Growth hormone mediates zone-specific gene expression in liver.

The CYP2B1/2 gene pair is an example of many liver genes that exhibit a characteristic zonated expression pattern in the liver. The factors governing this zonation are poorly understood. We observed that after hypophysectomy the expression of CYP2B1/2 protein and mRNA in the normally silent periportal (upstream) region was high, in both male and female rats. Treatment with growth hormone counteracted the effect of hypophysectomy, limiting expression to the perivenous (downstream) region, completely in females and partially in males. This shows that a hormone zone-specifically regulates gene expression in the liver.

Glucagon stimulates phosphorylation of different peptides in isolated periportal and perivenous hepatocytes.

The perivenous and periportal zones of the liver acinus differ in enzyme complements and capacities for gluconeogenesis, glycolysis and other metabolic processes. The biochemical factors governing this metabolic zonation are still poorly understood. Glucagon-mediated protein phosphorylation is an important factor in the regulation of hepatic metabolism. Here we show, by comparing the 32P-labelling pattern of isolated periportal and perivenous hepatocytes, that glucagon promotes the phosphorylation of zone-specific peptides as well as three common peptides (glycogen phosphorylase, glycogen synthase and pyruvate kinase) in the two cell types. We propose that the zone-specific phosphorylation of peptides is an important factor governing the shortterm zonation of metabolic processes in the liver.

Triiodothyronine downregulates the periportal expression of alpha class glutathione S-transferase in rat liver.

Most drug-metabolizing phase I and phase II enzymes, including the glutathione S-transferases (GST), exhibit a zonated expression in the liver, with lower expression in the upstream, periportal region. To elucidate the involvement of pituitary-dependent hormones in this zonation, the effect of hypophysectomy and 3,3',5-triiodo-L-thyronine (T3) on the distribution of GST was studied in rats. Hypophysectomy increased total GST activity both in the periportal and perivenous liver region. Subsequent T3 treatment counteracted this effect in the perivenous zone. However, analysis for either mu class M1/M2-specific (1,2-dichloro-4-nitrobenzene) or alpha class A1/A2-specific (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) GST activity revealed that T3 treatment did not significantly affect the perivenous activity of these GST classes. In contrast, T3 was found to significantly counteract the increase of alpha class GST activity caused by hypophysectomy in the periportal zone. To establish whether this effect was T3-specific, hepatocytes were isolated from either the periportal and perivenous zone by digitonin/collagenase perfusion and cultured either as pyruvate-supplemented monolayer or as co-culture with rat liver epithelial cells. Only in the latter it was found that T3 suppressed the A1/A2-specific GST activity and alpha class proteins predominantly in periportal cells. The data demonstrate that T3 is an important factor responsible for the low expression of alpha GST in the periportal region. T3 may be involved in the periportal downregulation of other phase I and II enzymes as well.

Induction mechanisms of cytochrome P450 2E1 in liver: interplay between ethanol treatment and starvation.

Chronic ethanol exposure causes marked induction of the ethanol-inducible cytochrome P450 (CYP) 2E1 isozyme in the centrilobular liver region, where alcoholic damage commonly is initiated. In contrast to most other CYP forms, which are ligand-activated at the transcriptional level, ethanol induction of CYP2E1 has been found to be post-translational. However, transcriptional activation of the CYP2E1 gene was recently described in fed animals maintained at very high ethanol levels. To further evaluate mechanisms of ethanol-mediated CYP2E1 induction we compared the effect of short-term heavy-ethanol treatment and fasting on CYP2E1 mRNA, protein and catalytic activity. High blood-ethanol levels (20-70 mM) were maintained for 3 days by regular alcohol intubations to fed or fasted rats. During this period, the amount of liver CYP2E1 apoprotein increased a maximum of 20-fold and catalytic activity 16-fold, both in fed and fasted animals, whereas starvation alone caused only a 4- to 5-fold increase. By comparison, the amount of CYP2E1 mRNA, as assayed both by Northern blot and slot blot, was significantly increased (5- to 6-fold) by ethanol only in fasted rats; this increase was smaller than that observed after fasting alone (8- to 9-fold). Analysis of cell lysates isolated from the periportal and perivenous region revealed that the increase in CYP2E1 mRNA by fasting occurred in the perivenous region. Thus no evidence was obtained for an increased pretranslational CYP2E1 gene expression as a consequence of the continuous presence of ethanol at intoxicating levels for 3 days. CYP2E1 mRNA elevation seems to be strongly associated with starvation while alcohol treatment increases the amount of enzyme, primarily by ligand-dependent stabilization of the synthesized protein. Our results indicate that transcriptional activation of CYP2E1 requires the long-term presence of highly intoxicating ethanol levels. It is conceivable that such activation occurs via indirect physiological responses related to those triggered by starvation.

No significant expression of CYP2E1 in rat liver stellate cells.

The putative role of the ethanol-inducible cytochrome P450(CYP)2E1 in stimulating collagen synthesis by rat liver stellate cells was studied. Analysis of carefully isolated stellate cells revealed that their content of immunoreactive CYP2E1 protein and of CYP2E1 mRNA, as determined by reverse transcription, polymerase chain reaction (RT-PCR), was very low, i.e. only 0-4% of that in hepatocytes. We conclude that it is improbable that such low expression of CYP2E1 in stellate cells would have functional importance.

Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes.

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.

Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes.

To study the mechanism of centrilobular damage developing in the centrilobular region after high doses of acetaminophen (APAP), its metabolism and toxicity were compared in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. Contrary to earlier reports, based on perfusions, no evidence for a periportal dominance of APAP sulfation could be observed. Glucuronidation, the dominant pathway of conjugation at high (5 mM) APAP concentration, was faster in perivenous cells. During primary culture, prolonged exposure (> or = 24 hr) to 5 mM APAP damaged perivenous cells, with a higher P450 2E1 level than periportal cells. When cells were isolated from ethanol-pretreated rats, to induce P450 2E1 levels specifically in the perivenous region, perivenous hepatocytes exhibited enhanced APAP vulnerability and extensive glutathione depletion. In contrast, corresponding periportal cells retained good viability. Isoniazid, an inhibitor of cytochrome P450 2E1, protected cells against APAP toxicity and prevented glutathione depletion. Induction of P450 2E1 also caused a 3-fold increase in the covalent binding of reactive intermediates from [14C]APAP, and this increase was mainly confined to perivenous cells. These results indicate that in rat liver there is only slight perivenous zonation of APAP conjugation and suggest that zone-specific APAP activation, mediated by the regional expression of ethanol-inducible cytochrome P450 2E1, is responsible for the characteristic centrilobular liver damage elicited by APAP.

Zonation of cytochrome P450 enzyme expression in rat liver. Isozyme-specific regulation by pituitary dependent hormones.

The effect of hypophysectomy and subsequent infusion of growth hormone (GH) or injections of triiodothyronine (T3) on the acinar expression pattern of four homonally regulated P450 isozymes was studied to elucidate the involvement of pituitary dependent hormones in regulating the characteristic centrilobular expression pattern of most members of the cytochrome P450 (CYP) gene family in rat liver. Hypophysectomy was previously observed to allow high expression of CYP2B1/2 and 3A1/2 in the normally silent periportal region. In the present study, it had much less effect on the zonation of the ethanol-inducible P450 2E1 form: only a moderate shift of 2E1 staining towards the periportal region was observed by immunohistochemistry. Subsequent injections with T3 moderately decreased CYP2E1 expression in the periportal region and no significant countereffect of GH was discerned. T3 treatment, previously observed to block only the periportal expression of CYP3A1/2, counteracted the increased CYP2B1/2 expression caused by hypophysectomy equally in the periportal and perivenous region. This was true both at the protein and mRNA level, as analysed from cell lysates obtained by in situ perfusion of livers by zone-restricted digitonin treatment. Thus, although hypophysectomy and subsequent GH and T3 treatment affect the total expression of CYP2B1/2, 2E1, and 3A1/2 similarly, the zonal effects were isozyme-specific. In contrast, the perivenous zonation normally seen for the dioxin-inducible P450 1A2 form was steepened rather than diminished by hypophysectomy, both in male and female rats. Administration of GH by the female-type continuous infusion had no effect in male rats, but partially counteracted the effect of hypophysectomy in females, suggesting an involvement of GH. In contrast to other CYP genes investigated, the female-characteristic expression of CYP2C12 was found to be completely non-zonated. Hypophysectomy and continuous GH administration dramatically affected the amount of mRNA of both P450 2C12 and the male-specific 2C11 form, but analysis of periportal and perivenous cell lysates indicated that these effects were not zone-specific. The distribution of the GH receptor was investigated to explain the zonal effects of GH. Immunohistochemically, a moderate perivenous dominance was observed, whereas the mRNA abundance of both GH receptor and GH binding protein was slightly higher in the periportal region. Thus, zonal regulation by GH does not appear to result from a GH receptor zonation; rather, a sinusoidal GH gradient may be involved. These data, combined with our previous results, indicate that pituitary-dependent hormones regulate the zone-specific expression of some P450 forms strongly (i.e. 2B1/2 and 3A1/2), and other forms are moderately regulated (i.e. 1A2 and 2E1), or are affected across the whole acinus (i.e. 2C11, 2C12).

Pretranslational induction of cytochrome P4501A enzymes by beta-naphthoflavone and 3-methylcholanthrene occurs in different liver zones.

Most of the cytochrome P450 (CYP) genes are expressed in an uneven, zonated pattern in the liver. Factors regulating this regionally restricted expression are not well known. In this study we have analysed cell lysates obtained from opposite zones of rat liver by infusing digitonin to the perfused liver to study the zonation of CYP1A1 and CYP1A2 induction. 3-Methylcholanthrene induced CYP1A1 protein in perivenous cells, while a low dose of beta-naphthoflavone caused periportal induction. Analysis of CYP1A1 mRNA from cell lysates by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and in situ hybridization experiments both demonstrated that this inducer-specific differently localized effect occurred at the pretranslational level. A corresponding difference in the regional pattern of CYP1A2 induction was seen: induction by beta-naphthoflavone reversed the constitutive perivenous pattern into a periportal CYP1A2 mRNA pattern while induction after 3-methylcholanthrene treatment was more panacinar. Attempts to identify the regiospecific factors involved were made by comparing the in vitro induction of CYP1A1 by beta-naphthoflavone and 3-methylcholanthrene in hepatocytes isolated from the periportal and perivenous region. However, after isolation, induction seemed to be independent of the source of the cells. Our results demonstrate the existence in the liver of regionally acting factors that mediate the induction of CYP1A1 and 1A2 in a local and inducer-specific fashion. These factors could be Ah receptor associated binding proteins operating in vivo, but no longer in isolated cells.

Drug metabolism by periportal and perivenous rat hepatocytes. Comparison of phase I and phase II reactions and their inducibility during culture.

Hepatocytes were aseptically isolated from either the periportal (pp; zone 1) or the perivenous (pv; zone 3) region by digitonin-collagenase perfusion and cultured on type I collagen for 4 to 9 days. In freshly isolated cells the pp:pv activity ratios of the acinar marker enzymes gamma-glutamyltranspeptidase (gamma-GT), alanine aminotransferase (ALAT) and glutamate dehydrogenase (GLDH) were 2.8, 1.6 and 0.76, respectively. During culture ALAT and GLDH activities gradually declined, but the pp-pv difference was retained for at least 4 days. In contrast, the difference in the gamma-GT activity was rapidly lost, due to its fast initial activation in pv cells. The initial 7-ethoxycoumarin O-deethylase (ECDE) activity was higher in pv cells; this difference was retained for several days of culture and was increased by induction in vitro with either phenobarbital (PB) or beta-naphthoflavone (beta NF). Although the basal UDP-glucuronyltransferase (UDPGT) activity with either p-nitrophenol (pNP) or hydroxybiphenyl (HBP) as substrate did not differ significantly, the in-vitro PB- or beta NF-induced activity was higher in pv cells. Both glucuronidation and sulfation of methylumbelliferone tended to be higher in pv cells. Glutathione S-transferase was initially significantly higher in pv cells and this difference was augmented after in vitro induction by PB or beta NF. After six days in culture all the observed pp-pv differences had disappeared. These results suggest that hepatocytes isolated from the perivenous region seem to maintain their initially higher capacity for phase I and phase II drug reactions during culture and also respond more strongly than periportal cells to in vitro induction.

Growth hormone-regulated periportal expression of CYP2C7 in rat liver.

Most drug- and steroid-metabolizing cytochrome P450 (CYP) enzymes are expressed in the mammalian liver in a characteristic zonated pattern, with high expression in the downstream perivenous (centrilobular) region. Here, we report that CYP2C7, a member of the rat CYP2 family, is expressed preferentially in the opposite, periportal region. CYP2C7 mRNA, as detected by reverse transcription-polymerase chain reaction, was detected almost exclusively in cell lysates obtained from the periportal region, indicating a very steep acinar gradient. The amount of immunoreactive CYP2C7 protein in periportal cell lysates was also higher than in samples from the perivenous region. This gradient was reversed by hypophysectomy, which markedly and selectively reduced the periportal CYP2C7 protein content. Subsequent growth hormone infusion by osmotic minipumps restored the zonation by selectively increasing the amount of periportal CYP2C7 protein. Although hypophysectomy suppressed CYP2C7 mRNA and growth hormone counteracted it, regulation at this level did not appear to occur in a zone-specific fashion. This indicates that growth hormone-mediated zonal regulation of CYP2C7 protein has additional translational or posttranslational components. Ethanol treatment, which has been shown to affect growth hormone levels, significantly induced CYP2C7 mRNA, but not zone specifically. Our results demonstrate that growth hormone up-regulates the CYP2C7 gene by enhancing the expression of the protein specifically in the periportal liver region. Growth hormone may up-regulate other periportally expressed liver genes in a similar fashion.

Aryl hydrocarbon receptor-associated genes in rat liver: regional coinduction of aldehyde dehydrogenase 3 and glutathione transferase Ya.

The tumor-associated aldehyde dehydrogenase 3 (ALDH3) and the glutathione transferase (GST)Ya form are coded by members of the Ah (aryl hydrocarbon) battery group of genes activated in the liver by polycyclic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The physiological role of the Ah receptor (AHR), its gene-activating mechanism and its endogenous ligands are still poorly clarified. We had previously observed that 3-methylcholanthrene (3MC) and beta-naphthoflavone (betaNF) induced the AHR-associated CYP1A1/1A2 pair in different liver regions, an effect not explained by the acinar distribution of the AHR protein. Here, we investigated AHR-associated regional induction by comparing the expression patterns of ALDH3 and GSTYa. Analysis of samples from periportal and perivenous cell lysates from 3MC-treated animals revealed that ALDH3 mRNA, protein and benzaldehyde-NADP associated activity were all confined to the perivenous region. In contrast, such regio-specific induction was not seen after beta-NF induction. Immunohistochemically, a peculiar mono- or oligocellular induction pattern of ALDH3 was seen, consistently surrounding terminal hepatic veins after 3MC but mainly in the midzonal region after betaNF. A ligand-specific difference in regional induction of GSTYa1 mRNA was also observed: The constitutive perivenous dominance was preserved after 3MC while induction by betaNF was mainly periportal. A 3MC-betaNF difference was also seen by immunohistochemistry and at the GSTYa protein level, in contrast to that of the AHR-unassociated GSTYb protein. However, experiments with hepatocytes isolated from the periportal or perivenous region to replicate these inducer-specific induction responses in vitro were unsuccessful. These data demonstrate that the different acinar induction patterns by 3MC and betaNF previously observed for CYP1A1 and CYP1A2 are seen also for two other Ah battery genes, GSTYa1 and ALDH3, but in a modified, gene-specific form. We hypothesize that unknown protein(s) operating in vivo and modifying the Ah-mediated response at the common XRE element located upstream of these genes is affected zonespecifically by 3MC and betaNF.

Induction of UDP-glycosyltransferase family 1 genes in rat liver: different patterns of mRNA expression with two inducers, 3-methylcholanthrene and beta-naphthoflavone.

Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), presently called UDP-glycosyltransferases, catalyse the detoxification of many toxic and carcinogenic compounds. Glucuronidation is also a major metabolic pathway for numerous drugs. The UGT1A6 gene (formerly known as UGT1*06 and UGT1A1) has been suggested to belong to the aryl hydrocarbon (Ah) gene battery, which consists of several genes encoding for drug-metabolising enzymes regulated by dioxin and other ligands of the Ah receptor. In this study, we analysed the localisation of UGT1A6 expression in rat liver by in situ hybridisation to mRNA. Two different RNA probes were used, one which was specific to UGT1A6 and the other against the C terminal sequence shared by all UGT1 genes. In this study, no UGT1A6 mRNA was detected in the control animals. However, other gene(s) of the UGT1 family were expressed in the perivenous region surrounding the central veins as detected by hybridisation with the probe against the common region of the UGT1 genes. Treatment with the lower dose (5 mg/kg) of 3-methylcholanthrene (3MC) induced expression of UGT1A6 perivenously. Treatment with the higher dose (25 mg/kg) of 3-Methylcholanthrene resulted in a more panacinar expression pattern. In contrast to the perivenous induction observed with 3-methylcholanthrene, treatment with 15 mg/kg of beta-naphthoflavone (BNF) resulted in strong induction in the periportal region. The results reveal an inducer-specific pattern of UGT1A6 expression similar to that demonstrated earlier for other Ah battery genes, namely CYP1A1, CYP1A2, GSTYalpha and ALDH3. The finding further supports the notion that common factors regulate the regional hepatic expression of Ah battery genes.

Effect of ethanol on the expression of hepatic glutathione S-transferase: an in vivo/in vitro study.

Ethanol, a human toxicant and a solvent in pharmacological research, is known to interfere with biotransformation of xenobiotics. We compared the in vivo and in vitro long-term effects of ethanol exposure on the expression of glutathione S-transferases (GST, EC 2. 5.1.18) in rat liver. Long-term in vivo ethanol treatment to achieve blood ethanol levels ranging between 10-50 mM was by liquid diet feeding. For in vitro experiments, rat hepatocytes co-cultured with rat liver epithelial cells were exposed to 17 and 68 mM ethanol for up to 10 days. Two weeks of liquid diet ethanol treatment increased total GST activity. Both Mu and Alpha classes and in particular the A1 and A2 subunits and the amount of their corresponding mRNAs were increased. Total GST activity was also increased in co-cultures after exposure to 68 mM ethanol for 10 days. However, the Mu class subunits M1 and M2 and the corresponding mRNAs were increased, rather than the Alpha class subunits. Thus, long-term exposure to ethanol induces hepatic GST both in vivo and in vitro, but different isoenzymes are affected. Consequently, extrapolation of in vitro data on GST expression and regulation to the in vivo situation must be judicious. During xenobiotic metabolism in cell culture, a shift in relative expression and induction of different GST forms may occur, resulting in either an under- or overestimation of effects.

Dose-dependent localization of TCDD in isolated centrilobular and periportal hepatocytes.

Dose-response relationships for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest a differential sensitivity of liver cell types to the induction of cytochrome P450 gene expression, and that the induction of hepatic protein CYP1A2 causes sequestration of TCDD. In addition, immunolocalization of hepatic CYP1A1/1B1/1A2 proteins is not uniform after exposure to TCDD. The mechanism for the regio-specific induction of hepatic P450s by TCDD is unknown, but may involve the differential distribution of participants in the AhR-mediated pathway and/or regional P450 isozymes, as well as, non-uniform distribution/sequestration of TCDD. Therefore, this study examined the effects of TCDD in unfractionated, centrilobular and periportal hepatocytes isolated from female Sprague-Dawley rats acutely exposed (3 days) to a single oral dose of 0.01-10.0 microg [3H]TCDD/kg. A dose-dependent increase in concentration of TCDD was accompanied by a dose-dependent increase in CYP1A1, CYP1A2, and CYP1B1 mRNA expression and associated enzymes in all liver-cell populations. Centrilobular hepatocytes showed a 2.7- to 4.5-fold higher concentration of TCDD as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Centrilobular hepatocytes also exhibited an elevated MROD activity as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Furthermore, centrilobular hepatocytes showed an elevated concentration of induced CYP1A2 and CYP1B1 mRNA as compared to periportal hepatocytes within the 0.01- and 0.3-microg TCDD/kg-treatment groups. This is the first study to demonstrate that a dose-dependent difference in distribution of TCDD exists between centrilobular and periportal cells that might be related to regional differences in P450 induction.

Alcohol withdrawal: delayed distribution of intubated ethanol in rats having seizures.

Tail blood ethanol levels two hours after acute oral administration of ethanol to rats during withdrawal from chronic ethanol feeding were much lower in rats with previous seizures than in non-convulsing rats. Since ethanol elimination rates were similar, this suggests that in the convulsive state the distribution of ethanol to the periphery is severely inhibited.

Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1.

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl4. In contrast, the mere presence of low concentrations (25-50 microM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

The calcium carbimide-ethanol interaction: lack of relation between electroencephalographic response and cerebrospinal fluid acetaldehyde.

Penetration of acetaldehyde into cerebrospinal fluid (CSF) was studied in healthy human volunteers during calcium carbimide-ethanol interaction. CSF was sampled via lumbar puncture and blood from a cubital vein. CSF and blood acetaldehyde concentrations varied from 1 to 41 and from 22 to 138 mumol/l, respectively. The results indicate that acetaldehyde penetrates the human blood-liquor barrier. Computer analysis of electroencephalograms (EEGs) recorded during the interaction showed reduction in alpha activity with a concomitant increase in delta activity. The changes were similar to those previously observed during 'normal' ethanol intoxication.

Suppression of alcohol drinking with brain aldehyde dehydrogenase inhibition.

Calcium cyanamide, an aldehyde dehydrogenase (ALDH) inhibitor used in the treatment of alcoholism, strongly suppressed voluntary ethanol drinking by rats. Such inhibitors have generally been believed to act primarily by limiting drinking through acetaldehyde accumulation after ethanol consumption. Administration of a low dose of 4-methylpyrazole (4-MP) that abolished acetaldehyde accumulation did not, however, remove the suppression produced by cyanamide. 4-MP alone did not affect the unsuppressed alcohol intake by Long Evans rats or the drinking by rats of the ANA strain developed for low levels of ethanol consumption. When given from the start with cyanamide, 4-MP did affect the development of the suppression, but probably by its effect in lessening the degree of brain ALDH inhibition: a high correlation (r = +0.825, p less than 0.001) was found between brain ALDH activity and ethanol consumption. The results suggest that cyanamide suppresses alcohol drinking also in the absence of acetaldehyde accumulation probably by some action related to its direct inhibition of brain ALDH.

Brain aldehyde dehydrogenase activity in rat strains with high and low ethanol preferences.

The activity of aldehyde dehydrogenase in subcellular fractions of whole brain homogenates from the AA and ANA rat strains developed respectively for high and low ethanol preferences has been studied. No significant strain or sex differences between naive AA and ANA rats were found. In ethanol-experienced rats some strain and sex differences were found, the most consistent being higher enzyme activity in AA females than in males both with aliphatic and aromatic aldehyde substrates. However, contrary to previous findings no relation between brain aldehyde dehydrogenase activity and drinking behavior was found in the AA and ANA rat strains.