Alan B. Combs

Prevention of acetaminophen-induced hepatotoxicity by dimethyl sulfoxide.

Dimethyl sulfoxide (DMSO) has previously been shown to protect against acetaminophen (APAP)-induced hepatotoxicity, but the mechanism of this effect was not clear. Treatment of mice with 1 mg/kg DMSO 4 h before 250 mg/kg APAP resulted in significantly less hepatotoxicity than with APAP alone, as measured by serum glutamic pyruvic transaminase (SGPT) content 24 h after APAP. Protection was also evident when 1 ml/kg DMSO was given 4, but not 8 h after 250 mg/kg APAP. The APAP-induced depletion of liver glutathione was prevented in mice pretreated with DMSO, although DMSO alone had no effect on liver glutathione levels. The hepatic concentration of cytochrome P-450 (P450) 4 h after treatment of mice with 1 ml/kg DMSO, was significantly decreased compared to saline-treated animals. However, while this DMSO pretreatment significantly decreased the activity of cytochrome P-450-linked aminopyrine-N-demethylase, it increased the activity of aniline hydroxylase. Covalent binding of [14C]APAP to hepatic protein in vivo was significantly decreased in mice pretreated with DMSO. Covalent binding of [14C]APAP to hepatic microsomal protein in vitro was not significantly altered after in vivo treatment with DMSO. However, the presence of DMSO in the in vitro incubation mixture significantly decreased covalent binding of [14C]APAP in a dose-dependent manner compared to microsomal fractions from untreated, saline-treated or DMSO pretreated animals. These data suggest that the DMSO-induced alterations in cytochrome P-450 content and activity may not be the cause of the observed protective action of this chemical. The ability to competitively inhibit APAP bioactivation or to directly scavenge free radicals produced during APAP metabolism, including the activated species which covalently binds to protein, may account for the hepatoprotection afforded by DMSO.

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.

In vivo effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and doxorubicin on the cardiac and hepatic glutathione systems

Doxorubicin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) are anti-cancer drugs which have been used together in combination therapy of certain cancers. Each drug has been reported to affect intracellular glutathione stores and together, doxorubicin and BCNU have been shown to exert synergistic toxicity and to deplete completely the glutathione content of isolated hepatocytes. Cardiac and hepatic glutathione reductase activity was significantly inhibited following treatment in vivo with BCNU. Treatment of mice with both doxorubicin and BCNU resulted in increased mortality compared to either drug alone. There was, however, no depletion of hepatic or cardiac glutathione levels in vivo beyond that seen with either BCNU or doxorubicin alone. Diethyl maleate, a known glutathione depletor whose effects are enhanced by BCNU in vitro, also was unable to increase GSH depletion after BCNU in vivo. These discrepancies between in vivo and in vitro studies may be due to the presence of more effective compensatory mechanisms in the whole animal, or to differences in the metabolism and inactivation of these drugs.

Effects of pharmacological interventions on emetine cardiotoxicity in isolated perfused rat hearts.

The cardiotoxicity of emetine continues to be a significant clinical problem. The purpose of this study was to investigate the effect of several mechanistic interventions, including ICRF-187, an iron-chelating agent which protects against doxorubicin toxicity, atropine, and fructose-1,6-bisphosphate (FBP) on the toxicity of emetine in our isolated, perfused rat heart model. The model includes functional, electrocardiographic, and biochemical determinations in the same preparation. Atropine and ICRF-187 had no effect on the time needed for emetine to induce ventricular asystole, while FBP significantly increased this time. Administration of 47 microM atropine, 300 microM FBP, or 1 mM FBP decreased the release of lactate dehydrogenase (LDH) into the coronary effluent, while ICRF-187 had no effect. These pharmacological interventions variably changed the amplitude of the biphasic response of the coronary flow to emetine. Finally, FBP was very effective in slowing the rate of QRS-waveform degeneration in the perfused hearts. Emetine caused PR- and QRS-prolongation which was not altered by FBP.

Emetine inhibits glycolysis in isolated, perfused rat hearts

This work was designed to test whether phosphofructokinase is a target for emetine action on the heart. The effects of 37, μM emetine on the activities of phosphofructokinase and hexokinase were measured in homogenates from perfused hearts. The action of increasing concentrations of emetine was determined in nonperfused heart homogenates. The effect of 37 μM emetine or control solutions on the concentration of fructose-6-phosphate and fructose-1,6-phosphate was measured. The effect of 37 μM emetine or control perfusion on the utilization of fructose-6-phosphate by phosphofructokinase in centrifugation supernatants of homogenates and in reconstituted 27,000g pellets was measured. Double-reciprocal plots of fructose-6-phosphate concentrations vs phosphofructokinase activities were plotted. Emetine decreased phosphofructokinase activity in homogenates from both perfused and nonperfused hearts. Emetine did not inhibit cardiac hexokinase activity. In homogenates from nonperfused hearts, the maximal inhibition with high concentrations of emetine was approx 50%. Emetine perfusion caused a simultaneous increase in the phosphofructokinase substrate fructose-6-phosphate and a decrease in the phosphofructokinase product fructose-1,6-bisphosphate. Phosphofructokinase and, consequently, glycolytic flux appear to be subcellular targets for emetine in the heart. Homogenate centrifugation studies indicate that emetine acts on bound rather than unbound phosphofructokinase. The inhibition may be uncompetitive in nature.

Influence of in vitro ubiquinone antagonists on doxorubicin toxicity in vivo

Doxorubicin is an anthracycline antibiotic with a very wide spectrum of anticancer activity. It has a great potential for clinical cardiotoxicity, however. One mechanism suggested for the cardiotoxicity is inhibition of ubiquinone-dependent enzymes. It was our purpose to study this possible mechanism using ubiquinone antagonists as probes. The effect on doxorubicin toxicity of three in vitro ubiquinone antagonists was tested in mice. Two of the antagonists, 2-hydroxy-3-n-dodecylmercapto-1,4-naphthoquinone and 2,3-dimethoxy-5-beta-naphthylmercapto-1,4-benzoquinone, enhanced doxorubicin toxicity in vivo as measured by survival. The latter was significantly toxic to mice, by itself. This effect was completely blocked by ubiquinone pretreatment, but only reduced by tocopherol pretreatment. Neither ubiquinone nor tocopherol was able to decrease the toxic interaction between doxorubicin and either of the ubiquinone antagonists. Cardiac and hepatic glutathione reductase and glutathione peroxidase activities were measured in studies using the 2,3-dimethoxy-5-beta-naphthylmercapto-1,4-benzoquinone. This compound appeared to cause a slight reduction in the activity of hepatic glutathione reductase. It appears that these antagonists are not useful to probe the relationship between doxorubicin cardiotoxicity and ubiquinone enzyme inhibition.

Effect of formulation factors on the matrix pH of nylon microcapsules

The application of nylon microencapsulation as a drug delivery system inherently demands that the microencapsulated matrix should not cause degradation of the encapsulated drug. The presence of alkaline hexamethylenediamine (HMD) in the microcapsule core will affect the final pH of the microcapsules and may influence the stability of some drugs. To follow the pH within the microcapsule core during manufacture, pH indicators were encapsulated. The final pH was found to depend on the formulation used and could be controlled by the addition of acid. Several other variables affecting the nylon wall formation were examined to determine optimum processing conditions. Increased agitation produced a decrease in microcapsule size. This cause an increase in nylon weight recovered. The increased recovery of nylon was due in part to nylon formation over a larger surface area. Varying the amounts of HMD and sebacyl chloride (SC) used also affected the total weight of nylon formed. In general, more nylon is recovered as the level of each reactant increases. However, the molar ratio of HMD:SC determined the total amount of nylon formed when SC was present in excess.

In vitro photodecomposition of uric acid in the presence of riboflavin

Abstract When uric acid solutions containing riboflavin-5′-phospate were exposed to visible light at ambient temperature, the concentration of uric acid decreased as monitored by measuring the change in uric acid absorption at 288 nm. Increasing the concentration of riboflavin appeared to increase the rate of disappearance of uric acid. The presence of potassium iodide in the reaction mixture retarded this photodecomposition.

Inexpensive Apple Macintosh-Based Electrocardiography in Small Animals

SummaryThe MacRecorder is a device used for recording digitized sound files by the Apple Macintosh line of microcomputers. Rather than recording sounds, the procedure described in this article involves the use of the MacRecorder as an analog-to digital converter to record electrocardiograms (ECGs) in rats and mice. This is much less expensive than is the usual use of costly analog-to-digital converter boards mounted in high-performance computers. Conical, wire cages were constructed to immobilize the animals and simultaneously to act as shields against electrical noise. Electrodes were placed subcutaneously in an animal. Then, the electrode wires were connected to the external microphone jack of the MacRecorder. In this manner, the MacRecorder digitized and recorded the electrical activity produced by the animals heart, rather than the electrical activity produced by a microphone. Simple techniques for manipulation of the resulting data files and for measurement of the ECG data were used. Demonstration d...