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Published as doi: 10.1096/fj.07-8346com.
 (The FASEB Journal. 2008;22:662-670.)
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(The FASEB Journal. 2008;22:662-670.)
© 2008 FASEB

The role of acetaldehyde in mediating effects of alcohol on expression of endogenous opioid system genes in a neuroblastoma cell line

Claudio D'Addario*,{dagger},1, Yu Ming*, Sven-Ove Ögren{dagger} and Lars Terenius*

* Department of Clinical Neuroscience and

{dagger} Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

1Correspondence: Karolinska Institute, CMM L8:01, Stockholm, 17176 Sweden. E-mail: claudio.daddario{at}ki.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethanol (EtOH) alters neural activity through interaction with various neurotransmitters and neuromodulators. The endogenous opioid system seems to play a key role in the activities of EtOH, since the opioid antagonist naltrexone (ReVia®) attenuates craving. We have investigated the transcriptional regulation of opioid system genes in response to EtOH exposure for up to 96 h in human neuroblastoma SH-SY5Y cells using quantitative real-time polymerase chain reaction. We observed a significant decrease in the expression of opioid peptide precursors (proopiomelanocortin, proenkephalin, and prodynorphin) and of the kappa opioid receptor after 48 and 72 h of EtOH exposure (10 and 40 mM). These alterations were not present when the EtOH metabolism was blocked by 4-methylpyrazole. To evaluate whether the effects evoked by EtOH were possibly due to the first product of EtOH metabolism, cells were exposed to 0.4 mM acetaldehyde. We observed the same pattern of changes for prodynorphin, proenkephalin, and the kappa opioid receptor as after 72 h exposure to EtOH. These results contribute to our understanding of EtOH action at a cellular level and provide evidence of the role of acetaldehyde in mediating some of the EtOH-induced effects.—D'Addario, C., Ming, Y., Ögren, S-O., Terenius, L. The role of acetaldehyde in mediating effects of alcohol on expression of endogenous opioid system genes in a neuroblastoma cell line.


Key Words: ethanol • gene expression • opioid peptides/receptors • 4-methylpyrazole


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALCOHOLISM IS A COMPLEX DISORDER likely to relate to several neurotransmitter systems within the brain. Several studies implicate the endogenous opioid system due to its central role in the reward system (1 , 2) . A strong argument to this proposition is the anticraving effect of the opioid antagonist naltrexone (ReVia®).

Recent studies using gene targeting show that mu opioid receptor (MOP) knockout (KO) mice display reduced ethanol (EtOH) consumption (3) , whereas delta opioid receptor (DOP) KO mice consume more EtOH than do wild-type controls (4) . It has also been observed that complete deletion of kappa opioid receptors (KOP) decreased preference for EtOH (5) . In humans, research efforts have focused on identifying genetic markers for alcohol abuse and dependence. Candidate gene studies indicate that MOP receptor gene variants show association with alcoholism (6) and also that polymorphism in the genes encoding for both the KOP and its ligand precursor prodynorphin (PDYN) are associated with the risk for alcohol dependence (7) .

In the past, it was commonly assumed that EtOH had a general, nonspecific toxic effect on many cellular targets and that these effects were mediated by the ability of EtOH to disrupt cell membranes. More recent evidence suggests that proteins constitute the primary molecular sites of action for EtOH (8) . EtOH can simultaneously alter the functions of specific membrane proteins that initiate cellular signal transduction processes (9) and thus affect neuronal excitability, reducing glutamate-mediated excitation and increasing GABA-, glycine-, and adenosine-mediated inhibition (10) .

EtOH neurotoxicity is a well-established phenomenon in both the developing and the mature nervous system. In recent years, a number of investigators have used tissue culture procedures in an effort to characterize this toxicity. It has been shown that cellular cAMP-signaling is affected by EtOH both in vivo and in vitro (11 , 12) , and changes were observed in adenynyl cyclase and Gs protein activities (13 , 14) . However, so far the mechanism for involvement of the downstream signaling is not clear.

To investigate the EtOH-induced alterations on the expression of endogenous opioid system genes, we used the human neuroblastoma SH-SY5Y cells, endogenously expressing these genes, which have previously been shown to respond to EtOH (15) . It has been shown that EtOH increases the expression of dopamine beta-hydroxylase and several other genes involved in norepinephrine (NE) production and increases releasable NE (16) . Moreover, changes in gene expression important for development of tolerance to and dependence on EtOH, such as the constitutive 70 kDa heat-shock protein and glucose-regulated proteins, have been observed using neuroblastoma cells as a model (17) . It has also been reported that EtOH exposure in other cells is associated with reduced mRNA, destabilization of mRNA, or alteration of signal transduction pathways that regulate gene expression or posttranslational processing (18 19 20 21 22) .

Intriguing results of a pilot study indicating differential effects evoked by EtOH on the opioid peptide systems stimulated a more extensive study. The aim of the study was to detect possible effects of low, clearly not intoxicating (10 mM), and high (40 mM) EtOH concentrations at different time points on the expression of genes in the endogenous opioid system using quantitative real-time polymerase chain reaction (PCR) in SH-SY5Y neuroblastoma cells.

Another aim was to study whether the effects evoked by EtOH were due to some of its metabolites. In these experiments cells were treated with a combination of EtOH and 4-methylpyrazole (4-MP), the known inhibitor of alcohol metabolizing enzymes. It is well known that EtOH produces a wide range of behavioral effects in experimental animals. In the past decades, it has been speculated that some of the behavioral effects of EtOH should be attributed to acetaldehyde (AcH) (23 24 25) , the first product of EtOH metabolism, formed by the oxidation of EtOH, which is catalyzed primarily by alcohol dehydrogenase. Studies on the effects of AcH in cultured cells have been limited due to the rapid evaporation of AcH from the culture medium (26 , 27) . However, we exposed the cells to different concentrations of AcH protecting evaporation, and we investigated again the effects on the endogenous opioid system genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and treatment
Human neuroblastoma (SH-SY5Y) cells were maintained in Eagle’s minimum essential medium (EMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 2% (v/v) glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, hereafter referred to as growth medium. Cells were incubated at 37°C in a humidified atmosphere of 5% carbon dioxide. In all experiments, cells were seeded into 6 cm diameter tissue-culture Petri dishes with growth medium (different seeding densities were optimized at the beginning of the experiments), allowed to reach 60% confluence before starting the treatment.

Condition for EtOH exposure
In alcohol treatments, EtOH was added to 2.5 ml of growth medium, yielding a final concentration of 10 or 40 mM. Cells were maintained in the presence of EtOH for different lengths of time. Plates were wrapped in Parafilm to prevent EtOH evaporation. The growth medium was removed daily and replaced by fresh growth medium containing the appropriate concentration of EtOH. Control cells maintained in EtOH-free growth medium received the same schedule of growth medium changes and were also wrapped in Parafilm. Cells were collected at 1, 24, 48, 72, and 96 h after initiation of the experiment by scraping in a small volume of ice-cold phosphate-buffered saline (PBS), centrifuged for 5 min at 2000 g, washed once in PBS, and stored at –80°C. Throughout the time course, five independent samples per time point were collected.

For the experiments with 4-methylpyrazole (4-MP, Sigma-Aldrich, St. Louis, MO, USA), cells were preincubated with 4-MP (1 mM) for 30 min and then cultured with EtOH (10 or 40 mM) for 48, 72, and 96 h as described above.

Condition for AcH exposure
Cells were exposed to 0.04, 0.4, and 4 mM of AcH for 48, 72, and 96 h and treated in the same way as in EtOH experiments. The concentrations were within the range of AcH exposure used in many experimental studies (28 , 29) . However, it is important to point out that 0.4 and 4 mM are relatively high concentrations, assuming that the expected AcH produced in situ reaches 0.1–0.2% of the initial concentration of EtOH (30) . Nevertheless, we chose to test these concentrations due to the high volatility and reactivity of AcH that makes it very unstable.

Media EtOH levels and cell viability
EtOH concentrations in media were measured by an NAD-ADH enzyme assay (Sigma) in an independent experiment. SH-SY5Y cells were incubated with 10 and 40 mM EtOH. Samples were collected from dishes with or without EtOH after 1, 4, 8, 16, and 24 h incubation. Cell-free control dishes were used to correct for loss of EtOH due to evaporation.

Cell viability of SH-SY5Y cells was determined by trypan blue exclusion. Cells grown in culture dishes (60x15 mm) were treated with 10 and 40 mM EtOH or 0.04, 0.4, and 4 mM AcH as described above. After treatment, the cells were trypsinized, washed, and harvested with sterile PBS. The cell suspension was mixed at the ratio of 1:1 with 0.4% trypan blue solution and incubated for 5 min at room temperature. The viable cells (i.e., those that excluded the dye) were counted in a hemocytometer with an inverted phase-contrast microscope.

Real-time quantitative RT-PCR
Total RNA was extracted using RNeasy Mini kit and DNase treatment (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The integrity of RNA and the absence of contaminating genomic DNA were checked by gel electrophoresis. RNA concentrations were measured by spectrophotometry and adjusted accordingly. Only RNA samples with an OD260/OD280 ratio >2 were used and converted to cDNA with the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA, USA) by using random hexamers (0.75 µg of total RNA in a final reaction volume of 20 µl). Relative abundance of each mRNA species was assessed by real-time RT-PCR using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on an DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA). Reaction volume was 20 µl, and 85 ng cDNA was used as the template for control and treated cells. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined and the point of early log phase of product accumulation is defined by assigning a fluorescence threshold above background, defined as the threshold cycle number or Ct. Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Relative expression of different gene transcripts was calculated by the delta-delta Ct (DDCt) method and converted to relative expression ratio (2–DDCt) for statistical analysis (31 , 32) . All data were normalized to the endogenous reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. In addition, results on RNA from treated samples were normalized to results obtained on RNA from the control, untreated sample. After PCR, a dissociation curve (melting curve) was constructed in the range of 60°C to 95°C (33) to evaluate the specificity of the amplification products. The primers used for PCR amplification are shown in Table 1 and were designed using Primer 3.


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  		Table 1. Primer sequences used for real-time PCR

The absolute expression levels of each gene investigated are shown in Table 2 and expressed as delta Ct (DCt).


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  		Table 2. Expression of target genes in wild-type SH-SY5Y cells quantified by real-time PCR

Statistical analysis
Statistical analysis was performed with GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Treatment effects were assessed with one-way ANOVA followed by Dunnett’s test. Data are presented as the mean values ± SE. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Media EtOH and cell viability
SH-SY5Y cells were incubated with 10 or 40 mM EtOH, and media were collected at the indicated time points and analyzed for alcohol content using an NAD-ADH assay kit. As shown in Fig. 1 , media EtOH levels measured in plates with and without SH-SY5Y significantly decreased over time for both 10 mM and 40 mM. Between 0 and 16 h, around 4–6% of the EtOH was metabolized by the cells. However, by 24 h, the cells had metabolized 10 and 18% of the EtOH in the media when cells were exposed either to 10 or 40 mM. After 48 h, 19 and 21% of the EtOH was metabolized and after 72 h, 23 and 41%, respectively. When cells had been treated previously with 4-MP and then exposed to EtOH no evidence of metabolism was found since the reduction of EtOH concentration was quite similar to that observed in the cell-free plates meaning loss due to evaporation.


Figure 1
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  		Figure 1. Metabolism of EtOH by SH-SY5Y cells. Cells were exposed to 10 and 40 mM EtOH for a total of 72 h. Media were collected at specific time points, and the concentration of EtOH was determined using an NAD-ADH assay kit. Cell-free dishes were used as a control to correct for loss due to evaporation of EtOH. Data are expressed as mean ± SE for 2 independent experiments.

Cell viability was assessed by trypan blue exclusion, and Fig. 2 shows that exposure of cells to EtOH did not induce cell mortality. Since SH-SY5Y neuroblastoma cells grow in clusters making direct counts of viable cells very difficult and since it has been observed that the addition of solutions containing trypsin caused cell mortality after 2 min (34) , total RNA was also used as an indirect indicator of cell growth. Incubation of SH-SY5Y cells up to 96 h with either of the EtOH concentrations (10 and 40 mM) did not cause changes in total RNA except for EtOH exposure at 40 mM for 96 h. When cells had been treated with AcH (0.04 and 0.4 mM), a reduction in total RNA was observed following 96 h of exposure and with 4 mM AcH reduction was recorded even after 48 h of exposure, suggesting a significant reduction in the number of cells (Fig. 3 ).


Figure 2
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  		Figure 2. Effects of EtOH (10 and 40 mM) and AcH (0.04, 0.4, and 4 mM) on SH-SY5Y cell viability. Cells in culture dishes (60x15 mm) were exposed for 48, 72, and 96 h, and viability was measured by trypan blue dye exclusion. Data are expressed as mean percent of untreated control ± SE for 4 independent experiments (n=5). Statistical significance was determined by one-way ANOVA and Dunnett’s test. Asterisks indicate significance when compared to untreated control (**P<0.01).


Figure 3
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  		Figure 3. Total cellular RNA of SH-SY5Y cells treated at different time points with EtOH (10 and 40 mM) (a) and AcH (0.04, 0.4, and 4 mM) (b). Data are expressed as mean ± SE. Asterisks indicate a significant difference from control group (**P<0.01; ANOVA and Dunnett’s test).

EtOH exposure and gene expression
After normalization to the internal reference GAPDH, a decrease was found after 48 h of 10 or 40 mM EtOH exposure in PDYN [0.30±0.16; 0.34±0.21 vs. control (1.0±0.16, P<0.05)], proenkephalin (PENK) [0.31±0.17, P<0.05 vs. control (1.0±0.1); 0.44±0.23 not significant], proopiomelanocortin (POMC) [0.34±0.14; 0.37±0.15 P<0.05 vs. control (1.0±0.14)], and KOP [0.13±0.07; 0.33±0.17 P<0.01 vs. control (1.0±0.12)] gene expression (Fig. 4 ). No changes were observed at these time points for the other genes investigated: MOP, nociceptin opioid receptor (NOP), and DOP (see Table 3 ).


Figure 4
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  		Figure 4. Quantitative real-time PCR results. Relative gene expression levels of PDYN (a), POMC (b), PENK (c), and KOP (d) in undifferentiated SH-SY5Y neuroblastoma cells following treatment with EtOH (10 and 40 mM) at different time points. Bars represent 2–DDCt value calculated by DDCt method. Expression was normalized to GAPDH, and means of mRNA levels are expressed relative to control cells ± SE. Asterisks indicate a significant difference from control group (*P<0.05; **P<0.01; ANOVA and Dunnett’s test).


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  		Table 3. Relative MOP, DOP, and NOP gene expression levels in undifferentiated SH-SY5Y neuroblastoma cells after treatment with EtOH (10 and 40 mM) at different time points

After 72 h of 10 or 40 mM EtOH exposure, we found a significant decrease in PENK [0.37±0.14; 0.31±0.13 P<0.05 vs. control (1±0.14)] and KOP [0.21±0.11, P<0.01; 0.43±0.19, P<0.05 vs. control (1±0.15)] mRNA. No alterations in any gene analyzed were observed at the other time points studied (1, 24, and 96 h) and also not in the cells pretreated for 30 min with 4-MP, and thereafter with EtOH at 10 or 40 mM for 48, 72, or 96 h (see Table 4 ).


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  		Table 4. Relative PDYN, PENK, POMC, and KOP gene expression levels in undifferentiated SH-SY5Y neuroblastoma cells after treatment for 30 min with 4-MP (1 mM) followed by EtOH (10 and 40 mM) at different time points

Since metabolism of EtOH is clearly taking place, we documented also the presence of alcohol dehydrogenase (ADH) in the cell culture. We evaluated the expression of class I, class II, and class III ADH, or here named ADH 5, and found the last one to be the only ADH expressed in SH-SY5Y cells (Table 2) . No alteration in the ADH 5 mRNA was observed on the different treatments and at the time points studied.

AcH exposure and gene expression
Exposure of cells to AcH at 0.4 mM for 72 h induced a significant decrease in the gene expression of PENK [0.39±0.21, P<0.05 vs. control (1±0.19)] PDYN [0.26±0.24, P<0.05 vs. control (1±0.15)] and KOP [0.37±0.10, P<0.05 vs. control (1±0.19)]. The same tendency for downregulation, even if not significant, was observed following treatment with 0.04 mM AcH for KOP mRNA after 72 h (Fig. 5 ).


Figure 5
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  		Figure 5. Quantitative real-time PCR results. Relative gene expression levels in undifferentiated SH-SY5Y neuroblastoma cells following treatment with AcH (0.04 and 0.4 mM) at different time points. Bars represent 2–DDCt value calculated by DDCt method. Expression was normalized to GAPDH, and means of mRNA levels are expressed relative to control cells ± SE. Asterisk indicates a significant difference from control group (*P<0.05; ANOVA and Dunnett’s test).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Different neurobiological studies suggest that some of the reinforcing effects of EtOH may be mediated by the endogenous opioid system of the brain (35 , 36) . Even if there is much evidence suggesting that EtOH alters the activity of this system, the cellular mechanisms involved in EtOH dependence are not well established, and interactions between alcohol and the endogenous opioid system remain unclear.

In this study, changes in the expression of the opioid receptors MOP, NOP, KOP, and DOP and the precursors of the opioid peptide ligands POMC, PENK, and PDYN were observed in response to EtOH or AcH exposure in human neuroblastoma SH-SY5Y cells, endogenously expressing these genes. It was not possible to detect pronociceptin (PNOC) gene expression in cells, so putative effects on this gene would have to be tested in another cell line.

With this exception, the use of this model system has many advantages. First of all the native transcript is directly assessed. Another advantage is the homogeneity of the cell population, which facilitates the analysis. Moreover, the SH-SY5Y cell line is derived from a human tumor, and this mitigates concerns about possible species differences in attempts to translate experimental findings to clinical situations. However, an observation made in a cell line does not necessarily reflect all regulatory events in normal tissues from different parts of the central nervous system (37) .

SH-SY5Y cells were exposed to EtOH in blocks of 24 h up to 96 h and differential effects on the endogenous opioid system genes were observed. Exposure for 48 h to EtOH at concentrations (10 and 40 mM) relevant in alcohol abuse could induce a significant decrease in POMC, PDYN, PENK, and KOP gene expression, and these alterations were still evident at 72 h for KOP and PENK mRNAs.

Other products of the EtOH metabolism could be involved in the EtOH effects on the mRNA expression of the opioid system and in particular in the regulation of POMC mRNA, since AcH did not modulate it. Acetate (acetic acid) is the major final product of EtOH metabolism. It has been observed that acetate levels increase after intake of EtOH, but acetate is then converted rapidly to water and carbon dioxide in the Krebs cycle, and thus the short persistence of acetate levels following alcohol consumption limits its applicability (38) . No alterations were observed in any gene investigated when the exposure to EtOH was prolonged up to 96 h. Expression of the other opioid receptor genes (MOP, DOP, and NOP) was resistant to change at all time points studied and at both EtOH concentrations used. Thus, it is unlikely that the effects observed are due to general toxicity induced by EtOH.

It is important to point out that the EtOH doses used in the study were chosen to represent blood alcohol concentrations that might result from low to high alcohol consumption (39) . The concentration of 10 mM (~0.5 mg/ml) EtOH is close to the legal maximum level for driving in different countries, whereas a blood alcohol level of 40 mM (~2 mg/ml) induces severe intoxication when there is very little control of mind or body.

Although all the opioid peptide systems have been implicated in the effects of EtOH, the role of opioid peptides has not been investigated extensively; most of the studies have focused on β-endorphin in the brain and pituitary.

POMC (precursor to β-endorphin)
Some studies report that long-term EtOH administration in rats leads to a significant increase in POMC mRNA and peptide levels (40 , 41) , whereas others report a reduction in the biosynthesis and release of POMC-derived peptides (42 , 43) . Reports on the effects of long-term EtOH administration on hypothalamic β-endorphin are also inconsistent (44 45 46 47 48 49) .

PENK and PDYN
Few data are available on interactions of EtOH with the enkephalin and dynorphin opioid systems, but available evidence indicates that both systems are influenced even if the effects seem to be species-, strain-, tissue-, or line-specific (50 51 52) .

Clear evidence indicates that the biosynthesis, content, and release of these classes of opioid peptides are altered following chronic EtOH treatment, although considerable disagreement exists as to the direction of changes. The inconsistencies are likely due to methodological differences: EtOH administration (i.e., liquid diet or vapor inhalation), quantity of EtOH consumed, duration of treatment, and species and strain differences. The conflicting results may also be associated with differences in the development of tolerance.

Opioid receptors
Alcohol intake may alter not only the activity of the endogenous opioid peptide system but also the density or affinity of specific opioid receptors in distinct regions of the brain. Such changes would alter the interactions of the opioid receptors with their respective ligands and, as a result, would alter the functional activity of the whole system. It has also been observed that opioid receptors could be affected following both short- and long-term EtOH administration (53) .

Again, published results are inconsistent, i.e., increases, decreases, and no changes in the activity of opioid systems have been reported. Consistent with our findings, previous studies suggest that chronic EtOH induces a downregulation of KOP receptors in selected rat brain regions (54) .

This is not the first study to suggest that EtOH can affect gene expression. However, the use of a defined cell population, and not a circuit of cells, allows us to state that the effect is differential. From our results, even if it is hard to generalize, it seems that the genes coding for opioid peptide precursors are more vulnerable than those coding for the corresponding receptors.

EtOH metabolism
The slow onset on the effects of EtOH led to the speculation that EtOH metabolites could be responsible for the effects observed. Thus experiments where SH-SY5Y cells were exposed to 4-MP, an inhibitor of EtOH oxidation, were conducted. Once cells were exposed to 4-MP and then to EtOH at the same concentrations reported above, the effects on opioid precursor mRNAs were not present any longer, indicating that the effect on the gene expression is specifically mediated by alcohol metabolism. To date, very few studies have investigated the effects of its first metabolite, AcH, on different neurotransmitter systems in relation to changes in behavior. Thus the neurochemical basis of the behavioral effects of AcH remains to be demonstrated.

AcH has been shown to bind to proteins forming stable adducts (55) . Moreover, AcH increases intracellular calcium levels, damages DNA, and further activates an apoptotic pathway (29 , 56) . Even if its mechanism in neurons is not well known, it could explain the alcohol-induced neurochemical alterations (57) and thus be responsible for many effects of EtOH. Practically nothing is known about the interactions of AcH with neuropeptides or neuropeptide receptors. So far it had been observed that AcH at low concentrations (12–50 µM) stimulates β-endorphin secretion in primary cultures of hypothalamic neurons (58 , 59) .

We report here that AcH at a higher concentration (400 µM) can induce a significant decrease of PDYN, PENK, and KOP mRNA after 72 h. This time point coincides with that also effective following EtOH exposure, at least for PENK and KOP expression. Thus it is possible to suggest that the changes observed in the expression of these genes are probably due to the action of this metabolite. A crucial observation is the time needed to reach the threshold concentration for the effect for both EtOH and AcH. AcH induces DNA changes with significant increase of low molecular DNA characteristic of necrotic or apoptotic cell death (60 , 61) . The reaction of AcH with DNA and thus the formation of adducts might be an important prerequisite that may be responsible for its genotoxic effects. At first glance it is hard to understand why the effect occurs in such a narrow time window. AcH is highly volatile, and therefore the delayed effects of AcH vs. EtOH could be due to an accumulation of a metabolite. To explain that the effect is transient, one may hypothesize that the development of metabolic adaptation occurs at later incubation times.

In conclusion, prolonged EtOH exposure induced long-term alterations in the expression of certain but not all endogenous opioid system genes. AcH, the first product of EtOH metabolism, seems to play the major role in these EtOH-induced effects. The importance of this metabolite(s) for EtOH effects needs to be clarified in further studies to generate knowledge about the neurochemical basis and to possibly develop new therapeutic approaches of alcohol abuse based on alterations in the metabolism of brain AcH.


   ACKNOWLEDGMENTS
 
This study was supported by grants from AFA Insurance and the Swedish Research Council.

Received for publication May 23, 2007. Accepted for publication September 13, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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