(-5) M NE, or 1 x 10(-5) M Coc with 1 x 10(-5) M NE. LDH release was elevated significantly after 24 h only with those cells exposed to 1 x 10(-3) M Coc alone and 1 x 10(-3) M Coc + 1 x 10(-5) M NE. Using NR retention as a score for lysosomal treatment of the cells with 1 x 10(-5) M Coc and 1 x 10(-3) M Coc alone did not decrease dye retention significantly. However, 1 x 10(-5) M NE combined with 1 x 10(-3) M Coc significantly reduced lysosomal dye retention as early as 1 h after treatment. After 24 h, 1 x 10(-5) M NE alone and 1 x 10(-5) M NE combined with 1 x 10(-5) M Coc significantly increased lysosomal fragility. Beating activity was altered in all treatment groups. Contractile activity was slow and irregular or completely absent with 1 x 10(-5) and 1 x 10(-3) M Coc, respectively. When NE (1 x 10(-5) M) was combined with both concentrations of Coc, there was distinct focalization of sharp, rapid contractions within the cells, which were asynchronous and/or arrhythmic in nature. Those cells exposed to 1 x 10(-5) M NE with 1 x 10(-5) M Coc for 24 h appeared hypercontracted with marked pseudopodia and cytoplasmic granule formation distinctly different from that exhibited by the cells exposed to 1 x 10(-5) M Coc alone. These data demonstrate that NE potentiates the adverse effects of Coc on contractile activity and morphology of spontaneously contracting neonatal myocardial cells maintained in culture.