Donald E. Richards

Arachidonic acid disrupts calcium dynamics in neonatal rat cardiac myocytes

OBJECTIVES The purpose of this study was to investigate the effects of prolonged arachidonic acid (AA) exposure on electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in cardiac myocytes and to identify intracellular biochemical events that may play a role in the actions of AA on [Ca2+]i dynamics. METHODS Electrically induced [Ca2+]i transients were investigated in cultured single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-[Ca2+]i binding. KCl-induced depolarization, caffeine and ryanodine were used to assess the effects of AA on Ca2+ handling by the sarcolemma and the sarcoplasmic reticulum. Prostanoid formation was measured with an ELISA technique. alpha-Tocopherol was used to determine if free radical formation was a factor in the AA effects on [Ca2+]i. RESULTS Exposure to 10-30 microM AA produced a concentration-dependent and reversible configuration change and eventually a cessation of [Ca2+]i transients. Continued exposure resulted in a Ca2+ overload (tonic [Ca2+]i greater than peak systolic [Ca2+]i). AA did not influence KCl-induced [Ca2+]i increase but did eliminate caffeine-induced [Ca2+]i transients. AA exposure stimulated the formation of 6-oxo-prostaglandin F1 alpha in a concentration-dependent manner, but thromboxane B2 formation was not influenced. alpha-Tocopherol pretreatment significantly delayed times till cessation of [Ca2+]i transients and Ca2+ overload, whereas ryanodine and cyclo-oxygenase inhibitors were without effect. CONCLUSIONS The present data provide evidence that the initial action of AA on [Ca2+]i transients during excitation-contraction coupling involves an effect of AA on sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ handling. AA-induced cessation of electrically induced [Ca2+]i transients and Ca2+ overload may involve the formation of free radicals.

Metabolism of bis(2-methoxyethyl) ether in the adult male rat: evaluation of the principal metabolite as a testicular toxicant.

The metabolism of the reproductive toxicant bis(2-methoxyethyl) ether was studied in male Sprague-Dawley rats, and the principal metabolite (2-methoxyethoxy)acetic acid and its metabolic precursor 2-(2-methoxyethoxy)ethanol were evaluated separately as testicular toxicants. For the metabolism study, rats were given single po doses of [1,2-ethylene-14C]bis(2-methoxyethyl) ether at 5.1 or 0.051 mmol/kg body wt. Within 96 hr, approximately 86 to 90% of the radioactivity was excreted in the urine. Urinary metabolites were separated by high-performance liquid chromatography and isolated for characterization by gas chromatography-mass spectrometry. The principal urinary metabolite, accounting for 67.9 +/- 3.3% of the administered high dose and 70.3 +/- 1.3% of the low dose, was identified as (2-methoxyethoxy)acetic acid. A second metabolite, representing 6.2 +/- 0.8% of the high dose and 5.8 +/- 0.8% of the low dose, was identified as methoxyacetic acid, a previously recognized testicular toxicant. In the toxicity study, (2-methoxyethoxy)acetic acid and 2-(2-methoxyethoxy)ethanol were administered to rats at 5.1 mmol/kg body wt by gavage as single daily doses for as many as 20 consecutive days. The testes of rats killed 24 hr after the administration of even numbered doses showed no gross or microscopic abnormalities. These results are in contrast to the previously reported testicular atrophy evoked after as few as 8 daily doses of the parent compound, bis(2-methoxyethyl) ether, tested under the same experimental conditions. Thus, the testicular toxicity reported for bis(2-methoxyethyl) ether could be explained by the presence of a minor metabolite, methoxyacetic acid.

Metabolism of ortho-, meta-, and para-toluidine in the adult male rat☆

Abstract The major route of excretion of 14C following the administration of a single oral 50 mg/kg dose of ortho-[methyl-14C]toluidine hydrochloride to male Sprague-Dawley rats was found to be urinary. Greater than 92% of the 14C was eliminated by this route within 24 hr of dosing. After treatment of rats with a single oral 500 mg/kg dose of ortho-, meta-, or para-toluidine, aminomethylphenols were excreted as acid-hydrolyzable conjugates. Two previously unreported urinary metabolites, 2-amino-4-methylphenol from meta-toluidine and 2-amino-5-methylphenol from para-toluidine, were identified in urine hydrolysates. In addition, unchanged toluidine was eliminated in amounts which varied with the isomer: 21% for ortho-; 2.5%, meta-; and 2.5%, para-toluidine. These differences may account for the previously reported observation that only the ortho-isomer causes tumors in the urinary bladder of the rat.

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: effects of ethanol on in vitro metabolism.

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190–220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [14C]diglyme at 1, 10, 30, and 50 μM for up to 48 h. For the in vivo study, rats were given single oral doses of [14C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme. Pretreatment of rats with ethanol resulted in a marked increase in the overall in vitro metabolism of diglyme. The major metabolic pathways for diglyme involve O-demethylation and cleavage of the central ether bond, and it is the latter pathway that leads to the formation of MAA, the metabolite associated with the reproductive and developmental toxicity of diglyme. The amounts of MAA formed in hepatocytes from ethanol-pretreated rats ranged from two to four times those formed in hepatocytes from untreated rats.

Bis(2-methoxyethyl) ether : metabolism and embryonic disposition of a developmental toxicant in the pregnant CD-1 mouse

An embryotoxic oral dose of bis(2-methoxyethyl) ether (DGDME), 3.73 mmol/kg body wt (500 mg/kg), administered on the 11th day of gestation to pregnant CD-1 mice was metabolized predominantly by O-demethylation to 2-(2-methoxyethoxy)ethanol with subsequent oxidation to (2-methoxyethoxy)acetic acid. Urinary excretion of this metabolite over 48 hr amounted to 63 +/- 2% of the dose. A smaller percentage of the administered dose was metabolized at the central ether linkage to produce 2-methoxyethanol, which was further metabolized by alcohol dehydrogenase to methoxyacetic acid. Urinary excretion of methoxyacetic acid, a potent developmental toxicant, amounted to 28 +/- 1% of the administered dose by 48 hr and was the second most prominent urinary metabolite. Unchanged DGDME and methoxyacetic acid were detected in the embryonic tissues from these animals, and embryos harvested after the initial 6-hr period showed detectable amounts of only methoxyacetic acid. The average amount of methoxyacetic acid per embryo was calculated to be 1.5 +/- 1.0 mumol (5.9 mmol/kg body wt) at the 6-hr termination time. This finding suggests that the reported teratogenic effects of DGDME are due to methoxyacetic acid formed, either in the fetus or by hepatic metabolism in the dam with subsequent distribution to the embryonic tissue. These results suggest that such developmental toxicity may occur with structurally similar aprotic ethylene glycol ethers in which metabolic O-dearylation would yield 2-methoxy-ethanol.

Testicular effects of bis(2-methoxyethyl) ether in the adult male rat.

The onset of testicular pathology in the rat and possible recovery over an 8-week period were evaluated after the administration of up to 20 daily oral doses of bis(2-methoxyethyl) ether (diglyme) at 5.1 mmol/kg bw (684 mg/kg bw). Primary and secondary spermatocyte degeneration and spermatidic giant cells were observed after six to eight treatments. In addition, the testes-to-body weight ratio was significantly reduced by the tenth day of treatment and continued to be depressed eight weeks after discontinuation of the treatment. Testicular LDH-X activity, a pachytene spermatocyte marker enzyme, was significantly decreased in animals by the eighteenth day of treatment with diglyme.

Calcium dynamics in cardiac myocytes as a target of dichloromethane cardiotoxicity.

The purpose of the present study was to determine if cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca2+ dynamics in cardiac myocytes. Neonatal rat ventricular myocytes were obtained from 2- to 4-day-old rats, and electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were investigated using spectrofluorometric analysis of fura-2-[Ca2+]i binding. In cultured myocytes, cumulative exposure to 0.64–40.96 mM DCM resulted in a concentration-dependent and reversible decrease in the magnitude of [Ca2+]i transients with IC10 and IC50 values of 7.98 and 18.82 mM, respectively. Total inhibition of [Ca2+]i transients and cessation of beating were observed at 40.96 mM DCM. Suffusion with DCM for 40 min did not cause morphological alterations of the myocytes. In a urethane-anesthetized rat model, left ventricular pressure was measured by introducing a tip catheter via the carotid artery into the left ventricle, the ECG was recorded by two needle electrodes applied subcutaneously to the chest wall, and arterial pressure was measured via the femoral artery. Oral administration of 3.1–12.4 mmol DCM/kg resulted in DCM blood concentrations between 1.0 and 1.6 mM, accompanied by a dose-dependent decrease in contractile force and heart rate without influencing blood pressure and ECG tracings. Moreover, DCM treatment provided significant protection against arrhythmia development due to CaCl2-infusion. In spite of the slight discrepancy between DCM blood concentrations and in vitro concentrations of DCM for [Ca2+]i transient inhibition, present data are consistent with the view that cardiac effects after DCM exposure are mediated by alterations of Ca2+ dynamics during excitation-contraction coupling.

H2O2‐induced oxidative injury in rat cardiac myocytes is not potentiated by 1,1,1‐trichloroethane, carbon tetrachloride, or halothane

Free radical-induced oxidative stress has been linked to ischemia-reperfusion injury of the myocardium. The .OH radical is considered the most damaging radical and can be increased in cells by treatment in vitro with H2O2. The purpose of the present study was to determine if aliphatic halocarbons enhance H2O2-induced oxidative injury in isolated cardiac myocytes from neonatal rats. Oxidative damage was assessed by measuring release of thiobarbituric acid-reactive substances (TBARS) from lipid peroxidation, loss of lactate dehydrogenase (LDH) through damaged sarcolemmal membranes, and alterations in intracellular calcium ([Ca2+]i) transients in electrically stimulated (1 Hz, 10 ms, 60 V) myocytes. H2O2 increased TBARS release and LDH leakage in a concentration-dependent (20-200 microM) manner. Continuous suffusion with H2O2 first altered the configuration of [Ca2+]i transients, then eliminated them, and finally caused [Ca2+]i overload (basal [Ca2+]i exceeded peak systolic [Ca2+]i of control). The time to [Ca2+]i overload was inversely associated with concentration, and the shortest time to overload was obtained with 100 microM H2O2. A 1-h preincubation of myocytes with the iron chelator deferoxamine inhibited all effects of H2O2. 1,1,1-Trichloroethane, carbon tetrachloride, or halothane at 1 mM significantly and reversibly reduced [Ca2+]i transients but did not influence TBARS release or LDH leakage. Simultaneous exposure of myocytes to H2O2 and halocarbons did not affect the myocyte response to H2O2 exposure. Results indicate that the three halocarbons tested do not enhance H2O2-induced oxidative injury in isolated cardiac myocytes.

The acute oral toxicity of isomeric monobutylamines in the adult male and female rat

Abstract The acute oral LD 50 values of n -butylamine, isobutylamine, sec. -butylamine, and tert. -butylamine were determined in both male and female Sprague-Dawley CD rats. Signs of toxicity observed after single oral doses of the monobutylamines included sedation, ataxia, nasal discharge, gasping, and salivation followed by convulsions and death at the higher dose levels. Gross pathological examination of animals that died after the monobutylamine treatment revealed pulmonary edema. The LD 50 values for the monobutylamines were calculated by the probit method of D. J. Finney (1971, Probit Analysis , 3rd ed., Cambridge Univ. Press, Cambridge). No significant sex-related differences were noted. The 14-day, po single-dose LD 50 values (mg/kg body wt) were: n -butylamine, male 365.4, female, 382.7; isobutylamine, male, 224.4, female, 231.8; sec. -butylamine, male, 157.5, female, 146.8; and tert. -butylamine, male, 82.3, female, 78.1.

Cardiotoxicity of dichloromethane in rats and in cultured rat cardiac myocytes.

The purpose of the present study was to examine whether cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca(2+) dynamics in cardiac myocytes. Electrically induced fluctuations of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) were investigated in neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2 binding. [Ca(2+)](i) transients were inhibited in a concentration-dependent and reversible manner with IC(10) and IC(50)values of 3.2 and 18.1 mm. Complete inhibition of [Ca(2+)](i) transients and cessation of beating were observed at 40.96 mm without morphological alterations. Left ventricular pressure in urethane-anaesthetized rats was measured by introducing a tip catheter by way of the carotid artery into the left ventricle and ECG (lead II) was recorded by two needle electrodes. Administration of 3.1, 6.2 or 12.4 mmol DCM/kg orally resulted in DCM blood concentrations between 1.0 and 1.6 mm accompanied by a dose-dependent decrease of contractility parameters. Moreover, DCM administration provided protection against arrhythmia development due to CaCl(2) infusion. These observations are consistent with the view that both the negative inotropic effects of DCM and the protection from CaCl(2)-induced arrhythmia are mediated by an inhibition of Ca(2+) dynamics in cardiomyocytes.