COMBINED CALCIUM CARBIMIDE AND ETHANOL TREATMENT INDUCES HIGH BLOOD ACETALDEHYDE LEVELS, MYOCARDIAL APOPTOSIS AND ALTERED EXPRESSION OF APOPTOSIS-REGULATING GENES IN RAT

Heidi Jänkälä1,3, C. J. Peter Eriksson5, Kari K. Eklund2, Matti Härkönen1 and Tiina Mäki4,*

1 Department of Clinical Chemistry,
2 Division of Rheumatology and
3 Department of Psychiatry, Helsinki University Central Hospital, Haartmaninkatu 4, SF-00290 Helsinki,
4 Clinical Laboratory of Jorvi Hospital and
5 Department of Mental Health and Alcohol Research, National Public Health Institute, POB 33, SF-00251 Helsinki, Finland

Received 30 May 2001; in revised form 30 October 2001; accepted 23 November 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The effects of ethanol and ethanol-derived acetaldehyde on rat myocardial apoptosis and expression of genes involved in the regulation of apoptosis and cell cycle arrest were studied. Combined ethanol and calcium carbimide treatment for 2, 5 or 8 days (E + CC) markedly increased blood acetaldehyde levels. Cytosolic DNA fragmentation was quantified in the 5-day treatment group. Increased amount of DNA-fragmentation, reflecting increased apoptosis, was evident in the E + CC group (23% increase compared to controls). mRNA levels of genes regulating apoptosis were measured by using quantitative PCR in the 2- and 8-day treatment groups. In the 2-day treatment group, p21 gene expression was increased by 25% and bax/bcl-2 mRNA ratio by 57% in E + CC, compared to the control, group. In the 8-day treatment group, p21 mRNA level was 24% lower, p53 mRNA level was 15% higher (P < 0.005), and bcl-2 mRNA level 36% higher in E + CC-treated, compared to the control, group. Interestingly, both ethanol and calcium carbimide treatments alone increased bax mRNA levels, as compared to the control group at 2 and 8 days. These results indicate that acetaldehyde might regulate the expression of apoptosis-linked genes and that apoptosis of myocardial cells may be involved in the development of alcoholic heart disease.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
It is currently estimated that 36% of all cases of dilated cardiomyopathy are due to excessive ethanol intake (Dancy et al., 1985Go). Almost one-third of chronic alcoholics exhibit cardiac dysfunction and a large proportion of those develop alcoholic heart muscle disease (AHMD) (Urbano-Marquez et al., 1989Go). The prognosis is poor, since 40% of these patients die over a mean follow-up period of 4 years (Preedy and Richardson, 1994Go). Up until now, neither the mechanism nor the specific toxic agent that produces AHMD has been established. One potential candidate is acetaldehyde, the major metabolite of ethanol. Acetaldehyde has been shown to have significant actions on cardiac function in both animal and human studies (Patel et al., 1997Go; Ren et al., 1997Go). A 4-fold increase in cardiac acetaldehyde levels clearly increased the rate of AHMD in rats (Liang et al., 1999Go).

In the past decade, several studies have documented that myocyte cell loss is a critical factor in the development and progression of ventricular dysfunction and failure, and apoptosis of myocardial cells has been associated with the progression of cardiomyopathy (Narula et al., 1996Go). In clinical studies, cardiomyocyte apoptosis is associated with the progression score of dilated cardiomyopathy (Saraste et al., 1999Go). Interestingly, both acetaldehyde and ethanol have been shown to accelerate apoptotic cell death in various cells (Baroni et al., 1994Go; Zimmerman et al., 1995Go; Oberdoerster and Rabin, 1999Go).

The p53 protein is a transcriptional activator involved in cell cycle control and repair of cellular DNA (Haunstetter and Izumo, 1998Go). The p53 protein has been demonstrated to enhance apoptosis in numerous systems, including cardiac myocytes responding to stress (Leri et al., 1998Go). Another cell cycle controlling protein, p21, is necessary for the G1 arrest in cells treated with DNA-damaging agents (El-Deiry et al., 1993Go) and cells lacking p21 are defective in DNA repair (McDonald et al., 1996Go). The p21 protein is particularly abundant in cardiac muscle (Spandidos and Dimitrov, 1985Go) and it has been implicated in the hypertrophic response of cultured cardiac myocytes (Kovacic-Milivojevic et al., 1997Go). It is proposed that, in situations of stress, p21 plays a fundamental role in the decision fork between cell death and survival, with enhanced p21 expression contributing to the ability of the cell to endure stress (Gorospe et al., 1997Go).

Bcl-2 family proteins may be either pro- or anti-apoptotic. Bcl-2 itself inhibits apoptosis in response to a wide variety of signals (He et al., 1997Go) and over-expression of bcl-2 can protect cardiac myocytes (Kirshenbaum and de Moissac, 1997Go). Bax, on the contrary, is a pro-apoptotic protein of the bcl-2 family (Kockx et al., 1998Go) and decreased bcl-2/bax ratio has been shown to increase the probability for myocardial cell apoptosis (Condorelli et al., 1999Go).

Tumour necrosis factor (TNF)-{alpha}, a pleiotropic cytokine, has also been shown to induce apoptosis in rat cardiac myocytes in vitro (Krown et al., 1996Go). TNF-{alpha} has a hypertrophic or pro-apoptotic effect on cardiomyocytes (Yokoyama et al., 1997Go; Bozkurt et al., 1998Go) and is rapidly synthesized in heart in response to a stressful stimulus (Kapadia et al., 1995Go).

To elucidate the mechanism of AHMD, we have studied the effect of ethanol and acetaldehyde on several hypertrophy- and apoptosis-related genes in rats. A model of elevating the ethanol-derived blood acetaldehyde levels by the aldehyde dehydrogenase inhibitor, calcium carbimide, was used and compared with effects of ethanol as well as vehicle alone. In this model, we studied the expression of p53, p21, bcl-2, bax and TNF-{alpha} genes as well as the occurrence of DNA fragmentation in left ventricular myocardium.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Experimental animals
As described in our earlier study (Jänkälä et al., 2000Go), 96 2-month-old male Wistar rats (Laboratory Animal Center, University of Helsinki) were randomly assigned to 12 experimental groups. The experimental groups were as follows: controls (C), ethanol [E; 1 g/kg body weight intraperitoneally (i.p.) as 12% w/v, in saline once a day], calcium carbimide (CC; 100 mg/kg of diet) and E + CC (ethanol 1 g/kg i.p. once a day + calcium carbimide 100 mg/kg of diet). Each of these four experimental groups was divided into three subgroups based on duration of treatment, i.e. 2, 5 and 8 days. The calcium carbimide diet started in the morning on day 1, ethanol on day 2. This way, in the 2-day treatment group rats received calcium carbimide for 2 days and ethanol for 1 day, in the 5-day treatment group rats received calcium carbimide for 5 days and ethanol for 4 days, and in the 8-day treatment group for 8 days and 7 days. Blood samples for ethanol and acetaldehyde measurements were taken from the tail vein 2 h after the alcohol injection 1 day before decapitation. After decapitation, hearts were removed within 60 s, weighed and the free walls of the left ventricles were immediately frozen in liquid nitrogen for later RNA isolation. All tissue samples were stored at –80°C until mRNA analysis. The study protocols were accepted by the National Public Health Institution Animal Care and Use Committee, Helsinki, Finland.

Blood ethanol and acetaldehyde measurements
Tail blood samples were haemolysed and their ethanol and acetaldehyde contents were measured with headspace gas chromatography on the same day the samples were collected (Eriksson et al., 1977Go). The detection limits for blood levels were 100 µM for ethanol and 1 µM for acetaldehyde.

Apoptosis ELISA assay
For quantification of DNA fragmentation, specific determination of cytosolic mononucleosomes and oligonucleosomes was performed using a commercial quantitative sandwich enzyme-linked immunosorbent assay kit (Boehringer Mannheim, Germany). Ten mg of frozen heart left ventricular free wall tissue were homogenized at room temperature in 200 µl of the lysis buffer supplied with the kit, using a glass homogenizer. After incubation for 30 min at room temperature the homogenate was centrifuged at 200 g for 10 min. The activity in the supernatant was measured immediately, all samples in one assay, for mono- and oligonucleosomes according to the manufacturer's recommendations.

Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) using internal standards
Total RNA was extracted from frozen muscle samples by using a modification of the acid guanidium thiocyanate– phenol–chloroform extraction (RNAzol B; Tel-Test, Friendswood, TX, USA). Frozen muscles were weighed and homogenized with Polytron (Kinematika, Littau, Switzerland) and RNA isolation was performed according to the manufacturer's recommendations, except that the actual RNA extraction phase was performed twice to eliminate all genomic DNA contamination. The total RNA concentration was assessed spectrophotometrically (Gene Quant; Pharmacia Biotech, Finland).

One µg of total sample RNA was reverse-transcribed to cDNA with both oligo-dT (Pharmacia Biotech) and random hexamer primers using Superscript II enzyme (Life Technologies). To evaluate the efficiency of the reverse transcription reaction, i.e. the amount of cDNA synthesized, a known amount of control RNA was added to each reaction tube as an internal standard.

Control RNA was produced by in vitro transcription by first constructing synthetic DNA templates that contained two primer pairs complementary to those used to amplify cDNA of interest. DNA templates also contained sequences for the bacteriophage T7 promoter at the 5' end for transcription into RNA and a poly A tail at the 3' end to facilitate RT by oligo-dT. Control RNA product was separated from synthetic DNA templates after in vitro transcription using phenol/chloroform extraction and ethanol precipitation after incubation with RNase-free DNase (Promega, Madison, Wisconsin, USA) (Feldman et al., 1991Go). Control RNA were tested for DNA contamination by performing RT–PCR without reverse transcriptase enzyme.

The cDNA was amplified in a DNA Thermal Cycler (Perkin Elmer) using the following reagents in a 100 µl reaction: 1 x PCR buffer (pH 8.8, 1.5 mM MgCl2) (Finnzymes), 200 µM dNTS, 250 ng (bax, bcl-2, p21 and TNF-{alpha}), 150 ng (ANP and BNP), or 100 ng (p53), each 5' and 3' primers and 2.5 U Dynazyme DNA polymerase (Finnzymes, Finland). A trace amount of {gamma}32P-labelled 3' primer was added to provide ~1.5 x 106 c.p.m. per reaction to label the DNA. In this way, each synthesized DNA strand was radiolabelled, as both sample and control cDNA included sequences complementary to primer. The first cycle started with a 4-min denaturation at 96°C. In the following cycles, each step lasted for 1 min: denaturation at 96°C, primer annealing at 60°C (p21 and bax), 49°C (bcl-2), 55°C (p53) or 57°C (TNF-{alpha}), and the synthesis step at 72°C. Oligonucleotide PCR primers complementary to the rat genes encoding bax (Tilly et al., 1995Go), bcl-2 (Sato et al., 1994Go), p21 (El-Deiry et al., 1993Go), p53 (Soussi et al., 1988Go) and TNF-{alpha} (Shirai et al., 1989Go) are the following: 5'-AAC ATG GAG CTG CAG AG-3' (bax sense), 5'-CTT GAG CAC CAG TTT GC-3' (bax antisense), 5'-CTT TGC CAC GGT GGT GGA-3' (bcl-2 sense), 5'-TCC GTT ATC CTG GAT CC-3' (bcl-2 antisense), 5'-ATC GAG ACA CTC AGA GCC ACA-3' (p21 sense), 5'-GAA GTC AAA GTT CCA CCG TTC T-3' (p21 antisense), 5'-GGG ACA GCC AAG TCT GT-3' (p53 sense), 5'-CTT GTA GAT GGC CAT GGC-3' (p53 antisense), 5'-CCA CGT CGT AGC AAA CCA CCA AG-3' (TNF-alpha sense), 5'-CAG GTA CAT GGG CTC ATA CC-3' (TNF-{alpha} antisense). Since internal controls also contained the complementary sequence, the PCR primers amplified both sample and control cDNA. Comparing the levels of radioactivity, the mRNA level of interest could then be calculated from the known amount of control RNA by using values from the exponential cycles (Feldman et al., 1991Go).

In using quantitative PCR, it is mandatory that co-linear amplification is obtained with internal standard. To ensure this and that measurements were performed during the exponential phase of amplification, 10 µl of each PCR reaction mixture were removed every fourth cycle during cycles 12–32. PCR product aliquots were electrophoresed on 4.25% (w/v) NuSieveGTG gel (FMC Bioproducts). The amplification products of the control cDNA and the cDNA of the sample were different in size, and therefore could be separated electrophoretically. Gels were stained with ethidium bromide and amplification products were visualized by UV light. Bands representing the gene and the control were excised from the gel separately and the radioactivity of each band was determined by Cerencov counting. The values from the cellular and internal control bands were plotted on a semi-logarithmic scale against the number of amplification samples. Parallel curves indicate that despite a difference in size, the two templates were amplified with comparable amplification efficiencies. Comparing the levels of radioactivity, the mRNA level of interest could then be calculated from the known amount of control RNA by using values from the exponential cycles (Feldman et al., 1991Go).

Drugs
Calcium carbimide (Dipsan®) was purchased from Cyanamide Canada Inc. (Montreal).

Statistical analysis
All the data are presented as means ± SEM. The differences between study groups were analysed by two-tailed Mann– Whitney U-test. P < 0.05 was considered to be significant.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Blood ethanol and acetaldehyde levels
The blood ethanol and acetaldehyde concentrations were measured one day before killing the animals and 2 h after ethanol injection. The concentrations of ethanol and acetaldehyde in the 2-day treatment group were: ethanol-treated (E) group: ethanol 2.6 ± 0.6 mM, acetaldehyde 2.1 ± 1.6 µM; combined ethanol + calcium carbimide-treated (E + CC) group: ethanol 10.6 ± 0.8 mM, acetaldehyde 205 ± 46 µM. In the 5-day treatment group: E: ethanol 2.0 ± 0.4 mM, acetaldehyde not detectable; E + CC: ethanol 9.2 ± 0.6 mM, acetaldehyde 107 ± 25 µM. In the 8-day treatment group: E: ethanol 5.7 ± 0.7 mM, acetaldehyde not detectable; E + CC: ethanol 12.8 ± 0.5 mM, acetaldehyde 418 ± 77 µM. In other experimental groups (C, CC) ethanol and acetaldehyde levels were not detectable. The concentrations of ethanol and acetaldehyde of the 2- and 8-day treatment groups have been published previously (Jänkälä et al., 2000Go).

Body and heart weights
The body and heart weights of the 2- and 8-day experiments have been published previously (Jänkälä et al., 2000Go). Total body weight increased in the control groups. Calcium carbimide treatment alone as well as combined with ethanol treatment had a statistically significant inhibitory effect on total body weight increase. This is confirmed by the present data showing that, also in the 5 days experiment, calcium carbimide treatment alone or combined with ethanol inhibited the total body weight increase observed in controls (6.3 ± 0.8% and 0.4 ± 1.3%, respectively vs 12.7 ± 1.0%; P < 0.005 and P < 0.001, respectively). Calcium carbimide treatment alone had a negative effect on heart weight, but heart to body weight ratio did not change significantly (Jänkälä et al., 2000Go).

Quantification of cytosolic DNA fragmentation by sandwich enzyme immunoassay
As seen in Fig. 1Go, at 5 days, both ethanol alone and calcium carbimide alone increased the relative amount of apoptotic nucleosomes slightly (15 and 11%, respectively) but not significantly. Combined treatment with ethanol and calcium carbimide, however, increased this index significantly (P < 0.05) by 23% above that in control rats.



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Fig. 1. Effects of ethanol, calcium carbimide and combined ethanol + calcium carbimide treatment on the amount of apoptotic nucleosomes in heart left ventricules in the 5-day treatment group.

Data are shown as means ± SEM, n = 8 per group. C, control; E, ethanol (1 g/kg body weight); CC, calcium carbimide (100 mg/kg of diet). *P < 0.05 as compared with the control group.

 
bax mRNA levels
As shown in Fig. 2aGo, ethanol treatment (1 g/kg/day) increased left ventricular bax mRNA levels by 37%, though not significantly, whereas calcium carbimide treatment (100 mg/kg diet) increased it significantly by 31% (P < 0.05), compared with the control group at 2 days. Combined ethanol and calcium carbimide treatment increased bax mRNA level by 56% compared to the control group (1.62 ± 0.10 vs 1.04 ± 0.10 x 107; P < 0.01). In the 8-day experiment, ethanol- and calcium carbimide-treated groups both had 37% higher bax mRNA levels compared with the control group (P < 0.05 and not significant, respectively). Combined ethanol and calcium carbimide treatment elevated bax mRNA level by 38%, compared with controls (1.44 ± 0.10 vs 1.04 ± 0.06 x 107; P < 0.005) (Fig. 2bGo).



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Fig. 2. Effects of ethanol, calcium carbimide and combined ethanol + calcium carbimide treatment on heart left ventricular bax mRNA levels.

(a) Two-day treatment group; (b) 8-day treatment group. Data are shown as means ± SEM, n = 8 per group. C, control; E, ethanol (1 g/kg body weight); CC, calcium carbimide (100 mg/kg of diet). *P < 0.05, **P < 0.01, ***P < 0.005 as compared with the control group.

 
bcl-2 mRNA levels
In the 2-day treatment group, there were no statistically significant changes in bcl-2 mRNA levels (C: 0.94 ± 0.06 x 107; E: 0.98 ± 0.07 x 107; CC: 1.02 ± 0.08 x 107; E + CC: 0.98 ± 0.07 x 107). In the 8-day experiment, ethanol treatment slightly increased bcl-2 mRNA concentration (by 14%) and calcium carbimide treatment by 22%; neither increase was significant. However, combined E + CC treatment significantly increased bcl-2 mRNA level by 36% as compared to the controls (1.01 ± 0.10 vs 0.74 ± 0.09 x 107; P < 0.05) (Fig. 3aGo).



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Fig. 3. Effects of ethanol, calcium carbimide and combined ethanol + calcium carbimide treatment on heart left ventricular (a) bcl-2 mRNA levels in the 8-day treatment group or (b) bax/bcl-2 ratio in the 2-day treatment group.

Data are shown as means ± SEM, n = 8 per group. C, control; E, ethanol (1 g/kg body weight); CC, calcium carbimide (100 mg/kg of diet). *P < 0.05 as compared with the control group.

 
bax to bcl-2 mRNA levels ratio
In the 2-day experiment, ethanol and calcium carbimide treatments alone induced a non-significant increase in the bax/bcl-2 mRNA levels ratio by 39 and 29%, respectively, compared to the control group. Combined E + CC treatment increased the ratio by 57%, as compared to the controls (1.72 ± 0.19 vs 1.10 ± 0.17; P < 0.05) (Fig. 3bGo). At 8 days, ethanol, calcium carbimide or E + CC exerted no significant effects on bax/bcl-2 mRNA ratios, as compared to the control group (data not shown).

p21 mRNA levels
There were no statistically significant changes in p21 mRNA concentrations in either ethanol- or calcium carbimide-treated groups at 2 days (Fig. 4aGo). Combined ethanol and calcium carbimide treatment increased p21 mRNA level by 25% compared with the control group (1.16 ± 0.05 vs 0.93 ± 0.06 x 107; P < 0.05) (Fig. 4aGo). In the 8-day experiment (Fig. 4bGo), the ethanol- and calcium carbimide-treated groups both had 11 and 26% lower p21 mRNA levels, respectively, compared with control group, though neither decrease was significant. However, combined ethanol and calcium carbimide treatment significantly decreased p21 mRNA level by 24% compared with controls (0.74 ± 0.07 vs 0.98 ± 0.05 x 107; P < 0.05) (Fig. 4bGo).



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Fig. 4. Effects of ethanol, calcium carbimide and combined ethanol + calcium carbimide treatment on heart left ventricular p21 mRNA levels. (a) Two-day treatment group; (b) 8-day treatment group.

Data are shown as means ± SEM, n = 8 per group. C, control; E, ethanol (1 g/kg body weight); CC, calcium carbimide (100 mg/kg of diet). *P < 0.05, **P < 0.01 as compared with the control group.

 
p53 mRNA levels
In the 2-day treatment group, there were no statistically significant changes in p53 mRNA levels (C: 1.88 ± 0.08 x 106; E: 2.04 ± 0.10 x 106; CC: 1.74 ± 0.08 x 106; E + CC: 1.94 ± 0.14 x 106). In the 8-day experiment, ethanol or calcium carbimide treatment alone had no significant effect on p53 mRNA levels (1.91 ± 0.10 and 2.04 ± 0.09 x 106, respectively). Combined E + CC treatment, however, increased significantly p53 mRNA level by 15% as compared to the controls (2.26 ± 0.08 vs 1.97 ± 0.05 x 106; P < 0.005) (Fig. 5Go).



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Fig. 5. Effects of ethanol, calcium carbimide and combined ethanol + calcium carbimide treatment on heart left ventricular p53 mRNA levels in the 8-day treatment group.

Data are shown as means ± SEM, n = 8 per group. C, control; E, ethanol (1 g/kg body weight); CC, calcium carbimide (100 mg/kg of diet). ***P < 0.005 as compared with the control group.

 
TNF-{alpha} mRNA levels
TNF-{alpha} mRNA levels tended to be lower in all the experimental groups as compared to controls. In the 8-day treatment groups, the level remained 9% lower in the ethanol group than in the control group (0.67 ± 0.08 vs 0.73 ± 0.08 x 105). However, none of these differences was significant.


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Neither the mechanism nor the specific toxic agent that induces AHMD has been established. One potential candidate is acetaldehyde (AcH) which is the first metabolite of ethanol. Liang et al. (1999) demonstrated that a 4-fold increase in cardiac acetaldehyde levels greatly enhanced the rate of onset of AHMD in a transgenic rodent model. In our earlier studies with rats as well as with isolated rat heart model (Langendorff), we have demonstrated the induction of cardiac hypertrophy-associated genes by ethanol or acetaldehyde (Jänkälä et al., 2000Go, 2001Go). Previous studies by others have also shown that AcH can either increase heart rate, cardiac output, coronary blood flow and left ventricular pressure, or depress myocardial contraction, alternate intracellular calcium mobilization and inhibit conduction (Brown et al., 1996Go; Patel et al., 1997Go; Ren et al., 1997Go). Interestingly, many patients with AHMD have circulating antibodies to cardiac acetaldehyde-protein adducts (Harcombe et al., 1995Go). AcH has also been shown to inhibit cell growth (Zimmermann et al., 1995). Typically, growth inhibition is accompanied by an increased probability of apoptosis (Wu et al., 1997Go). We have previously reported that growth of rats is greatly inhibited by combined ethanol and CC treatment (Jänkälä et al., 2000Go).

In the present study, a 5-day treatment with either ethanol or calcium carbimide alone tended to increase the relative amount of apoptotic nucleosomes, though these increases were not statistically significant. However, combined E + CC treatment with a concomitant increase in blood acetaldehyde concentration increased the index significantly by 23%. This result suggests that acetaldehyde might have a deleterious effect on the heart by inducing apoptosis. In accordance with this, it has been shown that, in cultured ovary cells, chronic exposure to acetaldehyde increases the rate of cell death in both a cumulative and a dose-dependent way (Zimmerman et al., 1995Go). In the present study, combined E + CC treatment for 8 days also increased p53 mRNA levels by 15% as compared to the controls. It is known that elevated p53 can result in either growth arrest or apoptosis (White, 1996Go). In the present study, apoptosis is activated, but probably also growth is inhibited, as significantly lower heart and body weights were observed in the 8-day treatment group.

Atrial natriuretic peptide (ANP) gene expression is a sensitive indicator of cardiac damage (Liang et al., 1999Go). We have previously reported that combined E + CC treatment induces rat left ventricular ANP gene expression after 2 and 8 days, probably by an acetaldehyde-dependent mechanism (Jänkälä et al., 2000Go). Thornburn et al. (1993) reported that over-expression of activated p21 in cultured neonatal rat cardiocytes results in activation of ANP gene transcriptional activity. The p21 protein is necessary for the G1 arrest in cells treated with DNA-damaging agents (El-Deiry et al., 1993Go), thus exerting a protective function in situations of stress (Gorospe et al., 1997Go). In the present study, in the 2-day experiment, p21 mRNA was markedly up-regulated in the E + CC group. After 5 days of experiment, the same treatment was shown to induce apoptosis in heart left ventricles, suggesting that p21 is involved in this process. However, in our study, elevated p21 could not protect myocytes from apoptosis. This finding is in agreement with a number of studies in which marked up-regulation of endogenous p21 has been reported to be coupled with induction of apoptosis in various cell types, including myocytes (El-Deiry et al., 1994Go; Shao et al., 1995Go; Blagosklonny et al., 1996Go). We postulate that acetaldehyde-induced DNA damage or biochemical stress is too overwhelming to be compensated by p21 up-regulation, and is thus followed by induction of apoptosis. In the 8-day experiment, E + CC-treated rats had 24% lower p21 mRNA levels than controls. This tendency was also evident in the ethanol- and, in particular, in the calcium carbimide-treated groups. Bcl-2 over-expression is related to suppression of p21 (Bukholm et al., 1997Go), and therefore the reversal of p21 induction seen in our present study may be a result of the observed up-regulation of bcl-2.

Bax is a DNA-damage inducible gene that promotes cell death, whereas bcl-2 suppresses apoptosis in part by heterodimerization with bax (Oltavi et al., 1993Go). In the present study, at 2 days, bax mRNA levels as well as the bax/bcl-2 ratio were significantly increased in the E + CC group, compared with controls. This was followed by an elevated number of apoptotic nucleosomes at 5 days. After 8 days, both bax and bcl-2 were higher in the E + CC group, than in controls, and the bax/bcl-2 ratio was only slightly higher than in controls. Similarly, with p21 activation, increased production of bcl-2 may be considered as an active attempt to control apoptosis-inducing signals and enhance survival (Hockenbery et al., 1993Go). An important property of the bcl-2 protein is to suppress expression of p21 (Upahhyay et al., 1995Go), which is in agreement with our 8-day experiment where increased bcl-2 mRNA levels accompanied decreased p21 mRNA levels. It is noteworthy that the mRNA levels of genes bcl-2, bax and p21 were very close to each other in the control groups. Interestingly, ethanol as well as calcium carbimide alone (at 2 and 8 days) induced bax gene expression, but neither of these agents changed the bax/bcl-2 ratio significantly. It has been postulated that it is the increased bax/bcl-2 ratio that increases the probability for a myocardial cell to undergo apoptosis (Condorelli et al., 1999Go).

TNF-{alpha} has been demonstrated to have a hypertrophic or pro-apoptotic effect on rodent cardiomyocytes (Yokoyama et al., 1997Go; Bozkurt et al., 1998Go). In the present study, TNF-{alpha} mRNA tended to be lower in all of the treatment groups in the 2-day experiments and in the ethanol group in the 8-day experiment than in control groups, but these differences were not statistically significant. Gutierrez-Ruiz et al. (1999) showed that ethanol or acetaldehyde exposure for 72 h reduced TNF-{alpha} mRNA expression in HepG2 cells, after an initial (24 h) increase. TNF is also thought to be involved in the genesis of alcoholic liver injury (McClain et al., 1993Go). However, in this study with a shortest experiment of 2 days, we found no evidence that TNF-{alpha} induction would be involved in the mechanism of acetaldehyde- or ethanol-induced myocardial apoptosis.

In the present study, ventricular mRNA levels tended to be increased/decreased in some ethanol- or calcium carbimide (for 2 or 8 days)-treated groups as compared to controls. It is noteworthy that the rats treated with ethanol alone had detectable but low acetaldehyde levels in blood. Also, Eriksson (1985) showed that calcium carbimide treatment alone elevates endogenous acetaldehyde levels. Such endogenous elevation most likely occurred also during the present conditions, although the acetaldehyde levels did not reach the detection limit of 1 µM. However, we cannot exclude the possibility that results in the E + CC group were at least partly due to combinatorial effects of the two agents other than generation of acetaldehyde. Only studies performed on tissue and cellular levels will establish whether acetaldehyde is the actual triggering molecule.

In summary, this study showed that combined treatment with ethanol and calcium carbimide raises blood acetaldehyde levels, induces left ventricular apoptosis and alters the expression of genes involved in the regulation of apoptosis, growth and repair of cellular damage. In our study, bcl-2 and p53 mRNA up-regulation occurred later than the induction of p21, ANP and bax genes. These results suggest that, in this model, apoptosis is induced by a p53-independent mechanism. It also seems that apoptosis occurs in a TNF-{alpha}-independent way. We suggest that acetaldehyde might participate in the development of AHMD by inducing apoptosis and disrupting the homeostasis of growth and cell cycle progression. It is noteworthy that ethanol alone or calcium carbimide alone was also capable of increasing the level of bax mRNA, but their effect on the bax/bcl-2 ratio was insignificant. Since this experimental setting is not comparable to prolonged alcohol misuse, further studies are needed to clarify whether prolonged exposure to ethanol is sufficient to induce apoptosis in heart left ventricles.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This work was supported by grants from The Finnish Foundation for Alcohol Studies, Finnish Foundation for Promoting Clinical Chemistry, the Helsinki University Central Hospital EVO-grant and the Maud Kuistila Foundation.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed at: Jorvi Hospital, Clinical Laboratory, Turuntie 150, SF-02740 Espoo, Finland. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Baroni, G. S., Marucci, L., Benedetti, A., Mancini, R., Jezequel, A. M. and Orlandi, F. (1994) Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver. Journal of Hepatology 20, 508–513.[ISI][Medline]

Blagosklonny, M. V., Schulte, T., Nguyen, P., Trepel, J. and Neckers, L. M. (1996) Taxol-induced apoptosis and phosphorylation of bcl-2 protein involves c-raf-1 and represents a novel c-raf-1 signal transduction pathway. Cancer Research 56, 1851–1854.[Abstract]

Bozkurt, B., Kribbs, S. B., Clubb, F. J. Jr, Michael, L. H., Didenko, V. V., Hornsby, P. J., Seta, Y., Oral, H., Spinale, F. G. and Mann, D. L. (1998) Pathophysiologically relevant concentrations of tumor necrosis factor alpha promote progressive left ventricular dysfunction and remodelling in rats. Circulation 97, 1382–1391.[Abstract/Free Full Text]

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