Evidence of apoptosis in chronic alcoholic skeletal myopathy

Evidence of apoptosis in chronic alcoholic skeletal myopathy

Evidence of Apoptosis in Chronic Alcoholic Skeletal Myopathy ´ NDEZ-SOLA ` , MD, JOSE´-MARI´A NICOLA ´ S, MD, JOAQUIM FERNA ´ , MD, GLORIA GARCI´A, MD...

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Evidence of Apoptosis in Chronic Alcoholic Skeletal Myopathy ´ NDEZ-SOLA ` , MD, JOSE´-MARI´A NICOLA ´ S, MD, JOAQUIM FERNA ´ , MD, GLORIA GARCI´A, MD, FRANCESCA FATJO ´ N ESTRUCH, MD, ESTHER TOBI´AS, EMILIO SACANELLA, MD, RAMO ´ RQUEZ, MD EVA BADIA, PHD, AND ALVARO URBANO-MA Apoptosis is a common mechanism of programmed cell death that has been implicated in the pathogenesis of alcohol-induced organ damage. Experimental studies have suggested alcohol-mediated apoptosis in cardiac muscle. The relationship between skeletal and cardiac muscle damage in alcoholism led us to consider the possible role of apoptosis in the pathogenesis of skeletal myopathy. We prospectively evaluated apoptosis in skeletal muscle biopsies of 30 consecutively selected male high-dose well-nourished chronic alcohol consumers and 12 nonalcoholic controls. Alcohol consumption, evaluation of muscle strength by myometry, and deltoid muscle biopsy with immunohistochemical and morphometric analysis were performed. Apoptosis was assessed by TUNEL, BAX, and BCL-2 immunohistochemical assays. Chronic alcoholics compared with controls showed a significantly higher apoptotic index in TUNEL (2.35% ⴞ 0.25% versus 0.18% ⴞ 0.03%, P < 0.001), BAX (9.16% ⴞ 2.00% versus 0.66% ⴞ 0.22%, P < 0.001), and BCL-2 muscle assays (8.08%

ⴞ 0.20% versus 0.83% ⴞ 0.20%, P ⴝ 0.001), respectively. In addition, these apoptotic indexes were higher in alcoholics with skeletal myopathy compared with in those without skeletal myopathy (3.04% ⴞ 0.36% versus 1.65% ⴞ 0.26%, P ⴝ 0.004 for TUNEL; 17.00% ⴞ 2.78% versus 1.33% ⴞ 0.22%, P < 0.001 for BAX; and 15.13% ⴞ 3.2% versus 1.03% ⴞ 0.33%, P < 0.001 for BCL-2 assays, respectively). We conclude that apoptosis is present in the skeletal muscle of high-dose alcohol consumers, mainly in those affected by myopathy. However, the specific pathogenic mechanism of apoptosis in chronic skeletal myopathy in alcoholics remains to be elucidated. HUM PATHOL 34:1247-1252. © 2003 Elsevier Inc. All rights reserved. Key words: apoptosis, ethanol, skeletal muscle. Abbreviations: SEM, standard error of the media; TNF, tumoral necrosis factor; NF, nuclear factor; TUNEL, deoxyribonucletidyltransferase-mediated-dUTP biotin nick-end labeling; NADH, nicotinomide adenine-dinucleotide terazolium reductase.

Apoptosis is a common mechanism of programmed cell death involved in the pathogenesis of a variety of disorders.1,2 Apoptosis has recently been implicated in the mechanisms leading to alcohol-induced organ damage.3,4 In fact, previous studies showed alcohol-induced apoptosis in hepatocytes,4,5 endothelial cells,6,7 thymocytes,8 lymphocytes,9 and neural cells.10,11 Ethanol may activate apoptosis through diverse mechanisms, with the increase in tumoral necrosis factor (TNF-␣),12 up-regulation of FAS,11,13 activation of nuclear-factor ␬-␤ (NF-␬␤),6,14 activation of mitochondrial caspases,7,9 and disruption of intracellular [Ca]2⫹ transients15 being the most frequently implicated. Chronic skeletal myopathy is a frequent disease that is observed in 30% to 50% of high-dose chronic alcohol consumers, producing progressive muscle weakness and atrophy.16,17 It has been related to a direct dose-dependent toxic effect of ethanol.18 However, its pathogenesis has not been clearly established3 and is probably multifactorial and related to disruption

of calcium-induced intracystolic (i.c.) transients,15 oxidative muscle damage,19 and protein synthesis and degradation processes.20 In experimental studies, Ja¨nka¨la¨ et al21 recently described the induction of myocardial apoptosis in rats through combined calcium carbimide and ethanol treatment. Because myocardial and skeletal muscle have structural and functional similarity and we previously observed a correlation in heart and skeletal muscle involvement in chronic alcoholics,22, we hypothesize that apoptosis plays a role in the pathogenic mechanism of alcohol-induced skeletal muscle damage. The present study was designed to assess the specific role of apoptosis in ethanol intake in the pathogenesis of skeletal muscle damage in high-dose, wellnourished chronic alcohol consumers. We evaluated deltoid muscle immunohistochemical activities related to apoptosis with assessment by the TUNEL assay to detect the presence of apoptosis and BAX and BCL-2 activities to detect induction or protection from preapoptotic mechanisms, respectively.

From the Department of Medicine, Hospital Clı´nic, Institut d’Investigacions Biome`diques August Pi i Sunyer, University of Barcelona, Barcelona, Spain. Accepted for publication July 16, 2003. Supported by research grants 02/533, 02/535, and G03/140 from Fondo de Investigaciones Sanitarias, Spain; Red CIEN from ISC III, Spain; and CUR 2001/SGR-279 from Generalitat de Catalunya, Spain. Address correspondence and reprint requests to Joaquim Ferna´ndez-Sola`, MD, Department of Internal Medicine, Hospital Clı´nic, Villarroel 170, 08036 Barcelona, Spain. © 2003 Elsevier Inc. All rights reserved. 0046-8177/03/3412-0005$30.00/0 doi:10.1016/j.humpath.2003.07.017

MATERIALS AND METHODS Patient and Control Selection Over a 1-year period, we evaluated 612 patients who were seen in the Alcoholism Unit of the Hospital Clı´nic of Barcelona. This unit deals with ambulatory patients who seek assistance in terminating their dependence on alcohol and who do not suffer from another obvious disease. Patients with overt alcohol-related diseases such as uncompensated liver disease, heart failure, peripheral neuropathy, malnutrition, or other disorders were referred to other clinics. On each Monday, we consecutively selected the first male patient aged

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less than 65 years who consulted this unit for assistance and who reported continuous ethanol consumption of ⱕ100 g/d for a minimum of the prior 10 years. According to previous studies, the development of alcoholic skeletal myopathy is frequent in this situation.16,18,22 Of 55 consecutive alcoholics initially selected, 7 refused to participate in the study, another 6 refused to undergo open muscle biopsy, and 8 presented caloric or protein malnutrition parameters. We also excluded 1 HIV-positive subject and excluded 3 other subjects for using illicit drugs. Thus, 30 active alcoholics were finally included in the study. During the same period, 67 nonalcoholic middle-aged male patients were studied in the Muscle Research Unit of the Hospital Clı´nic because of persistent muscle fatigue with normal values of creatine kinase (⬍ 150 IU/L). Thirty were of ages similar (mean ⫾ 2 years) to those of the alcoholics and did not show evidence of significant muscle weakness as evaluated by myometry (deltoid muscle strength of ⬎22 kg in the nondominant arm). These subjects reported a weekly consumption of ethanol of ⬍20 g, without binge intake, and were not affected by other causes of skeletal myopathy. To obtain a more accurate diagnostic, 20 of these patients accepted submission to an open deltoid muscle biopsy. In 8 cases, minimal nonspecific muscle changes were detected (muscle fiber atrophy, fiber variability, or isolated internal nuclei). Thus, 12 of these patients were found to have a normal muscle biopsy without any detectable alteration in the conventional histologic or immunohistochemical study and were included as controls. None of the patients or controls received any medication during the 3 previous months or during the investigation. Patients and controls were all Caucasian men of Spanish descent who lived with their families in or around Barcelona and had histories of stable employment. None were indigent. The study protocol was approved by the institutional review board of the Hospital Clı´nic. All subjects voluntarily agreed to participate in the study and gave informed consent for the various procedures.

Clinical and Nutritional Evaluation A detailed history of ethanol intake was obtained for each patient by using a structured questionnaire (time-line follow-back method), and data were confirmed in consultation with family members, as previously reported.16,18 Because the report of the quantity of ethanol consumed by alcohol abusers is known to be somewhat unreliable, we assumed the alcoholic intake reported to be not totally accurate. However, in our experience, because alcoholics tend not to exaggerate (and often underreport) the amount that they continue to drink, we considered the reports to be minimal estimates. Assessment of nutritional status was performed considering anthropometric caloric and protein nutritional parameters, as reported elsewhere.16,22 Patients were considered to have caloric malnutrition if their body weight was ⬍90% of their ideal weight or if the calculated lean body mass was ⬎10% below the control value. Protein malnutrition was diagnosed when the patients had abnormal values for 3 of the following parameters: hemoglobin, lymphocyte count, total protein, albumin, prealbumin, retinol-binding protein, or transferrin.

Skeletal Muscle Studies On admission, we measured deltoid muscle strength in the nondominant arm with an electronic myometer (Penny and Giles, RingWood, UK), which measures muscular force

TABLE 1. Histological Criteria and Degree of Alcoholic Myopathy (in Absence of Other Causes of Skeletal Myopathy) Degree of Myopathy Mild

Moderate Severe

Criteria Scattered myocytolysis or at least 4 of the following criteria: Fiber atrophy Inflammation Fiber regeneration Fiber size variability Internal nuclei ⬎ 10% of fibers Moth-eaten fibers ⬎ 10% of fibers Subsarcolemmal deposition ⬎ 10% of fibers Type I fiber predominance Fat deposition in endomisia Tubular aggregates Scattered myocytolysis and at least 4 of the previous criteria Myocytolysis in ⱕ5% of the fibers

against a fixed resistance.18 An open-biopsy specimen was taken from the deltoid muscle of the nondominant arm of each patient and control. Cryostat sections of deltoid-muscle biopsy specimens were stained with hematoxylin and eosin, modified Gomori trichrome, the periodic acid–Schiff reaction, and oil–red O and were stained for ATPase (at pH 4.3, 4.6, and 9.4), nicotinamide adenine-dinucleotide terazolium reductase (NADH) and nonspecific esterase activity. Fiber atrophy was evaluated considering mean area and transversal diameter by computer-assisted morphometry with NIH image software (National Institutes of Health, Washington, DC), according to a method described elsewhere.23 The specimens were randomly coded and were blindly evaluated by 2 independent observers with no knowledge of the clinical data of the patients. The histologic diagnosis of myopathy was established by means of a validated scale according to the modified criteria of Mastaglia and Walton,16,23 which includes evaluation of fiber size, specific fiber-type atrophy, the presence of myocytolysis, inflammation, and metabolic or oxidative disturbances of myofibers (Table 1). All samples were also graded on a semiquantitative scale as absent, mild, and moderate-severe.

Apoptosis Immunohistochemical Studies Skeletal muscle samples obtained from surgical biopsy were immediately frozen at ⫺80°C. Semi-thin cryostat sections of 6 ␮m were obtained with ultra-microtome and fixed by paraformaldehyde 4% PBS solution at pH 7.40. After PBS washing, sections were permeabilized with 0.1% Triton X-100 sodium citrate solution for 2 minutes at 4°C. Incubation with TUNEL (deoxyribonucleotidyl-transferase-mediated-dUTPbiotin nick-end labeling) was performed with the In Situ Cell Death Detection Kit (Boehringer, Manheim, Germany) for 60 minutes at 37°C inside a wet chamber, as described by the manufacturer. After washing with phosphate-buffered saline, a second incubation with Converter-Alkaline phosphatase was performed for 30 minutes under the same conditions. The reading process was performed, by adding Fast Red chromogen for 10 minutes, under microscopic control. Incubation with BAX and BCL-2 reagents were performed in semithin cryostat sections of 6 ␮m fixed by ketone at ⫺4°C. After primary antibody exposition (Biogenex, San Ramon, CA; AR 347-5R for BAX and AM 287-5R for BCL-2), link and labeling reactants were sequentially exposed for 20 minutes each (Kit

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TABLE 2. Epidemiological, Nutritional, and Laboratory Data of Chronic Alcoholics and Controls Data Age (y) Daily ethanol intake (g) Duration of alcoholism (y) Total lifetime dose of ethanol (kg ethanol per kg body weight) Deltoid muscular strength (kg) Creatine kinase (IU/L) Ideal body weight (% of real) Lean body mass (kg) Total protein (g/L) Albumin (g/L) Aspartate aminotransferase (IU/L) Alanine aminotransferase (IU/L) Gammaglutamyl transpeptidase (IU/L)

Alcoholics (n ⫽ 30)

Controls (n ⫽ 12)

52.8 ⫾ 1.3 159 ⫾ 10 24.3 ⫾ 1.2

54.3 ⫾ 1.2 NA NA

21.0 ⫾ 1.1 18.9 ⫾ 0.8 175 ⫾ 35 100.6 ⫾ 2.4 49.7 ⫾ 0.8 64.8 ⫾ 1.2 36.6 ⫾ 0.9 58.9 ⫾ 7.6 41.3 ⫾ 3.6 210.2 ⫾ 5.5

NA 25.4 ⫾ 1.2* 123 ⫾ 10 101.5 ⫾ 2.3 51.8 ⫾ 2.1 65.5 ⫾ 1.1 37.2 ⫾ 0.8 33.6 ⫾ 5.2* 24.2 ⫾ 3.3* 34.6 ⫾ 2.8**

NOTE. Data expressed as mean ⫾ SEM. Abbreviation: NA, not applicable. *P ⬍ 0.05 and **P ⬍ 0.001 compared with alcoholics.

Detection Supersensitive, Biogenex; QA000-5L), with a final chromogen (Fast Red) incubation of 10 minutes. Negative control slides were made in all the procedures by using the same process without reagent incubation. Positive controls were performed by using human palatinal amygdalar tissue, in which lymphoid cells take high TUNEL, BAX, and BCL-2 activities. The apoptotic index (AI) was determined by means of the percentage of apoptotic cells and calculated by dividing the total number of positive-staining myocyte nuclei in the TUNEL assay by the total number of the myocyte nuclei evaluated and multiplying this value by 100, as reported elsewhere by Narula et al.24 Cell nuclei at the edges of the tissues or in the interstitium were not counted. The count was made on at least 5 different fields (magnification ⫻250) of each sample, with at least 500 nuclei per field, according to the method described by Sandri et al.25 Positive cells for BAX and BCL-2 staining were considered in the presence of diffuse cytoplasmic hyperenhancement and subsarcolemmal or perinuclear deposition of the reagent, and were expressed as a percentage of positive to total evaluated cells. Interstitial deposition of these reagents and peripheral zones of the samples were not considered in the evaluation. BCL-2/BAX ratio was considered as a marker of global apoptosis activation, as suggested elsewhere.26

Statistical Analysis Descriptive analysis was performed in terms of mean ⫾ SEM. Differences between groups were evaluated by using the 2-tailed Student’s t test. Bonferroni’s correction was applied in multiple comparisons to adjust the type I error. Pearson’s correlation coefficients between the variables were calculated. All tests were performed with a bilateral significance level of P ⬍ 0.05. The software used for analysis was SPSS 10.0 (SPSS, Chicago, IL).

RESULTS A total of 30 high-dose chronic alcoholic men and 12 nonalcoholic controls were finally included in the study. Table 2 describes the main epidemiologic and

biochemical parameters of the alcoholic patients and controls. The mean age of the alcoholics was 52.8 years ⫾ 1.3 years. They reported a mean daily ethanol consumption of 159 g ⫾ 10 g, during a period of 24.3 years ⫾ 1.2 years, leading to a cumulated lifetime dose of ethanol of 21.0 kg ⫾ 1.1 kg of ethanol per kilogram of body weight. Twelve (40%) also smoked 1 to 2 packets per day during a mean of 10 years. The members of the control population were similar in age and smoking habits to the alcoholics but did not report any ethanol consumption. Serum biochemical studies were normal in alcoholics, except for a significant increase in aminotransferases and gammaglutamyl transpeptidase. No alcoholic or control individual presented caloric or protein malnutrition, and overall caloric and protein nutritional data were comparable between the patients and controls. Similar to what has been previously reported,16,18,22 deltoid muscle strength was significantly decreased in alcoholics with respect to controls (18.9 kg ⫾ 0.8 kg versus 25.4 kg ⫾ 1.2 kg, P ⬍ 0.05; Table 2). According to the established criteria (Table 1), we observed histologic changes of myopathy in 15 alcoholics (50%), considered to be mild in 9 cases and moderate-severe in the remaining 6 patients. Alcoholics with skeletal myopathy showed no differences in age, daily, and lifetime dose of ethanol consumption and duration of alcoholism compared with the case of alcoholics without skeletal myopathy (Table 3). However, the former presented significantly decreased deltoid muscle strength, as evaluated by myometry, than did the latter (17.1 kg TABLE 3. Epidemiological and Muscle Immunohistochemical Studies of Apoptosis in Chronic Alcoholics According to the Presence of Skeletal Myopathy Chronic Alcoholics

Parameter Age (y) Daily ethanol intake (g) Duration of alcoholism (y) Total lifetime dose of ethanol (kg per kg body weight) Deltoid muscle strength (kg) Creatine kinase (IU/L) TUNEL* BAX** BCL-2** BCL-2/BAX ratio

With Skeletal Myopathy (n ⫽ 15)

Without Skeletal Myopathy (n ⫽ 15)

Significance (P)

48.3 ⫾ 2.6

54.53 ⫾ 2.1

0.10

165 ⫾ 14

161 ⫾ 9

0.85

24.3 ⫾ 1.6

25.9 ⫾ 2.6

0.62

22.3 ⫾ 1.1

19.8 ⫾ 1.8

0.68

17.1 ⫾ 1.3

20.7 ⫾ 1.0

0.03

202 ⫾ 69 3.04 ⫾ 0.36 17.00 ⫾ 2.78 15.13 ⫾ 3.21 1.05 ⫾ 0.28

164 ⫾ 37 1.65 ⫾ 0.26 1.33 ⫾ 0.22 1.03 ⫾ 0.33 0.81 ⫾ 0.15

0.69 0.004 ⬍0.001 ⬍0.001 0.45

Results expressed as mean ⫾ SEM. *Results expressed as percentage of myocyte nuclei with positive reactivity. **Results expressed as percentage of myocytes with nuclear or cytoplasmic positive reactivity.

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FIGURE 1. TUNEL inmunohistochemical assay in skeletal muscle biopsy from a patient with skeletal myopathy. In the center (arrow), a myocyte apoptotic nuclei shows intense nuclear and perinuclear positive staining.

FIGURE 2. BAX inmunohistocemical assay in skeletal muscle biopsy from a patient with skeletal myopathy. Some nuclei take intense positive staining (arrows).

⫾ 1.3 kg versus 20.7 kg ⫾ 1.0 kg, P ⫽ 0.03). Deltoid muscle strength was not different in alcoholics with mild degree of skeletal myopathy compared with those without myopathy (16.8 kg ⫾ 1.3 kg versus 18.9 kg ⫾ 0.8 kg, respectively). However, alcoholics with moderate-severe myopathy showed significantly lower deltoid muscle strength than the other groups of alcoholics (14.2 kg ⫾ 1.3 kg versus 16.7 kg ⫾ 0.8 kg, respectively, P ⫽ 0.04). Serum creatine kinase values did not differ in alcoholics versus controls or between alcoholics with or without skeletal myopathy. However, these values were significantly higher in alcoholics with moderatesevere myopathy compared with those without myopathy (215 IU/L ⫾ 83 IU/L versus 135 IU/L ⫾ 38 IU/L, respectively, P ⬍ 0.001). Except for aminotransferases and gammaglutamyl transpeptidase, no significant differences were found in other biochemical and nutritional parameters comparing the different groups of alcoholics according to the presence and degree of skeletal myopathy. In the evaluation of the presence of muscle apoptosis with the TUNEL immunohistochemical assay (Fig 1), the apoptotic index (AI) of alcoholics was significantly higher than that of controls (2.35% ⫾ 0.25% versus 0.18 ⫾ 0.03%, P ⬍ 0.001). Similarly, BAX and BCL-2 AI (Figs 2 and 3) were significantly higher in chronic alcoholics than in controls (9.16% ⫾ 2.00% versus 0.66% ⫾ 0.22%, P ⬍ 0.001 for BAX; and 8.08% ⫾ 0.20% versus 0.83% ⫾ 0.20%, P ⫽ 0.001 for BCL-2 assay). The BCL-2/BAX ratio was slightly higher in alcoholics compared with controls (0.93 ⫾ 0.05 versus 1.33 ⫾ 0.36), without acquiring significant differences (Table 4). When comparing chronic alcoholics with skeletal myopathy to those without myopathy, all 3 AI were significantly higher in the former (3.04 ⫾ 0.36 versus 1.65 ⫾ 0.26, P ⫽ 0.004 for TUNEL; 17.00 ⫾ 2.78 versus 1.33 ⫾ 0.22, P ⬍ 0.001 for BAX; and 15.13 ⫾ 3.2 versus 1.03 ⫾ 0.33, P ⬍ 0.001 for BCL-2 assays, respectively). BCL-2/BAX ratio was not significantly different among alcoholics with skeletal myopathy compared with those without myopathy (1.05 ⫾ 0.28 versus 0.81 ⫾

0.15, respectively; Table 4). In addition, alcoholics without skeletal myopathy presented higher muscle AI in TUNEL and BAX, but not in BCL-2 assays, than controls (P ⬍ 0.001, P ⫽ 0.05, and P ⫽ 0.64 for TUNEL, BAX, and BCL-2 assays, respectively). DISCUSSION In the present study, we describe activation of apoptotic-regulating mechanisms (BAX and BCL-2) and also a higher apoptotic index (AI) measured by detection of DNA fragmentation by immunohistochemical TUNEL assay in skeletal muscle from high-dose chronic alcoholic men and controls. In addition, alcoholic patients with skeletal myopathy presented higher apoptotic indexes compared with alcoholics without skeletal myopathy. We selected well-nourished alcoholics and controls to avoid the potential confounding effect of malnutrition on skeletal muscle. Notably, this is the first study reporting evidence of apoptosis and activation of

FIGURE 3. BCL-2 immunohistocemical assay in skeletal muscle biopsy from a patient with skeletal myopathy. Nuclear (solid arrow) and cytoplasmic (dashed arrow) positive staining is seen.

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TABLE 4. Muscle Immunohistochemical Studies of Apoptosis in Chronic Alcoholics and Controls Immunohistochemical Study

Chronic Alcoholics (n ⫽ 30)

Controls (n ⫽ 12)

Significance (P)

TUNEL* BAX** BCL-2** BCL-2/BAX ratio

2.35 ⫾ 0.25 9.16 ⫾ 2.00 8.08 ⫾ 0.20 0.93 ⫾ 0.05

0.18 ⫾ 0.03 0.66 ⫾ 0.22 0.83 ⫾ 0.20 1.33 ⫾ 0.36

⬍0.001 ⬍0.001 0.001 0.23

Results expressed as mean ⫾ SEM unless otherwise noted. *Results expressed as percentage of myocyte nuclei with positive reactivity. **Results expressed as percentage of myocytes with nuclear or cytoplasmic positive reactivity.

apoptotic mechanisms in skeletal muscle in chronic alcohol consumers either with or without skeletal myopathy, and it reflects the possible pathogenic implication of apoptosis in alcohol-mediated skeletal muscle damage. The relatively high percentage of muscle apoptosis detected in the present study by TUNEL assay in alcoholics (2.35%) is in concordance with that observed in experimental studies.21 However, some degree of overdetection may be present, because the TUNEL assay may detect some reversible DNA fragmentation as an expression of increased activity of DNA repair in living cells.27 Another aspect to consider is that skeletal myocytes are syncytial multinuclear cells, in which the presence of nuclear apoptosis involving 1 nucleus does not necessarily suppose evolution to myofiber death. This situation is clearly different from that of other tissues with mononucleated cells such as hepatocytes or lymphocytes. In fact, the formation of DNA strand breaks precedes structural damage and is considered to be a marker of progression of the apoptotic process leading to cell necrosis.28 Apoptosis is a widespread pathogenic process that may be activated by a variety of stimuli, operating over several membrane receptors and signal transduction pathways. It leads to activation of a cascade of proteases (endonucleases, caspases) that execute a program of cellular self-destruction by cleaving cellular structures at specific sites in a strictly controlled manner.1,2 The local balance of proapoptotic (ie, BAX) and antiapoptotic stimuli (ie, BCL-2) decides the survival or death of each individual cell.29 Chronic alcohol consumption is one of the mechanisms that is known to potentially activate the apoptotic process, a fact previously described in a variety of cell types.4-11 The BCL protein family acts as the main regulator of the apoptotic pathway and may be either proapoptotic or antiapoptotic. BCL-2 overexpression inhibits apoptosis in response to a variety of signals30 and may decrease the final percentage of preapoptotic cells leading to apoptosis and necrosis, as observed in cardiac myocytes.24 On the contrary, BAX is a proapoptotic protein of the same BCL-2 family31 and induces cell death by acting on mitochondria. BAX binds to the permeability transition pore complex, involved in the

regulation of mitochondrial membrane permeability. A decreased BCL-2/BAX ratio has been shown to increase the probability of myocardial cell apoptosis.26 In the present study, BCL-2/BAX ratio, an index of global apoptotic activation, was slightly higher in alcoholic patients compared with in controls and also in alcoholics with skeletal myopathy as compared with those without myopathy, but these differences did not reach statistical significance. This fact supposes similar activation of proapoptotic and antiapoptotic BAX and BCL-2 factors, with other factors potentially influencing at some degree this alcohol-induced apoptotic process. We also observed a clear activation of TUNEL, BAX, and BCL-2 in high-dose chronic alcoholics, with good parallelism in the AI in these 3 assays, probably as expression of a common activation mechanism. Because these AI were higher in alcoholics than in controls, mainly in alcoholics with skeletal myopathy, independent of other factors, it seems reasonable to consider ethanol consumption to be a factor that is implicated in apoptosis activation in chronic alcoholics. Despite the evidence of higher TUNEL, BAX, and BCL AI in chronic alcoholics compared with in controls, we did not find significant correlation with age, muscle strength, biochemical or nutritional variables, or parameters of alcohol consumption. The lack of a significant correlation between AI and parameters of ethanol consumption may be due to the limitation in sample size and the homogeneous quantity of alcohol consumed by this group. It is still possible that BCL-2 and BAX activation is not directly related to alcohol-induced myocyte death, with other factors such as caspase, cytokine, or insulin-like growthfactor activation potentially influencing in this process.9,26,32 It is of note that the results of the present study do not apply to low-dose ethanol consumption and do not allow apoptosis to be designated as the only pathogenic mechanism leading to skeletal muscle damage in alcoholism, a situation that is supposedly multifactorial.33 The studies of apoptosis in this study were restricted to the described immunohistochemical methods (TUNEL, BAX, and BCL-2 assays) in frozen muscle samples. Other mechanisms of apoptosis were not tested. In summary, we observed a significantly increased apoptotic nuclear index (TUNEL assay) and altered the mechanisms that regulate apoptosis (BAX and BCL-2 assays) in skeletal muscle from high-dose chronic alcoholics, with this situation being more evident in alcoholics with skeletal myopathy. These findings suggest the implication of apoptosis in chronic skeletal muscle damage in alcohol misuse and require further studies to determine the specific mechanisms and factors influencing its development.33 REFERENCES 1. Thomson B: Apoptosis in the pathogenesis and treatment of disease. Science 267:1456-1462, 1995 2. Barinaga M: Forging a path to cell death. Nature 273:735-737, 1996 3. Molina P, McClain C, Valla D, et al: Molecular pathology and

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