TEENAGE ALCOHOL AND INTOXICATION DEBUT: THE IMPACT OF FAMILY SOCIALIZATION FACTORS, LIVING AREA AND PARTICIPATION IN ORGANIZED SPORTS

E. T. Hellandsjø Bu,*, R. G. Watten1, D. R. Foxcroft2, J. E. Ingebrigtsen3 and G. Relling4

The Bergen Clinics Foundation, Box 297, 5804 Bergen and University of Sport and Physical Education, Oslo,
1 Department of Health and Social Science, Lillehammer College and Institute of Psychology, Trondheim, Norway,
2 Oxford Centre for Health Research & Development, Oxford Brookes University, Oxford, UK,
3 Institute for Science in Sport, Norwegian University of Science and Technology, Trondheim and
4 Vestmo Treatment Centre for Substance Abuse, Ålesund, Norway

Received 27 November 2000; in revised form 19 June 2001; accepted 25 July 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
— The aim of the present study was to examine the age distribution of alcohol and intoxication debut and factors associated with this among a representative sample of Norwegian teenagers. A sample of 3368 teenagers aged 12–18 years was recruited from 34 Norwegian secondary schools to complete an 87-item questionnaire under examination conditions; 5.2% (168/3239) reported drinking alcohol for the first time when 10 years or younger, 25.2% (816/3239) when they were 13 years or younger and 60.1% (1948/3239) when 16 years or younger, with 39.9% having never drunk alcohol; 1.3% (44/3239) were first intoxicated by 10 years or younger, 12.8% (418/3239) when 13 years or younger and 37.5% (1649/3239) when 16 years or younger. Pupils with early alcohol or intoxication debut (<14 years) tended to come from single-parent families, from cities, experienced less family support and a more highly organized family life, reported more frequent peer and parental drinking, and did not participate in sports. They also showed a substantially elevated total yearly current alcohol consumption, compared to the group with alcohol debut at 14 years or later (8.1 and 2.5 l pure alcohol, respectively). Poor family support but high family organization, living in single-parent families and in cities, and more frequent parental and peer drinking are associated with early alcohol debut, whereas participation in organized sport activities may delay drinking and intoxication debut in teenagers. Sports organizations should be included in drinking prevention programmes.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most countries, children and young adolescents live in abstaining sub-cultures. Drinking alcohol is looked upon as a transition-marker, i.e. in terms of social behaviour, drinking represents a symbolic expression of the status-transformation from adolescence to adulthood. Drinking alcohol is therefore part of normal human development, an integrated part of the socialization process that usually occurs within the context of the family and society. Several reviews of the alcohol literature have stressed this point of normative adolescent drinking behaviour (e.g. Jessor and Jessor, 1975Go; Sharp and Lowe, 1989Go; Johnston et al., 1992Go; Lowe and Foxcroft, 1993Go; Miller and Plant, 1996Go, 1999Go). Therefore, it is not surprising that young adolescents' use of alcohol is rather widespread. Cross-sectional surveys (Hibell et al., 2001Go) among 15–16-year-old students in 30 European countries by The European School Survey Project on Alcohol and other Drugs (ESPAD) documented that an absolute majority of the 15–16-year-old students in the ESPAD countries have consumed alcohol at least once in their lifetime, and about two-thirds of the students have had an alcoholic beverage in the past 30 days. Hibell et al. (2001) also documented significant increases in alcohol consumption for this age group in most of the ESPAD countries between 1995 and 1999. Very few countries show decreasing figures, although some were largely unchanged. For instance, in Norway (Norwegian part of ESPAD) the proportion of students who had been drinking alcohol 20 times or more during the last 12 months increased, but was in 1999 still somewhat lower than the average for all ESPAD countries (78 vs 83%), while the proportion of students reporting drunken experiences also increased during the same period, but was somewhat higher (58 vs 52%). Hibell et al. (2001) also reported that many students in most ESPAD countries, especially boys, had tried alcohol at a fairly young age, but consumption had not led to intoxication to the same extent. The largest proportions of students who have had an early experience of drunkenness (13 years or younger) were found in high prevalence countries like Denmark (42%), UK (38%), Greenland (35%), Finland and Russia (33% each). In Norway, the proportion of drunkenness in this age group was reported to be 17%.

The ESPAD studies in 1995 and 1999 are the most detailed and comprehensive contemporary studies of European teenagers, but somewhat surprisingly, no data on variables related to age of alcohol debut were presented. Age of alcohol debut is related to adult alcohol consumption and several studies have documented that an early debut is associated with later elevated alcohol consumption, problem drinking or health and social problems associated with increased drinking (Margulis et al., 1977; Stacy and Elvy, 1982Go; Clapper and Lipsitt, 1992Go; Single and Wortly, 1993Go). More recently, in their longitudinal study, Pedersen and Skrondal (1998) found alcohol debut to be an excellent predictor of subsequent alcohol consumption and alcohol problems. Employing structural equation modelling, they found that a 10% delay in debut age would lead to a 35% decrease in subsequent expected alcohol consumption. Thus, from a public health point of view, factors associated with early alcohol debut apparently warrant more research attention.

Generally, when discussing determinants of young adolescents' drinking habits, several investigators have acknowledged the importance of family socialization (e.g. Barnes et al., 1986Go; Weber et al., 1989Go; Barnes, 1990Go; Foxcroft and Lowe, 1991Go; Miller, 1997Go; Miller and Plant, 1999Go). For example, Barnes et al. (1986) and Barnes (1990) found that adolescents' drinking was partly explained by parental models of drinking behaviour. Heavier drinking parents were more likely than other parents to have adolescents who were also heavy drinkers. Miller (1997) also found adolescents living in single-parent families, especially girls, more likely to have either tried, or to have frequently used, drugs. Based on a considerable amount of literature on the influence of family factors on young adolescents' drinking patterns in combination with theoretical work on the sociology of family by Parsons (1963), Barnes (1990) later constructed a comprehensive model of factors influencing adolescents' drinking behaviour. The most important family factors described by Barnes (1990) were family support and control in addition to parental models of drinking. Lamborn et al. (1991) also found that adolescents from indulgent homes had a strong sense of self-confidence, but reported a higher frequency of substance misuse, school misconduct and less school engagement.

Another important socialization domain, largely neglected in studies on drinking debut, is the sports domain (Edwards, 1973Go; McPherson et al., 1989Go). Most teenagers are engaged in a number of sports such as European football (or soccer; hereafter denoted football), handball, golf, swimming etc. For instance, in the authors' home country (Norway) 44% of the children and youth aged 8–24 years are actively engaged in organized sports and are members of the Norwegian Olympic Committee and Confederation of Sports (NOCCS), actually one of the largest private organizations in Norway. The highest percentages most active are found in the 8–11 and 12–15 year groups — also the most relevant age groups in terms of alcohol debut. One of the declared aims of the NOCCS is to promote a healthy life-style, including sensible or no use of alcoholic drinks: the separate sports organizations within the NOCCS have strict rules for drinking in sports contexts. In addition to social norms and rules of conduct, sports organizations are also aware that alcohol has several detrimental effects upon human physiology, most notably energy metabolism, the vascular and muscular systems and functional integrity of the nervous system. These general systemic effects are clearly acting against the primary aims of athletic training: increased strength, endurance, speed and precision of athletic tasks. Thus, teenagers engaged in organized sports might have a later onset of drinking, compared to teenagers not engaged in sports. On the other hand, several studies have documented considerable alcohol consumption among sports participants (O'Brian, 1993Go; Pedersen, 1993Go; Anderssen et al., 1994Go; Watten, 1995Go; Bu, 1999), which again might reflect specific social norms in certain sports groups (McPherson et al., 1989Go). However, keeping in mind the extensive global network organized sports represent, the impact of this large social domain upon alcohol debut should be more closely investigated.

The current study investigated the alcohol and intoxication debut age in a large, nationwide sample of young Norwegian adolescents of both sexes in terms of age when they first drank alcohol and the age when they reported being drunk for the first time. In addition to the relation between alcohol debut and sociodemographic variables, we investigated the impact of perceived parental and peer group drinking habits, family socialization factors and participation in organized sports activities. We also examined differences in current alcohol consumption between groups with early, compared to those with late, alcohol debut.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The total sample consisted of 3500 young adolescents between the ages of 12 and 18 years. Males and females were equally represented across the age groups. The participants were recruited from the middle and southern part of Norway and came from five different counties: Rogaland, Buskerud, Nord-Trøndelag, Sør-Trøndelag, and Møre and Romsdal. The sample was drawn from a socio-demographic cross-section of schools equally represented by town and rural areas. In total, 34 schools and seven different age groups (i.e. seven classes) were randomly selected and approached for participation. The State Office for Education contacted the principal for each school selected to participate in the project. Two schools with pupils in the lowest age group (12 years) refused to take part in the study, mainly on the ground that the teachers considered the pupils to be too young for this type of study. The final sample therefore consisted of 34 schools with 3368 pupils (1672 females and 1696 males). Within each school, a random sample of classes from different school years was drawn (by simple ballot technique). All eligible students in each school were approached, yielding a stratified single-phase cluster sample. Respondents were distributed across the 7 age groups as follows: 12 years: n = 217; 13 years: n = 504; 14 years: n = 584; 15 years: n = 585; 16 years: n = 555; 17 years: n = 472; 18 years: n = 451.

Measures
Drinking behaviour, sociodemographic factors and family variables were measured using the Adolescent Drinking and Family Life Questionnaire (ADFLQ) (Foxcroft and Lowe, 1991Go, 1992Go). Using established translation methods (Sartorius and Kuyken, 1994Go), the ADFLQ was independently translated into Norwegian and back-translated, and any discrepancies were discussed and resolved. The ADFLQ was completed in a classroom setting with a teacher present.

Demographic variables included age, gender, family composition, and living area: (1) living in or nearby a town (more than 100 000 people); (2) a small town (less than 100 000 people), and (3) in the countryside.

Alcohol debut was measured on a 5-point interval scale: (1) younger than 8 years; (2) 8–10 years; (3) 11–13 years; (4) 14–16 years; (5) have never tasted alcohol. Intoxication debut was registered according to the same interval scale as alcohol debut.

Family socialization was assessed using the Family Life Questionnaire section of the ADFLQ, a 69-item instrument assessing family life as perceived by the adolescents. There are three main scales: (1) family support; (2) family control; (3) family organization. The support scale consists of 20 items (e.g. ‘in my family we really help and support one another’; Cronbach's alpha: 0.88), the control scale consists of 14 items (e.g. ‘it's important to follow rules in my family' Cronbach's alpha: 0.88) and the organization scale consists of five items (e.g. ‘In my own family we make sure our rooms are neat and tidy’; Cronbach's alpha: 0.88). The respondents indicated whether they strongly agreed (4) or strongly disagreed (1) with each item.

Statistical analyses
Predictors of age for alcohol and intoxication debut were investigated with logistic regression analyses in a forced entry forward step procedure. The dependent variables (alcohol and intoxication debut) were debut age dichotomized as <14 years and >=14 years. Logistic regression analysis was undertaken in order to investigate the association between the dependent variables and the independent variables (family socialization, family integration, siblings, parents' and peers' drinking behaviour and attitudes towards drinking, place of residence and sports participation). The independent variables were entered in one block, employing a forward step procedure based on the Wald statistic. After entry one-by-one into the model, the variables were tested for removal according to the significance of the likelihood-ratio criterion. The variable with the largest probability greater than the specified POUT (probability of F to remove) value was removed and the model was re-estimated and evaluated for removal. The testing procedure was stopped when no more variables met the removal criteria.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Table 1Go presents the distribution statistics of alcohol debut in the sample. At the time of the study 39.9% had never tasted alcohol. Of those who had, the majority had done so between 14 and 16 years, but 20% reported their age of first drink as between 11 and 13 years, and more than 5% reported tasting alcohol below the age of 10 years. In all, 25.2% of the sample reported drinking alcohol by the age of 13 years. Early alcohol debut was predominantly a male phenomenon. For the group who reported first drinking alcohol before the age of 14, 57.9 and 42.1% were males and females, respectively ({chi}2 = 25.6, df = 1, P < 0.0001). Intoxication debut occurred later than drinking debut. Only 12.8% of the total sample had been intoxicated before they were 14 years old, and the male-to-female ratio for intoxication debut was very similar to drinking debut (57.5 and 42.5%, for males and females, respectively; {chi}2 = 12.6, df = 1, P < 0.01). Of the 816 respondents who reported that their first alcoholic drink was before the age of 14, only half (49.8%) also reported being intoxicated before 14.


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Table 1. Distribution of age for alcohol and intoxication debut
 
Table 2Go shows the location respondents reported having their first drink and their first intoxication. Most of the teenagers drank alcohol for the first time at a friends's house (42.7%), but also outdoors, such as in streets, parks and open air. Gender differences were observed; of those who had their first drink at home, a significantly higher proportion were males, whereas more females than males tended to debut at a friend's house or in pubs, clubs or dancing places ({chi}2 = 44.6, df = 1, P < 0.001). Compared to respondents with alcohol debut at 14 years or older, those with earlier debut tended to have their first drink at home ({chi}2 = 49.3, df = 1, P < 0.001).


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Table 2. Places where alcohol debut occurred (without presence of parents or guardians) ranked by percentage for the groups with early (<14 years) and late (>=14 years) alcohol debut
 
Figures 1 and 2GoGo show alcohol consumption in terms of estimated yearly consumption of beer, wine, liquor and total yearly consumption for the groups who made their alcohol debut (Fig. 1Go) and intoxication (Fig. 2Go) early (<14 years) and late (>=14 years).



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Fig. 1. Alcohol consumption in terms of beverage preferences and the total yearly alcohol consumption in litres of pure alcohol for respondents with an early alcohol debut (i.e. 13 years or younger; n = 816) and those with a later alcohol debut (14 years or older; n = 2423).

cons., consumption.

 


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Fig. 2. Alcohol consumption in terms of beverage preferences and the total yearly alcohol consumption in litres of pure alcohol for the respondents with an early intoxication debut (i.e. 13 years or younger; n = 418) and those with a later intoxication debut (14 years or older; n = 2821).

cons., consumption.

 
Young adolescents, who reported their first drink or first intoxication below the age of 14, exhibited substantially higher current alcohol consumption than young adolescents who first drank alcohol after 14 years of age. Teenagers with early debut drank almost four times more beer, wine and liquor than their counterparts with late debut.

Figures 3 and 4 GoGodepict the distribution of drinking throughout the whole week for subjects with early and late drinking (Fig. 3Go) and intoxication (Fig. 4Go) debut, respectively. There was a significantly higher percentage of teenagers with early drinking debut who reported drinking through the week, compared to adolescents with later debut. Most drinking occurred at the weekend, specifically on Friday and Saturday.



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Fig. 3. The distribution of drinking frequency as percentages of respondents drinking alcohol each day from Monday to Sunday for those with an early alcohol debut (i.e. 13 years or younger; n = 816) and those with a later alcohol debut (14 years or older; n = 2423).

cons., consumption.

 


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Fig. 4. The distribution of drinking frequency as percentages of respondents drinking alcohol each day from Monday to Sunday for those with an early intoxication debut (i.e. 13 years or younger; n = 418) and those with a later intoxication debut (14 years or older; n = 2821).

cons., consumption.

 
Table 3Go shows descriptive statistics on the ADFLQ for the adolescents with early and late alcohol and intoxication debut. There were significant differences between the groups with early or late alcohol and intoxication debut on several of the family and lifestyle factors. The group with early alcohol debut had lower scores on the family support dimension [F(1,3362) = 27.5, P < 0.0001], but elevated scores on the family organization dimension [F(1,3353) = 6.4, P < 0.01]. There were no significant differences on the control dimension. Compared to the late alcohol debut group, the early alcohol debut group also reported a higher drinking frequency for their fathers [F(1,3197) = 44.0, P < 0.0001], mothers [F(1,3225) = 16.3, P < 0.0001] and friends [F(1,3222) = 29.3, P < 0.0001]. They also had older siblings [F(1,3269) = 12.9, P < 0.001]. There were no significant differences on younger siblings. Moreover, the early alcohol debut group tended to live in single parent families ({chi}2 = 15.1, df = 2, P < 0.01), a large city ({chi}2 = 19.6, df = 5, P < 0.01), and fewer took part in organized sports ({chi}2 = 30.9 df = 1, P < 0.001).


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Table 3. Descriptive statistics for the Adolescent Drinking and Family Life Questionnaire: family socialization, family status, parental and peer drinking behaviour, residential area and sports participation for young adolescents with early (<14 years) and late (>=14 years) alcohol and intoxication debut
 
As can be seen in Table 3Go, the situation of the groups with early and late intoxication debut is almost identical with the pattern demonstrated by the drinking debut analyses. The only exception is family organization, with no significant differences for the intoxication groups. The mean differences, however, were very small.

Table 4Go presents the results from logistic regression analyses and shows the predictors of early alcohol debut. Early alcohol debut was positively associated with friends' and father's drinking frequency, single parent family, elevated scores on the family organisation dimension, and the number of younger siblings. Taking part in organized sports and elevated scores on the family support dimension were negatively associated with early alcohol debut.


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Table 4. Prediction of early (<14 years) and late (>=14 years) alcohol debut by logistic regression coefficients
 
Table 5Go shows the results of logistic regression analyses for early and late intoxication debut. Not surprisingly, the significant predictor variables for intoxication debut are almost the same as the predictor variables for alcohol drinking debut. Taking part in organized sports and reporting elevated scores on the family support dimension were negatively associated with intoxication debut, whereas living in large cities, father's drinking frequency, number of older siblings, single parent family, and elevated scores on the family control dimension were all positively associated with early intoxication debut.


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Table 5. Prediction of early (<14 years) and late (>=14 years) intoxication debut by logistic regression coefficients
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our findings indicate that 20% of Norwegian teenagers (12–18 years) have tasted alcohol by the age of 13, 60.1% had tasted alcohol by the age of 16, and 39.9% had never drunk alcohol. We also confirmed findings from several other studies showing that boys have an earlier alcohol debut than girls, both in terms of first drink and first intoxication (Kandel and Yamaguchi, 1985Go; Kandel and Ravis, 1989Go; Hibell et al., 2001Go). Although the socializing role of the family in terms of drinking habits has been emphasized in a number of studies, the majority of our teenagers (>40%) had their first drink and experienced their first intoxication at a friend's house. This is approximately twice as many as those who first drank at home or in other non-family environments, such as pubs, clubs, streets, or parks. Thus, the traditional, physical family settings were not important in terms of places where drinking started. However, teenagers with early debut (<14) were more likely to start their drinking career at home than youngsters debuting later. There was, however, an interesting gender difference here; girls were more likely to make their drinking debut outside their home.

The family socialization styles seem to be important for onset of drinking. Most important was the support dimension. The group showing early drinking and intoxication debut perceived significantly lower family support than those with later debut. Family support is often used interchangeably with family cohesion and is regarded by family therapists as a major dimension of family functioning (Minuchin, 1974Go; Maccoby and Martin, 1983Go; Foxcroft and Lowe, 1992Go; Miller and Plant, 1996Go, 1999Go; Miller 1997Go). In short, family support reflects emotional qualities, i.e. the emotional binding family members have toward each other. Families reporting high support scores are characterized with a positive emotional atmosphere, an accepting attitude and good emotional attachment. Attachment has been shown to be a crucial factor in childhood development (Bowlby, 1988Go). However, at the extreme level, support can be dysfunctional by compromising boundaries between individual family members resulting in an enmeshed family system (Minuchin, 1974Go). Elevated scores on the control and the organization dimensions, on the other hand, have been linked with early drinking and intoxication debuts, probably through the effect upon perceived autonomy leading to an oppositional and ‘protesting’ attitude; an attitude which might bring the adolescents more into contact with the existing, ‘anti-adult’ and experimenting youth culture.

In addition to family dynamics, our results also showed that teenagers' drinking behaviour was related to those of parents and peers. Parental models seemed to be important both for age of first drinking and age of first intoxication. Mothers' and fathers' drinking frequencies, as reported by their offspring, were positively associated with teenagers' alcohol debut; youngsters with early debut had parents with more frequent drinking (Table 3Go). It should be noted that, in the logistic regression analyses, the effect of the mother's drinking frequency disappeared. The reason might be statistical, since the drinking frequencies of the mothers and the fathers were strongly positively correlated. It should also be noted that the observed, actual drinking frequency was more important than parents' and peers' attitudes and norms for drinking (Bowlby, 1988Go). Thus, parents' and peers' influence seems to take place through role modelling, rather than through drinking norms. These findings support previous empirical data from the alcohol expectancy literature (Barnes et al., 1986Go), which documents the importance of young peoples' cognitive models of alcohol, models that are normally developed during the socialization process.

The third family factor related to onset of drinking was the number of siblings. Teenagers with early debut tended to have older siblings than teenagers with later debut; a factor pointing to the possible impact of role modelling, as mentioned above. Also parental status was important. Our results confirmed previous findings (Miller, 1997Go) suggesting that adolescents in single-parent families were more likely to drink and were over-represented in the group with early alcohol debut. Living area was a fourth influential factor. Early alcohol debut was associated with living in large cites. There were no significant differences between small cities or rural areas, suggesting that the (Norwegian) urban culture in large cities seems to promote early alcohol and intoxication debuts.

Participation in sports was an important factor delaying alcohol debut. Having developed interests in certain sports and regularly participating in sports was associated with later alcohol debut. There might be several explanations for this finding. First, most Norwegian sports clubs have strict rules for use of alcohol. For instance, according to the rules of the Norwegian Football Association, the largest organization within the NOCCS, drinking alcohol is not allowed when travelling with the soccer team, less than 48 h before a soccer match, and in all arrangements with other soccer teams. Second, from a family–sociological point of view, sports organizations are also important as a socialization domain. The changing family structures in most Western countries, with an increase in the number of divorces and single-parent families (consisting mostly of single women and children), make sports organizations more important as a socialization domain. Sports organizations may offer role models and social networks previously provided by the traditional family, also giving rise to additional sources for emotional attachment. Third, sports participation by young athletes occupies a considerable amount of young peoples' time. For instance, in a recent nationwide investigation of Norwegian adolescents (aged 12–18 years), 77.5% trained two or three times a week or more (Aas et al., 1995Go). The majority of those who competed at the national level trained daily. Thus, in addition to sport clubs' strict rules for drinking, the total amount of time directed at sports activities will reduce the possibility of taking part in norm-breaking activities, such as experimenting with alcohol or other substances.

The finding that participation in organized sports delays alcohol debut is interesting and points to possible primary prevention strategies. One element here is information about the adverse effects of alcohol upon the human body. Young athletes are probably more sensitive to this kind of knowledge, than are non-active youngsters. These effects can be summarized as: detrimental effects upon psychomotor skills, no improvement in work capacity and impairment of temperature regulation, with the consequence of decreased performance levels. Hangover effects are equally negative and might be summarized as impacting on: the citric acid cycle, aerobic metabolism, the lactate–pyruvate ratio, dehydration, and reduction in available carbohydrates. Impaired psychological functions, mood variations and increased sensitivity towards outside stimuli add to these detrimental physiological effects. Moreover, there are hazards of acute alcohol intake, such as temporarily weakening of ventricular contractions (Sandberg, 1990Go; O'Brian, 1993Go). It is especially important to convey such information to young athletes at the start of their sports career. Second, in addition to a number of other factors, the enormous social network organized sports represent make them well suited for large-scale, transnational prevention programmes in close cooperation with, for instance, the World Health Organization. Sports organizations should therefore be included systematically in alcohol primary prevention programmes at the national and global level.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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