Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism.

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 microM) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of 3H2O from 5-3H-glucose); (2) oxidation of glucose and pyruvate (production of 14CO2 from U-14C-glucose, 1-14C-glucose, 3,4-14C-glucose, 1-14C-pyruvate, and 2-14C-pyruvate); and (3) oxidation of palmitate (release of 3H2O from 9,10-3H-palmitate). We found that addition of carnitine (50 microM) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids.

Ca(2+)-dependent mechanisms of cell injury in cultured cortical neurons.

The contributions of several Ca(2+)-dependent processes to neurotoxicity were examined in primary cultures of rat cortical neurons. The Ca2+ ionophore ionomycin induced a rapid loss of axonal morphology and concomitant release of inositol phosphates that preceded morphological alterations of neuronal cell bodies, choline and arachidonate release, and protein degradation. These events were followed by a degree of neuronal lysis proportional to the external Ca2+ concentration and exposure time. The phospholipase inhibitor neomycin decreased both arachidonate release and the phospholipid hydrolysis catalysed by phospholipases C and D. Proteolysis was abated by the protease inhibitor leupeptin, but not by lysosomal proteolysis inhibitors. Neuronal lysis was inhibited partially by either leupeptin or neomycin and almost completely by both in combination. However, neither agent, alone or in combination, affected the morphological derangements. The diacylglycerol lipase inhibitor RHC-80267 reduced arachidonate release, but not neuronal lysis. Phospholipase A2 inhibitors had no effect on either arachidonate release or lysis. Treatment of mixed cultures of neurons and glia with a Ca(2+)-dependent glutamate challenge caused similar morphological changes and a delayed neuronal lysis that was also diminished by leupeptin and neomycin, but not by inhibitors of lysosomal proteolysis. These data describe several distinct stages of Ca(2+)-dependent injury to cortical neurons, a key feature of which is the stimulation of protease, and phospholipase C and D activities. The initial stage is characterized by a rapid loss of axonal morphology and increased phosphatidylinositol hydrolysis. An intermediate stage involves changes in cell body morphology plus the degradation of neuronal protein and phosphatidylcholine. In a later stage, the loss of plasma membrane integrity denotes neuronal death.

Chlorpromazine protection against Ca(2+)-dependent and oxidative cell injury. Limitations due to depressed mitochondrial function.

Chlorpromazine (CPZ), a phenothiazine, demonstrated both cytoprotective and toxic effects on cardiomyocytes. CPZ markedly reduced cytotoxicity caused by two toxic challenges, each with a distinct cytotoxic mechanism. Lethal cell injury was induced in cultured neonatal cardiomyocytes by either: (1) ionomycin, a Ca2+ ionophore that caused Ca(2+)-dependent cell injury; or (2) ethacrynic acid (EA), a glutathione (GSH) depletor that killed cells primarily via peroxidative damage. Pretreatment with 50 microM CPZ reduced the extent of ionomycin-induced cell death, as measured by lactate dehydrogenase (LDH) leakage, but enhanced the loss of intracellular ATP and collapsed the mitochondrial transmembrane potential (delta psi). In EA-treated cultures, 50 microM CPZ also lowered LDH leakage and diminished the peroxidative damage responsible for the cytotoxicity, but again enhanced the loss of intracellular ATP and collapsed the delta psi. CPZ protection was incomplete and limited to a narrow concentration range that was essentially identical for both toxic challenges. Maximum protection was observed with 50 microM CPZ, yet the amount of residual damage was similar to the degree of injury caused by a mitochondrial uncoupler, carbonylcyanide-m-chlorophenylhydrazone alone. In the absence of either challenge, 50 microM CPZ did not affect cellular energy status or kill the cells, but a higher concentration of CPZ (150 microM) did deenergize unchallenged cardiomyocytes. These data demonstrate that CPZ can reduce cytotoxicity caused by either Ca(2+)-dependent events or oxidative stress. However, even at an optimally protective level, CPZ in combination with either ionomycin or EA deenergized the cells, although neither toxic challenge nor 50 microM CPZ alone seriously affected delta psi. It would appear that intracellular perturbations induced by either challenge promote a depression of mitochondrial function by CPZ, which limits the protective action of the drug. Since both of the challenges used contain toxicologic features exhibited by a wide variety of toxic insults, results of this study have both mechanistic and clinical implications.

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.

Microsomal activation of fluoranthene to mutagenic metabolites.

The in vitro metabolism of fluoranthene (FA) was assessed by incubating 3-[3H]FA, the synthesis of which is described, with rat hepatic microsomal enzymes. Several metabolites including the FA 2,3-diol, FA 2-3,-quinone, 3-OH-FA, 1-OH-FA, and 8-OH-FA were isolated by high-pressure liquid chromatography and identified by comparison of chromatographic properties and uv-visible spectra with those of synthetic standards. The major metabolite produced over the FA concentration range studied (23-233 microM) was FA 2,3-diol, accounting for 29-43% of the total extractable metabolites. This diol was characterized further by high-resolution mass spectroscopy and H-NMR and determined to be identical in structure to the trans-2,3-dihydroxy-2,3-dihydrofluoranthene. The FA 2,3-diol, syn and anti 2,3-diol-1,10b-epoxides, FA 2,3-quinone, and FA 7,8-diol were all shown to be mutagenic toward Salmonella typhimurium TM677. The FA 1,10b-diol and syn and anti FA 1,10b-diol-2,3-epoxides were not mutagenic. The epoxide hydrolase inhibitor, 3,3,3-trichloropropylene oxide, markedly reduced the mutagenic potency of FA while concurrently inhibiting FA 2,3-diol production but not overall FA metabolism. These results suggests that a major metabolic activation pathway of FA resulting in the production of mutagenic species involves the formation of the FA 2,3-diol and the subsequent oxidation of this diol to a FA 2,3-diol-1,10b-epoxide. Another minor activation pathway with mutagenic endpoints may involve the formation of the 7,8-diol.

In vitro DNA-binding of microsomally-activated fluoranthene: evidence that the major product is a fluoranthene N2-deoxyguanosine adduct.

Incubation of 3-[3H]fluoranthene with a rat liver microsomal activation system in the presence of calf thymus DNA resulted in radioactivity bound to the DNA. The fluoranthene-modified DNA was enzymatically digested and a DNA adduct elution profile developed using h.p.l.c. The fraction containing the major fluoranthene--DNA adduct was further purified by h.p.l.c. and separated into two subfractions. Treatment of these with perchloric acid liberated guanine in both cases. Evidence that both were N-2 guanine derivatives was based on the pK values of these adducts determined before and after treatment with nitrous acid. The major adduct (70% of total modified deoxyribonucleosides) was further characterized by high resolution, fast atom bombardment mass spectroscopy which yielded a molecular ion consistent with a fluoranthene triol bound to the N-2 position of deoxyguanosine. Synthetic syn and anti 3-[3H]2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene were reacted directly with DNA and the 8-[3H]-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrofluoranthene was incubated with DNA and microsomes. The DNA from each of these reactions was enzymatically hydrolyzed and h.p.l.c. adduct profiles were developed. The major adduct formed from reaction of the anti 2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene with DNA and the major N-2 fluoranthene derived adduct had identical elution times on two different h.p.l.c. systems, similar pK values (before and after nitrous acid treatment) and the same u.v. spectra. In addition, derivatization of both adducts with ethyl methanesulfonate yielded identical products, as determined by h.p.l.c. analysis.

Glutathione conjugation and DNA-binding of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes.

Isolated rat liver hepatocytes, previously depleted of glutathione (GSH) by treatment with diethylmaleate, were allowed to incorporate [3H]glycine into their GSH. Incubation of 3H-labelled cells with 14C-labelled (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ((+/-)-BP-7,8-dihydrodiol) or (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+/-)-BPDE) revealed the formation of double labelled products. This together with evidence from amino acid analysis indicates formation of GSH-conjugates of the highly carcinogenic BP-derivatives. Incubation of hepatocytes isolated from 3-methylcholanthrene (MC) treated rats with 3H-labelled (+/-)-BP-7,8-dihydrodiol or (+/-)-BPDE resulted in binding of radioactivity to DNA. Reduction of the intracellular level of GSH to approximately 40% of the normal level resulted in an approximate 2-fold increase in the DNA-binding of either substrate. In addition there was a concurrent decrease in the amount of GSH-conjugates formed. These data clearly demonstrate that GSH participates in conjugation reactions with carcinogenic (+/-)-BP-7,8-dihydrodiol and (+/-)-BPDE and that the intracellular level of GSH is important in preventing reactive intermediates from reacting with the DNA in intact cells.

Active site specific inactivation of chymotrypsin by cyclohexyl isocyanate formed during degradation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Prolonged incubation of 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea with chymotrypsin resulted in covalent modification and concomitant inactivation of chymotrypsin via degradation of the nitrosourea to form cyclohexyl isocyanate. Cyclohexyl isocyanate was shown to be an active-site-specific inactivator of chymotrypsin. A cyclohexyl isocyanate to enzyme molar ratio of 0.63 was required to produce 50% enzyme inactivation, thus demonstrating the high specificity of inactivation. At 2.38 X 10(-4) M chymotrypsin this near stoichiometric inactivation was not significantly affected by the presence of 1, 5, and 10 mM L-lysine. Degradation of an excess of 1-(2-chloroethyl)-3-([1-14C]-cyclohexyl)-1-nitrosourea in the presence of enzyme yielded 1.11 +/- 0.07 mol of covalently bound [14C]cyclohexyl moiety per mol of enzyme inactivated. Short-term incubation demonstrated that the nitrosourea neither inhibited nor protected the enzyme from cyclohexyl isocyanate inactivation. Treatment of chymotrypsin with less than stoichiometric amounts of cyclohexyl isocyanate or titration of the active-site serine with phenylmethanesulfonyl fluoride followed by in situ degradation of excess 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea resulted in a decreased amount of covalently bound 14C proportional to the extent of inactivation by these reagents prior to 14C labeling. These results strongly suggest that cyclohexyl isocyanate, whether added directly or generated by CCNU degradation, reacted almost exclusively with the active site of the enzyme. The extent of this inactivation indicates that 70% of the CCNU degraded in such a manner as to form cyclohexyl isocyanate.