Elisa Dicker

Reproducibility and Complications in Gene Searches: Linkage on Chromosome 6, Heterogeneity, Association, and Maternal Inheritance in Juvenile Myoclonic Epilepsy

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.

The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes

Abstract The NADPH-dependent oxidation of ethanol by rat liver microsome preparations was studied in the presence of azide to inhibit the peroxidatic activity of catalase. Dimethylsulfoxide, benzoate, mannitol and thiourea, four compounds that react rapidly with hydroxyl radicals, each inhibited the oxidation rate of ethanol. Inhibition was competitive with respect to ethanol. In contrast, urea, a compound that reacts poorly with hydroxyl radicals, was essentially without effect. Dimethylsulfoxide at concentrations that inhibited the oxidation of ethanol had no effect on the xanthine oxidase-mediated oxidation of ethanol or on aniline hydroxylase or aminopyrine demethylase activity of microsomes. These results suggest that ethanol oxidation by microsomes can be dissociated from drug metabolism and that the mechanism of ethanol oxidation may involve, in part, the interaction of ethanol with hydroxyl radicals that are generated by microsomes during the oxidation of NADPH.

Malic enzyme 2 may underlie susceptibility to adolescent-onset idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescent-onset IGEs. We report a single-nucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both case-control and family-based association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2-centered nine-SNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1; 95% confidence interval 2.9-12.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genome-coded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present.

Increased oxygen radical-dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption.

Enzymatic and nonenzymatic mixed‐function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of the added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence or presence of several ferric complexes. The inactivation of enzymes was enhanced by the addition of menadione or paraquat to the microsomes, and rates of inactivation were higher with the microsomes from the ethanol‐fed rats. The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.—Dicker, E.; Cederbaum, A. I. Increased oxygen radical‐dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption. FASEB J. 2: 2901‐2906; 1988.

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats.

Abstract A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.

NADH-dependent generation of reactive oxygen species by microsomes in the presence of iron and redox cycling agents

NADH was found previously to catalyze the reduction of various ferric complexes and to promote the generation of reactive oxygen species by rat liver microsomes. Experiments were conducted to evaluate the ability of NADH to interact with ferric complexes and redox cycling agents to catalyze microsomal generation of potent oxidizing species. In the presence of iron, the addition of menadione increased NADPH- and NADH-dependent oxidation of hydroxyl radical (.OH) scavenging agents; effective iron complexes included ferric-EDTA, -diethylenetriamine pentaacetic acid, -ATP, -citrate, and ferric ammonium sulfate. The stimulation produced by menadione was sensitive to catalase and to competitive .OH scavengers but not to superoxide dismutase. Paraquat, irrespective of the iron catalyst, did not increase significantly the NADH-dependent oxidation of .OH scavengers under conditions in which the NADPH-dependent reaction was increased. Menadione promoted H2O2 production with either NADH or NADPH; paraquat was stimulatory only with NADPH. Stimulation of H2O2 generation appears to play a major role in the increased production of .OH-like species. Menadione inhibited NADH-dependent microsomal lipid peroxidation, whereas paraquat produced a 2-fold increase. Neither the control nor the paraquat-enhanced rates of lipid peroxidation were sensitive to catalase, superoxide dismutase, or dimethyl sulfoxide. Although the NADPH-dependent microsomal system shows greater reactivity and affinity for interacting with redox cycling agents, the capability of NADH to promote menadione-catalyzed generation of .OH-like species and H2O2 or paraquat-mediated lipid peroxidation may also contribute to the overall toxicity of these agents in biological systems. This may be especially significant under conditions in which the production of NADH is increased, e.g. during ethanol oxidation by the liver.

Ethanol oxidation by isolated hepatocytes from ethanol-treated and control rats; factors contributing to the metabolic adaptation after chronic ethanol consumption

Abstract Chronic consumption of ethanol by rats produced a fatty liver and resulted in a pronounced increase in the rate of ethanol oxidation by isolated hepatocytes. Despite the increase in ethanol oxidation, oxygen consumption with several substrates was not enhanced after chronic ethanol treatment. Ouabain, an inhibitor of the (Na + + K + )-ATPase activity, did not abolish the increase in the rate of ethanol oxidation. About 40–50 per cent of the increase in ethanol oxidation persisted after inhibition of alcohol dehydrogenase, mitochondrial oxygen consumption or the malate-aspartate shuttle. The addition of substrates for the malate-aspartate shuttle slightly increased the rate of ethanol oxidation in hepatocytes from control and ethanol-treated animals. The increased rate of ethanol oxidation was not abolished by the uncoupling agent dinitrophenol. which by itself had little effect on ethanol oxidation. In the presence of aspartate or α-glycerophosphate, dinitrophenol augmented the rate of ethanol oxidation; in the presence of glutamate, the rate of ethanol oxidation was doubled by dinitrophenol. However, the higher rate of ethanol oxidation after ethanol consumption was still found in the presence of various combinations of substrate shuttles, with or without dinitrophenol. Pathways independent of alcohol dehydrogenase may contribute, at least in part, to the increase in ethanol oxidation found after chronic ethanol consumption. It is concluded that ethanol oxidation may be enhanced after chronic ethanol consumption without the estabishment of a hypermetabolic state of the liver.

Effect of cyanamide on the metabolism of ethanol and acetaldehyde and on gluconeogenesis by isolated rat hepatocytes

Abstract Previous experiments demonstrated that acetaldehyde stimulated glucose production from pyruvate, whereas gluconeogenesis from glycerol, xylitol and sorbitol was inhibited [A.I. Cederbaum and E. Dicker, Archs Biochem. Biophys . 197 , 415 (1979)]. To determine the mechanism whereby acetaldehyde affects glucose production from these precursors, and to evaluate the role of acetaldehyde in the actions of ethanol, experiments with cyanamide were carried out. The oxidation of acetaldehyde by isolated rat liver cells was inhibited by cyanamide after a brief incubation period. Associated with this inhibition of acetaldehyde oxidation was an inhibition of ethanol oxidation by cyanamide and an increase in the amount of acetaldehyde which arose during the oxidation of ethanol. Ethanol oxidation was decreased because of the ineffective removal of acetaldehyde in the presence of cyanamide. Cyanamide had no effect on hepatic oxygen uptake. The increase in the β-hydroxybutyrate/acetoacetate ratio produced by acetaldehyde was completely prevented by cyanamide, whereas the slight increase in the lactate/pyruvate ratio was not prevented by cyanamide. Cyanamide partially reversed the ethanol-induced increase in the lactate/pyruvate ratio, but it completely prevented the ethanol-induced increase in the β-hydroxybutyrate/acetoacetate ratio. The ethanol-induced change in the mitochondrial redox state may, therefore, be due primarily to the mitochondrial oxidation of the acetaldehyde which arises during the oxidation of ethanol. The inhibitory effects of acetaldehyde on gluconeogenesis from glycerol, xylitol and sorbitol, as well as the stimulation of acetaldehyde of glucose production from pyruvate, were completely prevented by cyanamide. These results indicate that the effects of acetaldehyde on gluconeogenesis represent metabolic effects, rather than direct effects of acetaldehyde. Changes in the cellular NADH/NAD − ratio as a consequence of acetaldehyde metabolism are postulated to be responsible for these actions of acetaldehyde. Ethanol stimulated glucose production from pyruvate, while inhibiting gluconeogenesis from glycerol, xylitol and sorbitol. Cyanamide, which prevented the effects of acetaldehyde on gluconeogenesis, also prevented the effects of ethanol on gluconeogenesis. This prevention by cyanamide may be suggestive for a role for acetaldehyde in the actions of ethanol on gluconeogenesis. The possibility cannot be ruled out, however, that the prevention of the effects of ethanol by cyanamide may be due to the partial inhibition of ethanol oxidation by cyanamide. These results indicate that cyanamide is an effective inhibitor of acetaldehyde oxidation by isolated liver cells and therefore can be used to determine the mechanism whereby acetaldehyde affects metabolic function. Depending on the reaction under investigation, acetaldehyde can have direct or indirect effects on cellular metabolism.

Increased NADH-dependent production of reactive oxygen intermediates by microsomes after chronic ethanol consumption : comparisons with NADPH

Microsomes from chronic ethanol-fed rats were previously shown to catalyze the NADPH-dependent production of reactive oxygen intermediates at elevated rates compared to controls. Recent studies have shown that NADH can also serve as a reductant and promote the production of oxygen radicals by microsomes. The current study evaluated the influence of chronic ethanol consumption on NADH-dependent microsomal production of reactive oxygen intermediates, and compared the results with NADH to those of NADPH. Microsomal oxidation of chemical scavengers, taken as a reflection of the production of hydroxyl radical (.OH)-like species was increased about 50% with NADH as cofactor and about 100% with NADPH after chronic ethanol consumption. The potent inhibition of the production of .OH-like species by catalase suggests a precursor role for H2O2 in .OH production. Rates of NADH- and NADPH-dependent H2O2 production were increased by about 50 and 70%, respectively, after chronic ethanol consumption. A close correlation between rates of H2O2 production and generation of .OH-like species was observed for both NADH and NADPH, and increased rates of H2O2 production appear to play an important role in the elevated generation of .OH-like species after chronic ethanol treatment. Microsomal lipid peroxidation was elevated about 60% with NADH, and 120% with NADPH, after ethanol feeding. With both types of microsomal preparations, the characteristics of the NADH-dependent reactions were similar to the NADPH-dependent reactions, e.g., sensitivity to antioxidants and free radical scavengers and catalytic effectiveness of ferric complexes. However, rates with NADPH exceeded the NADH-dependent rates by 50 to 100%, and the increased production of reactive oxygen intermediates by microsomes after ethanol treatment was greater with NADPH (about twofold) than with NADH (about 50%). Oxidation of ethanol results in an increase in hepatic NADH levels and interaction of NADH, iron, and microsomes can produce potent oxidants capable of initiating lipid peroxidation and oxidizing .OH scavengers. These acute metabolic interactions produced by ethanol-derived NADH are increased, not attenuated, in microsomes from chronic ethanol-fed rats, and it is possible that such increases in NADH (and NADPH)-dependent production of reactive oxygen species play a role in the development of oxidative stress in the liver as a consequence of ethanol treatment.