Richard A. Deitrich

Genetic aspects of increase in rat liver aldehyde dehydrogenase induced by phenobarbital.

In the supernatant fraction of homogenized rat liver, the activity of aldehyde dehydrogenase that is dependent on nicotinamide adenine dinucleotide (E.C. 1.2.1.3) is increased up to tenfold after administration of phenobarbital for 3 days. The effect is genetically controlled and is inherited as an autosomal dominant characteristic. The mechanism is apparently unrelated to other druginduced increases in enzyme activity such as that which occurs in the hepatic microsomal systems for drug metabolism.

Putative role of brain acetaldehyde in ethanol addiction

The putative contribution of brain acetaldehyde (AcH) to ethanol (EtOH) tolerance and dependence (addiction) is reviewed. Although the role of AcH in EtOH addiction has been controversial, there are data showing a relationship. AcH can be formed in the brain tissues through the peroxidatic activity of catalase and by oxidation via other oxidizing enzymes such as cytochrome P-4502E1. Significant formation of AcH occurs in vitro in brain tissue at concentrations of EtOH that can be achieved by voluntary consumption of EtOH by rodents. AcH itself possesses reinforcing properties, which suggests that some of the behavioral pharmacological effects attributed to EtOH may be a result of the formation of AcH, and supports the involvement of AcH in EtOH addiction. Modulation of aldehyde dehydrogenase (ALDH) and brain catalase activity can change EtOH-related addictive behaviors presumably by changing AcH levels. Moreover, some condensation reaction products of AcH may promote some actions of EtOH and its consumption. On the basis of the findings, it can be concluded that AcH may mediate some of the CNS actions of EtOH including tolerance and dependence, although further exploration the involvement of AcH in EtOH addiction is warranted.

A critical evaluation of tetrahydroisoquinoline induced ethanol preference in rats

It has been reported that certain tetrahydroisoquinoline compounds, especially salsolinol and tetrahydropaveroline (THP) when infused into the lateral ventricle of rats brains results in increased preference for alcohol solutions. The effect is reported to be long-term, in that animals do not return to baseline drinking even months later. The current report provides a replicatin of the original experiments and also an extension of the work to complete dose-response curves for salsolinol and THP. Generally we have confirmed that rats of the Sprague-Dawley and Long-Evans strains do increase their alcohol intake in response to infused THP or salsolinol and that the effect is long lasting, up to 10 months. Such animals consume less alcohol at concentrations above 20% than below, in contrast to the previous reports where drinking was maintained at high concentrations of alcohol. While the animals will select alcohol in the face of a saccharin choice, they will not drink alcohol adulterated with quinine. We have failed to observe signs of dependence or withdrawal by these techniques and suggest that the original reports of these signs may have been a result of cellular damage caused by the long-term infusions. Additionally we have carried out extensive dose-response experiments with both salsolinol and THP. Doses of THP of 104 nmoles/day were inhibitory to alcohol drinking. We conclude that these compounds do shift these animals preference for alcohol relatively permanently, but not to the point of gross intoxication nor into the highly aversive range of alcohol concentration. We cannot confirm the reports that salsolinol or THP produce withdrawal symptoms when infused.

Inhibition of bovine brain aldehyde reductase by anticonvulsant compounds in vitro

Abstract The catalytic activity of partially purified NADPH-linked aldehyde reductase (alcohol: NADP oxidoreductase, EC 1.1.1.2) from bovine brain was markedly inhibited in vitro by anticonvulsant compounds. In general, the ability of these drugs to inhibit aldehyde reductase in vitro paralleled their anticonvulsant activity. Inhibition by various barbiturates, hydantoins or succinimides was non-competitive with either NADPH or aldehyde as the variable substrate, whereas the 2,4-oxazolidinediones produced a mixed type of inhibition. Inhibition by all ionizable compounds was found to vary with the pH of the reaction mixture, while the non-ionizable substances, paradione, trimethadione, methsuximide, 3-methyl-5-ethyl-5-phenylhydantoin, were not inhibitory. At pH 7.0 the inhibitor constants ( K i values) for phenobarbital, 5,5-diphenylhydantoin, 5,5-dimethyloxazolidinedione and ethosuximide were 1.2 × 10 −4 M, 1.7 × 10 −4 M, 4.7 × 10 −4 M and 5.5 × 10 −3 M respectively. The possibility that inhibition of brain NADPH-linked aldehyde reductase by these agents is concerned with their anticonvulsant actions is discussed.

Selective breeding for initial sensitivity to ethanol

Selective breeding for initial sensitivity to ethanol has been carried out by a number of investigators in order to investigate the mechanisms by which ethanol brings about a myriad of effects on the mammalian central nervous system. In addition the availability of these selectively bred animals provides clues to the causes of the genetic predisposition of humans to alcoholism. Eventually it is envisioned that the synteny between the mouse and human genomes will allow identification of specific genes responsible for acute effects of ethanol in both species as well as clues as to how alcoholism in humans can be better identified, prevented, and treated.

Blood and liver acetaldehyde concentrations during ethanol oxidation in C57 and DBA mice

Hepatic and blood acetaldehyde concentrations during ethanol oxidation were determined in C57 and DBA mice. Liver acetaldehyde was determined with the perchloric acid-thiourea method (no artefactual acetaldehyde formation). Levels ranging from 5 to 118 nmole/g were observed. At ethanol concentrations below 50-60 mumole/g, liver acetaldehyde concentrations were higher in DBA compared with C57 mice. A positive correlation was found between the ethanol and acetaldehyde concentration, when ethanol concentration was below 25 (DBA) or 70 mumole/g (C57). At higher ethanol concentrations the correlations tended to become negative. Artefactual acetaldehyde formation during the analytical procedures was obtained with the use of hemolysis, with or without thiourea, and semicarbazide methods for blood acetaldehyde determination. The magnitude of the artefactually formed acetaldehyde was of such order that no conclusions regarding the existence of true in vivo blood acetaldehyde concentrations could be drawn. Earlier reported mice blood acetaldehyde concentrations are suggested to be re-evaluated.

A convenient spectrophotometric assay for monoamine oxidase.

Abstract A rapid, convenient, and sensitive assay of monoamine oxidase activity in whole homogenates and partially purified preparations of monoamine oxidase has been developed. The assay depends upon the production of p-dimethylaminobenzaldehyde (ϵ = 2.77 × 104 at 355 mμ) from p-dimethylaminobenzylamine. Although the substrate is oxidized at a lower rate than benzylamine, the high extinction coefficient of the product at 355mμ makes the assay useful. The product of the reaction, p-dimethylaminobenzaldehyde, is not further metabolized, probably because it is a poor substrate for aldehyde and alcohol dehydrogenases. Thus, the assay can be carried out even in whole liver homogenates that are known to contain large amounts of enzymes that might be expected to interfere.

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Abstract: Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 μM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 μM 3,4‐dihydroxyphenylacetaldehyde and 10 μM indoleacetaldehyde as substrates, 40–50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15–30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion‐exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 μM and 10.2 μM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.

Effect of pyrazole in vivo on aldehyde metabolism in rat liver and brain.

Abstract The effect of administration of pyrazole in vivo on aldehyde reduction and oxidation has been studied in rat liver and brain. It was found that while pyrazole is capable of complete inhibition of NADH dependent reduction of a number of aldehydes in liver, it is only partially effective as an inhibitor of NADPH dependent aldehyde reduction and is ineffective as an inhibitor of NAD dependent aldehyde oxidations in liver. No effect of pyrazole on oxidation or reduction of aldehydes in brain was found. Pyrazole has a rapid onset of action in vivo and has an effective half life for inhibition of NADH dependent aldehyde reduction in rat liver of about 76 hr following a dose of 360 mg × kg−1 intraperitoneally.

Confirmation of quantitative trait loci for ethanol sensitivity and neurotensin receptor density in crosses derived from the inbred High and Low Alcohol Sensitive selectively bred rat lines

RationaleGenetically influenced alcohol sensitivity is thought to be an important risk factor for the development of alcoholism. An effective first step for identifying genes that mediate variation in alcohol sensitivity is through quantitative trait loci (QTL) mapping in model organisms.ObjectiveFourteen provisional QTLs related to alcohol sensitivity were previously mapped in an F2 derived from the IHAS1 and ILAS1 rat lines. The objective of the current study was to confirm those QTLs in an independently derived F2 and in congenics that were bred for two of the loci.Materials and methodsIHAS1 X ILAS1 F2 (n=450) were tested for alcohol-induced loss of righting reflex (LORR), blood ethanol concentration at regain of righting reflex (BECRR), sensitivity and acute tolerance on the Rotarod, and neurotensin receptor density (NTR1). Rats were genotyped at the 14 candidate loci and QTL mapping was conducted. Reciprocal congenic strains were bred for loci on chromosomes 2 and 5 and tested for LORR and BECRR.ResultsFour LORR QTLs were mapped at the suggestive or significant level (chromosomes 2, 5, 12, and 13). BECRR was mapped to chromosomes 5, 12, and 13 either in the original or current experiment. Results of the congenic experiment also support QTLs for LORR and BECRR on chromosomes 2 and 5. QTLs for NTR1 density and behavior on the Rotarod were not confirmed.ConclusionsQTL mapping in crosses derived from the IHAS1 and ILAS1 has successfully identified loci related to alcohol sensitivity. Recombinant congenics are now being bred to more finely map the confirmed QTLs.