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Lipid peroxidation in microsomal membranes produces a large number of aldehydes, alcohols, and ketones, some of which have been shown to be cytotoxic. This study has determined the kinetic parameters for the oxidation of aldehyde lipid peroxidation products by purified rat hepatic microsomal aldehyde dehydrogenase (ALDH). Livers were obtained from male Sprague-Dawley rats for preparation of microsomal ALDH which was purified 400-fold. Kinetic parameters, Vmax and V/K, were determined for saturated and unsaturated aldehydes of three to nine carbons in length in the presence of NAD+. Of the aldehydes examined, only acrolein and 4-hydroxynonenal were not oxidized by ALDH. The Vmax values (mumol NADH produced/min/mg protein) increased linearly with carbon chain length and ranged from 6.5 to 23 for the saturated series and 4.0 to 9.0 for the unsaturated aldehydes. The affinity constant V/K (nmol NADH produced/min/mg protein/nmol aldehyde/liter) also increased with carbon chain length and ranged from 12 to 9000 for the saturated aldehydes and 13 to 5300 for the unsaturated aldehydes. These results suggest that microsomal ALDH may serve a biological role for detoxification of reactive aldehydes produced by lipid peroxidation of microsomal membranes.

The mechanism of cocaine-induced cytotoxicity was investigated in hepatocytes isolated from both male C3H mice and male Sprague-Dawley rats. Cocaine was more cytotoxic to mouse hepatocytes than rat and induced reduced glutathione (GSH) depletion prior to marked increases in cytotoxicity in both systems. In both mouse and rat cells, GSH depletion was accompanied by GSSG production, but in rat cells, quantitative measures suggested that other mechanisms contributed to GSH depletion. No cocaine-induced depletion of protein-thiol groups or generation of protein-glutathione mixed disulfides could be detected in rat cells. Cocaine induced lipid peroxidation, using malondialdehyde (MDA) production as an index of the peroxidation process, in both mouse and rat hepatocytes. Inhibition of MDA production to below control levels using the antioxidant N,N'-diphenyl-phenylene diamine (DPPD) however, had no inhibitory effect on cocaine-induced cytotoxicity in either mouse or rat cells. These data suggest that neither generalized protein thiol depletion nor lipid peroxidation are critical determinants of cocaine-induced cytotoxicity in cellular systems.

In order to assess the genetic correlates of differences in ethanol-induced anesthesia, a set of 27 recombinant inbred (RI) strains was derived from an initial cross of the "long-sleep" (LS) and "short-sleep" (SS) selected lines of mice. In generations F24 and F25, samples of 534 and 580 mice from the LSXSS RI strains were tested for fall time, sleep time, and blood ethanol at awakening subsequent to intraperitoneal injection of a 4.1-g/kg body weight dose of ethanol. Approximately 2 weeks later, mice from F24 were also tested for body temperature lowering and blood-ethanol elimination rate (beta 60). Differences among the average ethanol-induced sleep-time scores of the RI strains are large (ranging from 36 to 171 min) and account for over 50% of the observed variance. Effects due to generation, sex, litters within strains, and the interaction between strain and generation are also significant, but account for relatively small proportions of the total variance. Quantitative genetic analyses of these data suggest that differences in sleep-time scores are polygenic; however, allelic differences at the albino (c) locus may have a pleiotropic effect. Genetic correlations between sleep time and blood ethanol at awakening (-0.79) and between body temperature 60 min after injection and beta 60 (+0.48) are significant. Because differences among the LSXSS RI strains are large and highly reliable, they should be valuable animal models for testing more searching hypotheses about the etiology of individual differences in ethanol-induced anesthesia.

The specific physiological roles of ALDH have not been determined. In this overview, we have identified malondialdehyde as one potential endogenous toxic aldehyde that is oxidized by ALDH. Undoubtedly, there are other cytotoxic aldehydes which occur in several different tissues that are detoxified by this same system of enzymes. Intuitively, the role of aldehyde dehydrogenases in the detoxification of xenobiotic aldehydes should also receive additional consideration and attention. An interesting feature of specific studies described in this overview is that a single aldehydic substrate, like malondialdehyde, may be a substrate for one ALDH isozyme and a potent inhibitor of ALDH present in another cellular compartment. Assuming that the ALDH susceptible to this inhibition performs other important physiological or biochemical functions, a potentiating effect would occur. Thus, the efficient metabolism of potentially cytotoxic aldehydes may be a protective mechanism against a number of different organ system pathologies. Another striking feature of ALDH is its sensitivity to in vitro and in vivo inhibition by a number of chemically and pharmacologically diverse agents. This characteristic suggests that ALDH may be a target enzyme for inhibition by several therapeutically important drugs or their metabolites. As a result, the perturbation of ALDH by specific therapeutic agents suggests a potential role of this enzyme in a number of different drug-induced toxicities.

The suggestion that psychopathologies are in part mediated by aberrant catecholamine metabolism has resulted in one of the more rapidly growing areas of pharmacogenetics. Collectively, the studies conducted to date indicate that psychopathological conditions have multiple causes which cannot be related to single genetic or biochemical deficits. However, through multidisciplinary research integrating behavioral, genetic, and biochemical approaches, a great deal of insight may be gained concerning the causes of psychopathological disorders and the use of drug therapy to modify the course of these illnesses.

Liver aldehyde dehydrogenase (ALDH) activity was found to be two-fold greater in pregnant (days 16-20 of gestation) mice than in non-pregnant controls. A subcellular fractionation revealed that the increase in ALDH activity was confined to the cystosolic fraction of the liver. Kinetic characterization of partially purified liver ALDH revealed similar Km values for either acetaldehyde or propionaldehyde in pregnant and non-pregnant animals. The similarity of apparent Km values suggests that the increase in cytosolic ALDH may involve induction of normally present ALDH isozyme, rather than originating from synthesis of a new enzyme. Induction of liver ALDH was found to significantly decrease blood acetaldehyde concentrations in pregnant mice receiving 2.0 or 3.0 g/kg ethanol when compared to the respective control (non-pregnant) animals. However, differences in blood acetaldehyde levels measured in pregnant and control animals were not apparent when blood ethanol concentrations were below 40 mumol/ml.

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.

Cocaine-induced hepatotoxicity was examined in vivo in a dose-responsive manner in C57BL/6Ibg, DBA/2Ibg, C3H/2Ibg, and Balb/cJ mice. Serum glutamic-pyruvic transaminase (SGPT) activities were determined 24 hours after intraperitoneal (IP) administration of cocaine (20 to 100 mg/kg). Significant elevations (100- to 150-fold) in SGPT were observed in male mice receiving cocaine. Significant differences in sensitivity to cocaine-induced hepatotoxicity were found among males of the inbred strains, with Balb being most sensitive and C57BL being least sensitive and C3H and DBA strains exhibiting intermediate sensitivity. Female mice of the four inbred strains were more resistant than males to cocaine-mediated hepatotoxicity, as indicated by only twofold to tenfold elevations in SGPT values. Among the females, sensitivity of the four inbred strains--as indicated by dose response curves--fell into two categories: the sensitive strains (C3H and C57BL) and the resistant strains (Balb and DBA). Pretreatment of males of the four inbred strains with the P-450 inducer phenobarbital resulted in enhancement of cocaine-mediated hepatotoxicity in the C57BL and Balb but not the C3H and DBA mice. Phenobarbital pretreatment of females of the four inbred strains resulted in enhancement of the hepatotoxic response to cocaine in the C3H, DBA, and Balb mice. Phenobarbital-pretreated C57BL females exhibited a 100% mortality rate after the acute cocaine dose, and thus no determination of hepatotoxicity could be established for them. These data demonstrate sex and strain differences in cocaine-induced hepatotoxicity and suggest that phenobarbital pretreatment does not uniformly enhance the hepatotoxicity of cocaine.

To investigate the formation and elimination of nicotine-1'-N-oxide (NNO) in mice treated with a single injection of nicotine, sensitive and selective methods were developed to quantitate this polar and heat-labile metabolite. The compound was isolated from tissue homogenates as a dodecyl sulfate ion pair with C18 extraction cartridges and analyzed on an amino bonded-phase high-performance liquid chromatographic column with a mobile phase consisting of isopropanol-water. Overall recoveries of NNO were 64-76% from biological media. Several methods of detection were evaluated; radiolabeling was necessary to achieve the sensitivity required for pharmacokinetic studies in mice. The cis and trans isomers of NNO were separated on a Partisil PAC column and enzymatic selectivity was evaluated for the formation of these isomers in mice.

The mechanisms by which chlorpropamide and tolbutamide disrupt acetaldehyde metabolism were studied in C57BL and DBA mice. Acute po administration of varying doses of tolbutamide or chlorpropamide 2.5 hr before a 3.0 g/kg ip dose of ethanol to C57BL and DBA mice resulted in significant elevations of blood acetaldehyde when measured 2.5 hr after ethanol dosing. Dose-response analysis revealed a significant (p less than .05) difference in ED50 values for the elevated blood acetaldehyde response to tolbutamide in DBA (60 mg/kg) and C57BL (100 mg/kg) mice. The ED50 value for potentiation by chlorpropamide of blood acetaldehyde concentration was similar (23 to 32 mg/kg) in both inbred strains. At higher doses of chlorpropamide, DBA mice displayed elevations of blood acetaldehyde nearly threefold greater than those measured in C57BL mice treated identically. Measurements of aldehyde dehydrogenase (ALDH) in hepatic subcellular fractions, obtained from both inbred strains treated with 100 mg/kg tolbutamide or chlorpropamide prior to a 3.0 g/kg dose of ethanol, revealed a 50 to 80% inhibition of the low-Km ALDH present in mitochondria. Chlorpropamide and tolbutamide did not inhibit ALDH in vitro, suggesting that metabolites of these hypoglycemic agents may be responsible for the genotypic-dependent alterations in in vivo acetaldehyde oxidation.

The activities of aldehyde dehydrogenase and aldehyde reductase were measured in microvessel fractions isolated from bovine brain. Aldehyde dehydrogenase specific activity in microvessels was enriched 1.5-fold over that measured in grey matter. The specific activity of aldehyde reductase was significantly lower in parenchymal vessles and microvessels than in grey matter. The presence of aldehyde dehydrogenase and aldehyde reductase in cerebral microvasculature is consistent with previous reports of enrichment of other enzymes involved in synthesis and degradation of monoamines. The enrichment of aldehyde dehydrogenase in cerebral microvasculature provides additional evidence for an enzymatic blood-brain barrier for biogenic aldehydes or ethanol-derived acetaldehyde.

The distribution of biochemical genetic variants was examined among eight inbred strains of mice, which served as contributors to a heterogeneous stock of mice (HS), and in short-sleep (SS) and long-sleep (LS) mice, selectively bred from the HS stock for differential ethanol sensitivity. Fifteen loci for enzymes of alcohol and aldehyde metabolism, as well as 12 other biochemical loci, were investigated. Thirteen of these loci exhibited allelic variation between strains, of which six were separately fixed in the SS and LS mice. Comparisons of genetic similarity coefficients, based upon the distributions of allelic variants for the loci examined, with behavioural sensitivities (sleep-time) to an acute dose of ethanol for the inbred and selected strains of mice, indicated no correlations between these data. This suggests that this collective group of loci are not useful indicators of the genes selectively bred in the SS and LS strains, which are responsible for the differential sensitivities to acute doses of ethanol.

Male mice selected for genetic differences in ethanol-induced sleep time, thereby designated long sleep (LS) an short sleep (SS), were treated with the Lieber-DeCarli liquid diet for 25 days. This chronic ethanol treatment produced an increase in liver/body weight and kidney/body weight in SS mice only. In addition, chronic ethanol treatment produced significant increases in both LS and SS treated mice in in vivo ethanol elimination, hepatic cytochromes P-450 and B5, NADPH cytochrome c reductase and hepatic and renal 7-ethoxycoumarin O-de-ethylase activity. Genotypic differences were observed in the magnitude of response of microsomal ethanol oxidation per mg of microsomal protein (SS greater than LS). Further, control LS and SS mice possessed substantially different activity of renal 7-ethoxycoumarin O-de-ethylase. Both lines exhibited similar induced renal 7-ethoxycoumarin O-de-ethylase activity after chronic ethanol ingestion. Ethanol binding spectra produced when ethanol was added to hepatic microsomes were examined using double reciprocal plots. Chronic ethanol ingestion produced genotypically related (LS greater than SS) increases in the absorbance change maximum per mg of microsomal protein. No significant changes in the spectral dissociation constant or absorbance change maximum per nM cytochrome P-450 were observed following ethanol treatment.

The disposition of nicotine, cotinine, and nicotine N-oxide was investigated in male C57BL, DBA, and C3H mice following an ip injection of nicotine (1.0 mg/ml). The half-lives (t1/2) of nicotine in blood were 5.9 to 6.9 min. The rapid elimination of nicotine was accompanied by a rapid accumulation of metabolites; maximal concentrations of cotinine in blood (204 to 364 ng/ml) were achieved in 10 min and nicotine N-oxide (23 ng/ml in C3H mice) in 15 min. The t1/2 in blood was 20.1 to 39.8 min for cotinine and 18.4 min for nicotine N-oxide. The t1/2 values for nicotine in brain were similar to those in blood, but the values for liver were slightly larger (6.3 to 9.2 min) and interstrain differences were significant. A large strain-related difference in the t1/2 for cotinine was found; the metabolite was eliminated from the blood of DBA mice at only about one-half the rate determined for the other strains. The t1/2 for nicotine N-oxide in liver ranged from 12.7 to 27.3 min; the values were significantly different with C57BL greater than DBA greater than C3H. Strain-related differences were also observed in response to chronic exposure to cigarette smoke. The t1/2 of injected nicotine appeared to be slightly decreased in C57BL and DBA mice but was increased by 60% in livers of C3H mice compared to a control group.

The role of various cytosolic aldehyde dehydrogenase (ALDH) isozymes in acetaldehyde metabolism was determined. Rats of known genotype (RR, reactor; rr, nonreactor) with respect to induction of a cytosolic enzyme by phenobarbital were used. Animals were treated with either phenobarbital (1.0 mg/ml in drinking water for 3 days) or 75 microng/kg (i.p.) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to ethanol treatment (2.5 g/kg). TCDD treatment is known to induce a second cytosolic ALDH isozyme in both RR and rr animals. Phenobarbital increased cytosolic ALDH activity 23-fold only in the RR animals, while TCDD increased activity 155-fold in both groups. Phenobarbital-treated RR rats maintained significantly lower blood acetaldehyde levels at 30, 90 and 150 minutes after ethanol injection compared to control RR animals. Blood acetaldehyde levels in phenobarbital-treated rr rats were not significantly different from rr controls. Administration of TCDD to RR or rr rats did not significantly alter blood acetaldehyde levels at 30, 90 or 150 minutes after ethanol treatment compared to appropriate controls. Similarly, phenobarbital significantly increased blood ethanol elimination rate (1.5 times greater than control) only in RR rats. Ethanol elimination rate was not significantly altered by TCDD in either group. Pargyline (100 mg/kg i.p.) did not significantly inhibit cytosolic ALDH activity, while total mitochondrial ALDH activity was inhibited by 28 and 57% in RR animals receiving TCDD and phenobarbital, respectively. Pargyline significantly increased blood acetaldehyde levels after ethanol administration. However, pretreatment with phenobarbital or TCDD significantly decreased elevated blood acetaldehyde levels resulting from pargyline treatment, with the greatest reduction in the phenobarbital-treated RR animals.

This study determined the accuracy of an AudioScope (AS) and pure-tone audiometer (PTA) screening at 40 dB HL from 500 to 4000 Hz in reference to a pure-tone threshold (PTT) test on 1210 ears of adult subjects 20 to 96 yr old. The overall results (averaged over age group and frequency) indicated that the AS and PTA screenings were equally accurate in reference to the PTT test. Averaged over the screening tests, the sensitivity was 91.4% with a false negative rate of 8.6% and the specificity was 93.5% with a false positive rate of 6.5%. As a function of age (averaged over frequency), the AS and PTA screenings had similar accuracy and decreased as age increased to about 60 yr old and then remained stable. As a function of frequency (averaged over age), each screening test had similar accuracy for each frequency. It was concluded that the results of a 40 dB HL AS or PTA screening are very accurate in reference to a PTT test.

A single injection of ethanol was shown to produce a relatively rapid increase in the in vitro activity of aniline hydroxylase and in microsomal ethanol oxidation. Stimulation of the microsomal ethanol oxidating system (MEOS) was dependent on the ethanol dose and time after dosing. Hepatic MEOS activities were significantly increased 2, 3 and 4 hr after a single 4.0 g/kg i.p. dose of ethanol and 4 hr after administration of 2.0 and 3.25 g/kg doses. Renal MEOS activity was also significantly increased 4 hr after a 4.0 g/kg dose of ethanol. Hepatic MEOS and aniline hydroxylase activities per gram of liver were significantly increased 2 hr after a 4.0 g/kg dose of ethanol, whereas microsomal cytochromes P-450 and b5, NADPH cytochrome c reductase, protein and NADPH-stimulated and ethanol/NADPH-stimulated lipid peroxidation were unchanged. Cycloheximide pretreatment did not block ethanol-induced stimulation of aniline hydroxylase or MEOS activity. Finally, an acute injection of ethanol (4.0 g/kg) produced significantly higher MEOS activities per gram of liver (121% of control) in mice chronically ingesting ethanol than in mice treated with saline. This investigation shows that MEOS activity can respond rapidly to a dose of ethanol and that synthesis of new protein is not responsible for this stimulation of activity.

Recent findings have shown that microsomal membrane lipid peroxidation generates a variety of reactive aldehydic products. The interaction of lipid peroxidation products with hepatic aldehyde dehydrogenases (ALDH) was studied using rat liver subcellular fractions. The well-documented membrane peroxidation product malondialdehyde (MDA) was studied to determine if ALDH isozymes play a role in metabolism of this aldehyde. The cytosolic and mitochondrial hepatic subcellular fractions were found to contain ALDH isozymes capable of oxidizing MDA. The kinetic properties of a cytosolic ALDH (Km of approximately 16 microM) suggest that this enzyme may be involved in the metabolism of MDA in vivo. Both the cytosolic and mitochondrial fractions also contained an ALDH isozyme with Km values in the millimolar range. Addition of the cytosolic fraction of rat liver produced a significant decrease in the accumulation of MDA during CCl4-induced microsomal membrane lipid peroxidation but did not protect cytochrome P-450 from destruction. The mitochondrial low Km ALDH isozyme was found to be a target enzyme for inhibition during in vitro microsomal lipid peroxidation. These studies show that a select ALDH isozyme is sensitive to inhibition during membrane lipid peroxidation whereas other isozymes may be involved in the metabolism of aldehydic peroxidation products.

Acrolein, the lowest member of the ethylenic aldehyde series, has been widely studied as a result of the diverse toxicities associated with it. Previous investigations into the enzymatic process responsible for the detoxification of acrolein implicated rat liver aldehyde dehydrogenase (ALDH) in the oxidation of this aldehyde. Contrary to these reports, in our investigation we were unable to detect the oxidation of acrolein to acrylic acid by Sprague-Dawley rat liver mitochondrial or cytosolic ALDHs measured spectrophotometrically by the production of NADH, or by HPLC analysis for the production of acrylic acid. However, in the course of these experiments, it was demonstrated that acrolein is a potent inhibitor of rat liver ALDHs. Mitochondrial and cytosolic high affinity ALDHs are particularly sensitive to the inhibitory effects of acrolein. The type of inhibition exhibited by these high affinity ALDHs is primarily irreversible, with a slight degree of reversible noncompetitive inhibition. The inhibition is rapid with a 91 and 33% reduction in control mitochondrial and cytosolic ALDH activities, respectively, with a 5-sec preincubation of 30 microM acrolein prior to the addition of the aldehyde substrate. Significant inhibition of total (high plus low affinity isozymes) mitochondrial and cytosolic ALDHs occurs only at relatively high acrolein concentrations (greater than or equal to 50 microM). The inhibition displayed by the total mitochondrial and cytosolic ALDHs is mixed-type, with both reversible noncompetitive and irreversible inhibition demonstrated.

Starting with a foundation population of 200 genetically heterogeneous mice (HS/Ibg), selective breeding has been initiated for a multivariate index of the ethanol withdrawal syndrome. This paper discusses the advantages of using multivariate indices to define pharmacological concepts. The seven variables included in our multivariate index are described. Because five of the seven variables showed a significant sex difference, separate principal component analyses were performed to establish selection indices appropriate for each sex. The study employs within-family mating to minimize inbreeding and includes replicate high, low, and control lines. These selectively bred lines should eventually provide a useful animal model for studying physiological and neurological mechanisms that may be involved in the ethanol withdrawal syndrome.

Cytosolic aldehyde dehydrogenase (AlDH) activity was increased in DBA and C57Bl mice following phenobarbital (Pb) treatment. The kinetic constants for the control and induced enzymes were similar in both inbred strains. The DBA mice showed enhanced rates of ethanol and acetaldehyde metabolism after Pb treatment, whereas the C57Bl mice did not. It was concluded that the cytosolic enzyme is more important for acetaldehyde metabolism in the DBA than in the C57Bl mice.

Mammalian liver contains a group of pyridine nucleotide linked aldehyde dehydrogenases [E.C. 1.2.1.3] which are present in high specific activity and possess wide substrate specificities. Malondialdehyde (MDA), a difunctional three-carbon aldehyde thought to be toxic, is generated during membrane lipid peroxidation in hepatocytes. The role of aldehyde dehydrogenase (ALDH) in the metabolism of MDA was tested in vitro with subcellular fractions and semipurified cytosolic preparations from rat livers. The cytosolic fraction accounted for virtually all of the MDA (50 microM) metabolizing activity observed in the postnuclear supernatant fraction. The rate of MDA disappearance was relatively low in the mitochondrial fraction and was not detectable in reaction mixtures which contained microsomes. Rat liver cytosol contained two ALDHs with MDA metabolizing activity. These enzymes were separated by DEAE-cellulose ion exchange chromatography and had apparent Km values of 16 microM and 128 microM for malondialdehyde. Mitochondria contained an ALDH enzyme with lower affinity (Km of 7.3 mM with NAD+) for malondialdehyde. These data show that rat liver contains at least three ALDH enzymes which oxidize malondialdehyde.

C57BL mice were treated (75 or 100 mg/kg) with pargyline or Lilly 51641 90 min prior to sacrifice. Liver and brain subcellular fractionation revealed that pretreatment with these drugs resulted in a significant inhibition of aldehyde dehydrogenase (ALDH) in liver cytosol and mitochondria, while brain ALDH in these same fractions was unaffected. Administration of pargyline or Lilly 51641 prior to ethanol treatment (3.0 g/kg) resulted in a significant elevation of blood acetaldehyde. Significant increases in brain acetaldehyde concentrations were not observed until blood acetaldehyde levels surpassed 200 nmol/ml. When mice were injected with ethanol (3.0 g/kg) and acetaldehyde (200 mg/kg), a similar relationship between blood and brain acetaldehyde concentrations was observed. Data presented in the present study indicate that there are very efficient enzymatic mechanisms responsible for acetaldehyde oxidation in brain and that at blood acetaldehyde concentratins normally occurring after ethanol ingestion, brain acetaldehyde levels would be extremely low.

The mammalian kidney is composed of numerous cell populations associated with the interstitial spaces, vasculature and various portions of the nephron. Not surprisingly, mammalian kidneys also exhibit an array of drug metabolizing activities, including the pyridine nucleotide-linked aldehyde dehydrogenase(s) (ALDH). To define the ALDH activity in segment 2 of the proximal tubule, a portion of the nephron which frequently shows drug-induced pathology, proximal tubules were isolated by purely mechanical methods from female rabbits. Isopycnic centrifugation of tubule-derived postnuclear supernates in linear sucrose gradients resulted in propionaldehyde (5 mM)-driven ALDH activity being distributed in a manner consistent with both a mitochondrial and cytosolic localization. Mitochondrial and cystosolic fractions yielded biphasic reciprocal plots when propionaldehyde was used as substrate. Km values of 282 microM and 4 mM were obtained from the mitochondrial enzymes, whereas the cytosolic enzymes gave KmS of 132 microM and 2.4 mM. The apparent Vmax values (nanomoles of NADH produced per minute per milligram of protein) are 16.3 and 37.8 for the mitochondrial enzymes and 23.5 and 19.0 for the cytosolic enzymes. Thus, the S2 proximal tubule cells contain ALDH activities at levels and subcellular sites comparable to those found in the liver. Because the proximal tubules constitute approximately 40% of the renal cortical mass, the high levels of ALDH activity observed in these cells may protect other cortical cells and more distal nephron components by detoxifying potentially cytotoxic aldehydes.

These experiments investigated the effects of pargyline, N-[2-(o-Chlorophenoxy)-ethyl]-cyclopropylamine (Lilly 51641), and nialamide on voluntary ethanol consumption of C57BL/6J mice. Both pargyline and Lilly 51641 reduced ethanol preference; in contrast, nialamide did not affect preference, despite the fact that it inhibited MAO activity by more than 90%. A subsequent experiment determined that both pargyline and Lilly 51641 produced substantially greater elevations in acetaldehyde levels than did nialamide. It is suggested that increased acetaldehyde is the mechanism responsible for the reduction in ethanol preference observed with pargyline and Lilly 51641.

Selective extraction and chromatographic techniques have been developed to measure low nanogram quantities of nicotine and cotinine in tissues. Analyses were performed by capillary column gas chromatography with a specific nitrogen-phosphorus detector and by gas chromatography-mass spectrometry. With close structural analogues for internal standards, high quantitative accuracy and precision were demonstrated for the range 5-1000 ng per g of tissue. The sensitivity limit was 2-3 ng/g for both compounds. The advantage of these techniques compared to previously published methods is increased selectivity; the other methods were developed for analysis of biological fluids and are not readily adaptable to more complex biological matrices such as tissue homogenates. With the newly developed techniques, we were able to perform a pharmacokinetic study of nicotine and cotinine in mouse liver following a single intraperitoneal injection of nicotine.

The oxidative and reductive metabolism of the acrolein-glutathione adduct, S-(2,aldehydo-ethyl)glutathione, by rat liver aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) was characterized. The glutathione-acrolein adduct is oxidized to the respective acid by two different forms of ALDH contained in rat liver cytosol which are distinct from two forms of ALDH present in the mitochondria also capable of oxidizing the aldehyde moiety of the adduct. Extensive kinetic characterization (Km, Vmax and V/K parameters) of the ALDH enzymes suggest that the glutathione-acrolein adduct is oxidized most efficiently by one form of mitochondrial ALDH which is 3.5 to 175 times more active (based on V/K comparisons) than the other forms of mitochondrial and cytosolic ALDH evaluated. The glutathione-acrolein adduct is also subject to reductive metabolism by rat liver ALH. However, the Km value (877 microM) for reduction of the adduct suggests that this would be a minor pathway of metabolism. Collectively, these results indicate that the glutathione-acrolein adduct formed after exposure to acrolein, or as a result of allyl alcohol oxidation and cyclophosphamide metabolism, can be oxidized by hepatic ALDH or ADH, respectively. However, the kinetic parameters for these pathways suggest that micromolar concentrations of this adduct may accumulate before these enzyme systems mediate significant oxidative or reductive pathways of detoxification. The proposition that the glutathione-acrolein adduct may play a role in acrolein-mediated hepatotoxicity is discussed.