Gary F. Merrill

Antioxidant properties of acetaminophen and cardioprotection

Abstract The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14 ± 0.06/min vs 0.03 ± 0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90 % repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31 ± 4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57 ± 6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias.

Estrogen diminishes postischemic hydroxyl radical production

Reperfusion of blood flow to an ischemic myocardium is imperative to survival; ironically, it may also manifest several pathophysiological conditions. The most important of these are reperfusion arrhythmias and tissue injury and/or death. The mechanisms involved in reperfusion arrhythmias remain to be fully elucidated; however, increasing evidence indicates that reperfusion-induced arrhythmias are a free radical-mediated phenomenon. Acute administration of conjugated equine estrogen to dogs attenuates ischemia- and reperfusion-induced arrhythmias. The cardioprotective effect of estrogens in postmenopausal women is well documented, and recent studies suggest that estrogens possess strong antioxidant properties, with equine estrogens most potent. In this study we show that administration of conjugated equine estrogen to fully anesthetized dogs abolishes the burst of .OH radicals typically produced on reperfusion of the myocardium. This indicates that estrogen might attenuate reperfusion-induced ventricular arrhythmias by virtue of its antioxidant properties, suggesting a novel cardioprotective effect of the hormone.Reperfusion of blood flow to an ischemic myocardium is imperative to survival; ironically, it may also manifest several pathophysiological conditions. The most important of these are reperfusion arrhythmias and tissue injury and/or death. The mechanisms involved in reperfusion arrhythmias remain to be fully elucidated; however, increasing evidence indicates that reperfusion-induced arrhythmias are a free radical-mediated phenomenon. Acute administration of conjugated equine estrogen to dogs attenuates ischemia- and reperfusion-induced arrhythmias. The cardioprotective effect of estrogens in postmenopausal women is well documented, and recent studies suggest that estrogens possess strong antioxidant properties, with equine estrogens most potent. In this study we show that administration of conjugated equine estrogen to fully anesthetized dogs abolishes the burst of ⋅ OH radicals typically produced on reperfusion of the myocardium. This indicates that estrogen might attenuate reperfusion-induced ventricular arrhythmias by virtue of its antioxidant properties, suggesting a novel cardioprotective effect of the hormone.

Coronary interactions between nifedipine and adenosine in the intact dog heart

Coronary vascular interactions between adenosine and the calcium entry blocker, nifedipine were studied in the open-chest, blood-perfused dog heart. Adenosine was administered either as a constant intra-coronary infusion or released endogenously during brief occlusions of the left anterior descending (LAD) coronary artery. Nifedipine was administered in therapeutic concentrations as a single i.v. bolus via the femoral vein. Prior to nifedipine treatment, adenosine (1.2 mumol/kg per min) produced a significant (P less than 0.05) 2-3 fold increase in LAD flow. This response was reduced markedly (P less than 0.05) in a dose-dependent manner by nifedipine (6-20 microgram/kg). Following administration of an average dose of 11 microgram/kg nifedipine, adenosine (1.2 mumol/kg per min) failed to elevate LAD flow significantly. Further, reactive hyperemia, produced by releasing a 30-s occlusion of the LAD, was significantly attenuated by these same nifedipine concentrations. The nifedipine-mediated attenuation could be partially overcome by prolonging the period of occlusion (60 s), or by increasing the rate of adenosine infusion. These results could not be accounted for by a nifedipine-mediated alteration of hemodynamics and suggest the possibility of pharmacological competition between adenosine and nifedipine at a vascular smooth muscle receptor.

Acetaminophen in the Post-ischemia Reperfused Myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 ± 3, 27 ± 3, and 20 ± 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 ± 2 [P < 0.05], 51 ± 3, and 65 ± 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12–15 units/liter in the three groups. It increased to 35–40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 ± 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5–2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3–8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant–mediated cardioprotection in the postischemia, reperfused myocardium.

Acetaminophen reduces mitochondrial dysfunction during early cerebral postischemic reperfusion in rats

Acetaminophen, a popular analgesic and antipyretic, has been found to be effective against neuronal cell death in in vivo and in vitro models of neurological disorders. Acute neuronal death has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. The potential impact of acetaminophen on acute injury from cerebral ischemia-reperfusion has not been studied. We investigated the effects of acetaminophen on cerebral ischemia-reperfusion-induced injury using a transient global forebrain ischemia model. Male Sprague-Dawley rats received 15mg/kg of acetaminophen intravenously during ischemia induced by hypovolemic hypotension and bilateral common carotid arterial occlusion, which was followed by reperfusion. Acetaminophen reduced tissue damage, degree of mitochondrial swelling, and loss of mitochondrial membrane potential. Acetaminophen maintained mitochondrial cytochrome c content and reduced activation of caspase-9 and incidence of apoptosis. Our data show that acetaminophen reduces apoptosis via a mitochondrial-mediated mechanism in an in vivo model of cerebral ischemia-reperfusion. These findings suggest a novel role for acetaminophen as a potential stroke therapeutic.

Acetaminophen attenuates doxorubicin-induced cardiac fibrosis via osteopontin and GATA4 regulation: reduction of oxidant levels.

It is well documented in animal and human studies that therapy with the anti‐cancer drug doxorubicin (DOX) induces fibrosis, cardiac dysfunction, and cell death. The most widely accepted mechanism of cardiac injury is through production of reactive oxygen species (ROS), which cause mitochondrial damage, sarcomere structural alterations, and altered gene expression in myocytes and fibroblasts. Here we investigated the effects of acetaminophen (APAP, N‐acetyl‐para‐aminophenol) on DOX‐induced cardiac injury and fibrosis in the presence or absence of osteopontin (OPN). H9c2 rat heart‐derived embryonic myoblasts were exposed to increasing concentrations of DOX ± APAP; cell viability, oxidative stress, and OPN transcript levels were analyzed. We found a dose‐dependent decrease in cell viability and a corresponding increase in intracellular oxidants at the tested concentrations of DOX. These effects were attenuated in the presence of APAP. RT‐PCR analysis revealed a small increase in OPN transcript levels in response to DOX, which was suppressed by APAP. When male 10–12‐week‐old mice (OPN+/+ or OPN−/−) were given weekly injections of DOX ± APAP for 4 weeks there was substantial cardiac fibrosis in OPN+/+ and, to a lesser extent, in OPN−/− mice. In both groups, APAP decreased fibrosis to near baseline levels. Activity of the pro‐survival GATA4 transcription factor was diminished by DOX in both mouse genotypes, but retained baseline activity in the presence of APAP. These effects were mediated, in part, by the ability of APAP, acting as an anti‐inflammatory agent, to decrease intracellular ROS levels, consequently diminishing the injury‐induced increase in OPN levels. J. Cell. Physiol. 228: 2006–2014, 2013.

Chronically Administered Acetaminophen and the Ischemia/Reperfused Myocardium

Male and female Hartley strain guinea pigs weighing 280 ± 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 ± 4 vs 21 ± 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 ± 14 (baseline) to 78 ± 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 ± 8 and 44 ± 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.

Acetaminophen is cardioprotective against H2O2-induced injury in vivo.

Here we report our ongoing investigation of the cardiovascular effects of acetaminophen, with emphasis on oxidation-induced canine myocardial dysfunction. The objective of the current study was to investigate whether acetaminophen could attenuate exogenous H2O2-mediated myocardial dysfunction in vivo. Respiratory, metabolic, and hemodynamic indices such as left ventricular function (LVDP and ±dP/dtmax), and percent ectopy were measured in anesthetized, open-chest dogs during intravenous administration of 0.88 mM, 2.2 mM, 6.6 mM H2O2. Following 6.6 mM H2O2, tissue from the left ventricle was harvested for electron microscopy. Left ventricular function did not vary significantly between vehicle and acetaminophen groups under baseline conditions. Acetaminophen-treated dogs regained a significantly greater fraction of baseline function after high concentrations of H2O2 than vehicle-treated dogs. Moreover, the incidence of H2O2-induced ventricular arrhythmias was significantly reduced in the acetaminophen-treated group. Percent ectopy following 6.6 mM concentrations of H2O2 was 1 ± 0.3 vs. 0.3 ± 0.1 (P < 0.05) for vehicle- and acetaminophen-treated dogs, respectively. Additionally, electron micrograph images of left ventricular tissue confirmed preservation of tissue ultrastructure in acetaminophen-treated hearts when compared to vehicle. We conclude that, in the canine myocardium, acetaminophen is both functionally cardioprotective and antiarrhythmic against H2O2-induced oxidative injury.

Antiarrhythmic Properties of Acetaminophen in the Dog

Mongrel dogs bred for research and weighing 25 ± 3 kg were used to test the hypothesis that acetaminophen has antiar-rhythmic properties. Only ventricular arrhythmias defined by the Lambeth Conventions were investigated. Dogs were exposed either to 60 mins of regional myocardial ischemia followed by 180 mins of reperfusion (n = 14) or were administered a high dose of ouabain (n = 14). Both groups of 14 dogs were further divided into vehicle and acetaminophen treatment groups (n = 7 in each). During selected 10-min intervals, we recorded the numbers of ventricular premature beats, ventricular salvos, ventricular bigeminy, ventricular tachycardia (nonsustained and sustained), and we recorded the heart rate, systemic arterial blood pressure, and left ventricular function. Neither heart rate nor the number of ventricular arrhythmias differed significantly under baseline conditions. Conversely, the combined average number of ventricular ectopic beats during ischemia and reperfusion was significantly less in the presence of acetaminophen (28 ± 4 vs. 6 ± 1; P < 0.05). Similarly, percent ectopy during reperfusion in vehicle- and acetaminophen-treated dogs was 1.4 ± 0.4 and 0.4 ± 0.2, respectively (P < 0.05). The number of all ventricular ectopic beats except ventricular salvos was also significantly reduced in the presence of acetaminophen. Similar results were obtained with ouabain. Our results reveal that systemic administration of a therapeutic dose of acetaminophen has previously unreported antiarrhythmic effects in the dog.

Acetaminophen in the Hypoxic and Reoxygenated Guinea Pig Myocardium

We investigated the effects of 0.35-mM acetaminophen and its vehicle on isolated, perfused guinea pig hearts made hypoxic and subsequently reoxygenated. Hearts were allowed 30 min postinstrumentation to reach baseline, steady-state values, and then were exposed to 6 min of hypoxia (5% O2, 5% CO2, balance N2) followed by 36 min of reoxygenation (95% O2, 5% CO2). We recorded hemodynamic, metabolic, and mechanical data in addition to assessing ultrastructure and the capacity of coronary venous effluent to reduce reactive oxygen species. We found that acetaminophen-treated hearts retained a greater fraction of mechanical function during hypoxia and reoxygenation. For example, the average percentage change from baseline of left ventricular developed pressure in acetaminophen- and vehicle-treated hearts at 6 min reoxygenation was 9 ± 2% and -8 ± 5% (P < 0.05), respectively. In addition, electron micrographs revealed greater preservation of myofibrillar ultrastructure in acetaminophen-treated hearts. Biochemical analyses revealed the potential of coronary effluent from acetaminophen-treated hearts to significantly neutralize peroxynitrite-dependent chemiluminescence in all recorded time periods. During early reoxygenation, the percentage inhibition of peroxynitrite-mediated chemiluminescence was 56 ± 10% in vehicle-treated hearts and 99 ± 1% in acetaminophen-treated hearts (P < 0.05). We conclude that acetaminophen has previously unreported cardioprotective properties in the nonischemic, hypoxic, and reoxygenated myocardium mediated through the reduction of reactive oxygen species.