* Laboratory of Experimental Pathology; Laboratory of Signal Transduction;
Laboratory of Respiratory Biology; and
Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received July 13, 2004; accepted November 8, 2004
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ABSTRACT |
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Key Words: ephedrine; caffeine; cardiotoxicity; apoptosis; coagulative necrosis; ischemia.
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INTRODUCTION |
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Pharmacologic activity of ephedrine may be mediated by its ability to cause the release of norepinephrine and epinephrine from adrenergic nerve terminals and thereby produce indirect stimulation of adrenoreceptors (Weiner, 1980). The cardiovascular effects of ephedrine are similar to those of epinephrine, but they persist for a longer period of time.
Ephedrine is both an - and ß-adrenergic agonist that enhances the release of catecholamines, such as norepinephrine, from sympathetic neurons. Ephedrine stimulates heart rate and cardiac output and causes systemic vasoconstriction (Tong and Eisenach, 1992
); as a result, it usually increases blood pressure (Persky et al., 2004
). The myocardial ischemia and myocardial cell death are thought to be due to vasoconstriction and cardiac stimulation induced by epinephrine (Weiner, 1980
). Single case reports (Enders et al., 2003
; Foxford et al., 2003
; Krome and Tucker, 2003
; Kumar and Jugdutt, 2003
; Lustik et al., 1997
; Miller and Waite, 2003
; Naka et al., 2003
), reviews of adverse-events data from 140 (Haller et al., 2002
) and 926 cases (Samenuk et al., 2002
), and placebo-controlled studies (McBride et al., 2004
) showed that the risk of hypertension, cardiac arrhythmia, coronary-artery constriction, and reduction in myocardial oxygen supply was associated with the intake of ephedrine-containing herbal supplements. Coronary-artery vasospasms have been reported with ephedrine administration during spinal cord surgery or abusive injection (Cockings and Brown, 1997
; Hirabayashi et al., 1996
).
Total alkaloid content of these herbal medicines has been estimated at 0.52.5%, with ephedrine accounting for 3090% of the alkaloids (Gurley et al., 1998; Lee et al., 2000
). Many of these ephedrine-containing herbal supplements contain a botanical form of caffeine, referred to as guarana-derived caffeine, from the plant Paullinia cupano (Haller et al., 2004
). The ephedrine-containing herbal supplements may also be taken with caffeine from coffee or tea averaging 75150 mg caffeine per cup (Rose'Meyer et al., 2001
). Ephedrine and caffeine act synergistically to increase heart rate and diastolic and systolic blood pressure (Astrup et al., 1991
). The pressor effects of caffeine have been attributed to increased catecholamine and renin release and/or antagonism of endogenous adenosine (Nurminen et al., 1999
; Rose'Meyer et al., 2001
; White et al., 1997
).
Ephedra sinica extract commonly used in dietary supplements consists of ephedrine, pseudoephedrine, methylephedrine, norpseudoephedrine, and norephedrine, of which ephedrine is the principal alkaloid component. A typical ephedrine-alkaloid-containing dietary supplement dose contains 20 mg ephedrine and
200 mg of guararna-derived caffeine (Haller et al., 2004
; Jacob et al., 2004
). Ephedrine alkaloids are rapidly absorbed, with maximum plasma concentration (tmax) occurring within 2.4 h and an ephedrine plasma half-life of
6.1 h (Haller et al., 2002
; White et al., 1997
; Wilkinson and Beckett, 1968
). A typical human ephedrine/caffeine dosage is
0.3 mg ephedrine/kg body weight (11 mg/m2 body surface area) and
3 mg caffeine/kg body weight (111 mg/m2 body surface area) (Table 1).
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MATERIALS AND METHODS |
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Barbeito-Lopez trichrome stain (BLTS). This histochemical staining method was applied for routine diagnosis of myocardial degeneration or necrosis (Milei and Bolomo, 1983; Milei and Storino, 1986
).
Caspase-3. Immunostaining for the localization of cleaved caspase-3 protein expression was performed, using a polyclonal antibody (Cell Signaling Technology, Beverly, MA) to detect apoptosis. Sections were first deparaffinized in xylene and hydrated through a series of ethanols to 1x Automation Buffer (1x AB) (Biomeda, Foster City, CA). Endogenous peroxidase was blocked using 3% H2O2 for 15 min at room temperature. Antigen unmasking was accomplished by heating sections in 200 ml of citrate buffer (pH 6.0) in DecloakerTM (both, Biocare Medical, Walnut Creek, CA). Following depressurization, slides were allowed to cool for 10 min before the reaction was stopped in running distilled water. Prior to the application of the primary antibody, a protein block (Dako, Carpinteria, CA) and an avidin-biotin block (Vector Laboratories, Burlingame, CA) were applied. The primary antibody, rabbit anti-cleaved caspase-3, was then applied for 1 h at room temperature at a dilution of 1:50. Nonimmune rabbit IgG (Jackson Immunoresearch Labs, West Grove, PA) was used as the negative control at equivalent conditions in place of the primary antibody. Localization of the primary antibody was detected using the LSAB + Kit (Dako, Carpinteria, CA). The antibody complex was visualized with a diaminobenzidine (Liquid DABTM, Dako) reaction occurring for 6 min in the dark. Finally, slides were rinsed in running tap water, counterstained with Harris Hematoxylin (Harelco, Gibbstown, NJ), dehydrated through a series of ethanols to xylene, and coverslipped with Permount (Fisher Scientific, Norcross, GA). The positive control tissue for the stain was necrotic acinar pancreatic tissue from a rat.
Anti-phospho-H2A.X. Immunostaining of hyperphosphorylated H2A.X provided another indicator of apoptosis (Talasz et al., 2002). A commercially available polyclonal antibody for H2A.X (Upstate Signaling Solutions, Waltham, MA) was used at a dilution of 1:250. The positive control tissue for the stain was testis. The procedure for staining of H2A.X was the same as that for caspase-3.
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RESULTS |
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Histopathological Findings
The incidences of the changes observed in the hearts are presented in Table 2. In the hearts of animals found dead or sacrificed in distress 45 h after the first dosing of ephedrine and caffeine (i.e., 5/7 of the 14-week-old treated rats), changes were observed chiefly in the interventricular septum and, to a lesser extent, the left and right ventricular walls. Massive interstitial hemorrhage occurred at the subendocardial myocardium of the left ventricle and interventricular septa (Figs. 2A and 2B). The hemorrhage was associated with degeneration of the surrounding myofibers that appeared hyalinyzed, vacuolated, and with loss of striations. In some of these cells the nuclei were pyknotic or had disappeared. The incidence and severity of hemorrhage in the hearts of 7-week-old ephedrine-and-caffeine treated rats were lower than in 14-week-old ephedrine-and-caffeine-treated rats, and none of the 7-week-old treated rats died acutely after the first dose of ephedrine-caffeine. The remainder of the pathological changes seen in the 7-week-old animals were, however, comparable in nature and severity to those seen in the 14-week-old animals.
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DISCUSSION |
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Different studies in the literature suggest that older rats, as well as older humans, have a diminished ability to respond to ischemia compared to younger individuals (Isoyama and Nitta-Komatsubara, 2002). This difference may result from an age-related diminished capacity to adapt to increased hemodynamic overload and ischemia and combat heart disease (Goyns et al., 1998
). Identifying the critical genes involved in the response to ischemia is an area of future exploration.
Reports of possible toxicity in humans induced by herbal supplements containing both 37% ephedrine and guarana-derived caffeine have indicated an increased risk of stroke, myocardial infarction, and sudden death (Samenuk et al., 2002). In one of the human sudden-death cases, histopathological examination of the heart revealed changes comparable to those seen in this study, consisting of multifocal and confluent myocardial necrosis with the occurrence of scarring in one to two weeks. Interpretative analysis indicated that myocardial necrosis was not specific and could have been due to primary myocytic toxicity or occurred secondarily to ischemia. Furthermore, the presence of hemorrhage and cellular necrosis was consistent with the effects of factors that collectively could have been responsible for vascular damage, vasoconstriction of small arterial vessels, and myocytic toxicity. These pathological features are reminiscent of experimental and clinical manifestations of adrenergic and sympathomimetic agents (Samenuk et al., 2002
).
The myocardial changes seen in our rats after 1 to 3 days of ephedrine/caffeine exposure consisted of acute interstitial hemorrhage, myocytic necrosis, apoptosis, and early inflammatory cell infiltration. Caspase-3 staining revealed positivity in multiple myofibers in the animals dying within 45 h of exposure. Caspase-3, which belongs to a family of highly homologous endopeptidases, plays a crucial role in executing apoptosis and has been found to be a reliable marker of apoptosis in other models of chemically induced cardiotoxicity (Li et al., 2002). Using an in vitro model of heart ischemia, investigators suggested that, within 1 h, heart ischemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3-like proteases (Borutaite et al., 2001
; Naka et al., 2003
).
The immunostaining for the DNA-phosphorylated histone H2A.X proved to be reliable in detecting apoptotic cells in which H&E staining had revealed nuclear fragments. Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin; histone modifications, such as phosphorylation, may affect chromatin function during both the cell cycle and apoptosis (Talasz et al., 2002). Hyperphosphorylation of H2A.X is a necessary step in apoptotic pathways, occurring during the induction of apoptosis concomitantly with the appearance of the condensation of chromatin, but before nucleosomal fragmentation (Rogakou et al., 2000
). The H2A.X histone becomes specifically phosphorylated at serine 13 within seconds after the induction of DNA double-strand breaks. The formation of H2A.X during apoptosis is a downstream consequence of caspase activation (Rogakou et al., 2000
). In our study, the caspase-3 staining identified early necrotizing myofibers, while the anti-H2A.X staining was effective in confirming the fragmentation of nuclear chromatin.
The BLTS detected myocardial degeneration in the rat as early as 45 h after ephedrine/caffeine exposure. In contrast, in the H&E-stained heart sections from the ephedrine/caffeine-treated rats, the early degenerating myofibers could not be easily identified, especially when they exhibited only loss of striation. In the BLTS-stained heart sections, degenerating myofibers were identified with a yellow color, in contrast to the green-blue coloration of the undamaged myofibers. Milei and Storino (1986) have also used BLTS to demonstrate heart toxicity in the rat following exposure to isoproterenol sulfate. In these studies, isoproterenol induced coagulative necrosis in rat hearts 30 min after myocardial ischemia, and BLT stained the cytoplasm of these necrotic cells a patchy pale-yellow color. In the coagulative necrosis that had become established after 24 h, the cytoplasm appeared golden yellow (Milei and Storino, 1986
).
In our ephedrine/caffeine rat studies, the combination of caspase-3, H2A.X, and the BLTS staining methods was shown to be particularly effective in locating early degenerating myofibers. We recommend, therefore, that, in evaluating the cause of sudden death where cardiac damage is suspected, this triple-combination staining be added to the routine H&E staining.
Although the myocardium can be damaged by various agents or factors, such as anoxia, ischemia, infectious agents, and physical and chemical agents, the pattern of response is relatively limited (Greaves, 2000). In ephedrine/caffeine-treated rats, myofiber degeneration, apoptosis, and necrosis reflected a typical range of acute damage, while the infiltration of macrophages represented the process of elimination of dying cells. If the animal did not die due to extensive heart failure, this stage could be followed in a few days by further scarring by fibrosis. The myocardial necrosis, morphologically and histochemically consistent with apoptosis, was frequently associated with acute inflammation. Inflammation has been documented in the case of myocardial ischemia (Vinten-Johansen, 2004
); however, experimental studies provide strong but somewhat conflicting evidence that neutrophils are involved in the myocardial response leading to lethal injury upon reperfusion. Whether the accumulation of neutrophils within an ischemic-reperfused area represents a response to injury or is an active process contributing to injury of the myocardial cells is not clear.
We hypothesize that, in our study, many of the 14-week-old rats exposed to a single dose of ephedrine and caffeine developed severe heart insufficiency, leading to sudden death. In contrast, when ephedrine alone was given to rats or mice for 13 weeks at doses up to 120 mg/kg/day, or for 104 weeks at doses up to 18 mg/kg/day (National Toxicology Program, 1986), heart toxicity was not observed. Even when mice were administered ephedrine alone at doses up to 600 mg ephedrine/kg/day for up to seven days, acute heart toxicity and death did not occur (Minematsu et al., 1991
). In our studies, administration of caffeine alone to rats did not cause acute heart toxicity and death. Other studies do show that long-term administration of caffeine alone may cause some cardiac toxicity (Johannsson, 1981
); however, the exposure to the combination of ephedrine and caffeine causes acute heart toxicity. The animals were dosed in the afternoon when the stomach would have been relatively empty, and this dosing on an empty stomach may have facilitated uptake of ephedrine and caffeine (Yuan, 1993
).
In humans to whom ephedrine alone (Cohn, 1965; Franciosa and Cohn, 1979
) or caffeine alone was administered (Vlachopoulos et al., 2002
), acute heart toxicity and death did not occur. In humans, however, consumption of products containing ephedrine and caffeine may cause acute heart toxicity and death (Samenuk et al., 2002
). The combined effects of caffeine, which can antagonize the adenosine receptors resulting in increased blood pressure (Nurminen et al., 1999
), and ephedrine, which is an
- and ß-adrenergic agonist (Nurminen et al., 1999
), work together to cause the acute and sometimes fatal heart toxicity. Our study design can serve as a model to elucidate further these combined mechanisms that function in this type of cardiotoxicity and can be used to develop preventive strategies.
The heart damage observed in this rat study is suggested to be caused by ischemia, followed by myofiber necrosis. Combined ephedrine and caffeine exposure probably induced intense diffuse vasoconstriction of the coronary arterial system, decreasing myocardial blood supply, leading to ischemia and apoptotic death of the cardiomyocytes. These findings support the recommendations of the FDA (United States Food and Drug Administration 2004) to ban the over-the-counter sale of dietary supplements containing ephedra.
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NOTES |
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1 To whom correspondence should be addressed at Laboratory of Experimental Pathology, MD B3-06, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709-9998. Fax: (919) 541-7666. E-mail: nyska{at}niehs.nih.gov
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