The Effects of Ethanol on Longevity. A Drosophila Mortality Model
Alcoholism is a major problem in our society. Absolute ethanol, ethyl alcohol (C2H5OH), is the addictive, intoxicating organic compound contained in alcoholic beverages (1). An alcoholic is a person who has progressed from use of ethanol to abuse to dependence (2). The stages leading to alcoholism are associated with both biological and psychosocial factors (2). In the 1970’s, studies showed that alcoholism runs in families (3). Some researchers think genes may directly cause alcoholism by affecting the body’s metabolism, while others believe the genetic role is less direct as it influences a person’s personality in a way that increases vulnerability to alcoholism (3).
Animal models are used to study alcoholism because it is possible to study larger numbers and more generations, to control the environment, and to conduct experiments not possible on humans. The common fruit fly, Drosophila melanogaster, is currently being used in ethanol studies. One recent study found that flies that carry a genetic mutation called “cheap date” produce low levels of cyclic AMP and are especially likely to get inebriated when exposed to ethanol vapors (4). The behavioral responses of fruit flies are very similar to humans under the influence of alcohol—they become hyperactive and uncoordinated, buzzing about before they become dazed and pass out.
When cells are stressed by environmental factors, such as sudden temperature increases, chemical agents, or radiation, there is a change in the expression of certain genes called the heat-shock response (5). New gene products are made depending on the kind of stress. This protective response is lower in older organisms than younger ones (5). The response has been observed in many organisms, including Drosophila (6).
Genetic studies of fruit flies began around 1909. In recent years, molecular
geneticists studying Drosophila have uncovered many similarities to human genes. Fruit flies have about 12,000 to 15,000 genes, compared with approximately 100,000 in the human genome (7). Humans and flies share a large number of homologous genes; vertebrates have about four homologues for every gene found in Drosophila (7).
The fruit fly is easily cultured in the laboratory. Its generation time is only two weeks at 21 degrees Celsius (8). The normal life span is about forty days for both males and females (5). Fruit flies are considered young, middle-aged, and old at ages 10, 28, and 44 days, respectively (9). If fruit fly responses to ethanol—such as attraction, consumption, sensitivity, tolerance, withdrawal, and developmental defects—can be demonstrated in the laboratory, then successful searches for gene mutations affecting the responses would explain the physiology of the events.
The purpose of this project was to determine the effects of ethanol on longevity of Drosophila melanogaster, as indicated by mortality rates. Because it was believed that exposure to ethanol vapors would increase mortality rates across the life span—with increased mortality as the frequency of exposure increased—two null hypotheses were developed as follows. Weekly exposure to ethanol vapors does not affect mortality rates of D. melanogaster at any age in the life span. Increasing the frequency of exposure to ethanol vapors does not affect mortality rates.
A culture of D. melanogaster was obtained and an adult sponsor purchased 95% ethanol for the project. The ethanol was transferred to laboratory bottle, labeled, and stored in a locked flammables’ cabinet. Different amounts of ethanol on a cotton swab were placed in vials containing flies for different amounts of time to determine a sublethal dose. The decision was made to use .2 ml of ethanol for a 15-minute time period because flies showed some behavioral responses, but were still mobile. After one week, the flies were transferred from the culture so that emerging flies would not be mixed in with the parents. The flies were allowed three days to emerge. Then, ten flies were transferred to each of 15 prepared vials (5 for control, 5 for once-a-week exposure, and 5 for twice-a-week exposure). The flies were one week old when ethanol vapor exposure began. The weekly schedules were repeated until all flies in the exposed groups were dead. The flies were transferred with an aspirator weekly to fresh vials to prevent new flies from entering the groups. The flies were briefly immobilized by placing them in the freezer for approximately 60 to 90 seconds, so that the aspirator and foam plug could be exchanged.
The number of flies in each of the vials was counted daily until all flies died or reached 63 days old. Flies that were still alive at the end of the study were disposed of in a detergent/water morgue. A total of 150 fruit flies were used for this project, and 740 vial counts were made over the nine-week period. A life table was constructed for the control group and the two groups exposed to ethanol vapors.
When comparing the number of flies alive in each group at different ages , the results show that the control group had the highest survival numbers at each week, except week three when the once-a-week group had slightly more flies alive. The twice-a-week group had surviving flies longer than the once-a-week group, but they started dying off earlier. This can be explained by the possibility that the flies which were exposed to ethanol more often became more tolerant to it after a while. Other studies have shown a conditioning effect leading to longer life spans in Drosophila for some environmental stresses (6). By age 7 weeks all of the once-a-week group had died, with all flies in the twice-a-week group dead by 8 weeks of age. However, 24 of the 50 control flies were alive at 8 weeks of age.
The logarithmic survivorship curve also shows these comparisons. All three groups had similar curves until the beginning of week four when the twice-a-week group declined sharply. The curve of the once-a-week group showed a similar decline one week later. However, the curves of the control group had only begun to gradually decline during weeks 7, 8, and 9.
The proportional mortality curve may more accurately represent the differences in mortality between the groups (Graph 3). It shows the proportion of the number alive at the beginning of the week, which died during that week. The values for the control group stayed mostly the same throughout the study. It peaked in week 7 and then went back down. A possible reason for the peak is that the flies were reaching old age near the end of the normal life span, but the oldest flies have lower proportional mortality rates as do many other organisms. The once-a-week group stayed about the same as the control until the beginning of week 5 when the alcohol started to affect them as they got older; they couldn’t tolerate it as well as when they were young. The twice-a-week group’s proportional mortality numbers were the highest of all the groups until the beginning of week 7, when the once-a-week group’s proportional mortality number started increasing.
The data was statistically analyzed. Analyses of variance were calculated to compare the number of flies dead in each group for each of eight weeks. The variance ratio tests showed significant differences at the p=.05 level of confidence between the total number of flies which died during weeks 5, 7, and 8. For weeks 2 through 4 and 6, the variation between the groups was not significant. To compare the differences between the mean number of flies alive in the ethanol-exposed groups, t-Tests were done for weeks 2 through 7. Only week four showed significant difference at the p=.05 confidence level.
The results of this study seem to indicate that ethanol does have an affect on the longevity of D. melanogaster. Weekly exposure to ethanol vapors did affect mortality rates during middle and old ages. Increasing the frequency of exposure did affect the mortality rates of middle-aged flies. The null hypotheses were rejected.
If these affects of ethanol on mortality rates can be linked to Drosophila genes and if there are human homologues, then this may mean that drinking alcohol regularly and drinking alcohol more often can have an affect on our longevity.
Future work may be done to see how exposure to ethanol affects the reproduction of the flies or the mortality of their offspring.
1. The Merck Index: Twelfth Edition. New Jersey: Merck & Co., Inc, 1996.
2. Youth Drinking: Risk Factors and Consequences. (1997, July). Alcohol Alert, No 37
3. The Genetic of Alcoholism. (1992 October). Alcohol Alert, No 18
4. Haney, Daniel Q. Mutant fruit flies open new understanding about effects of alcohol [online] http://www.onlineathens.com/1998/061298/0612.a3fruitflies.html,9/29/00
5. Medina, J. (1996). The Clock of Ages. Cambridge: Cambridge University Press.
6. Finkel, Toren and Holbrook, Nikki. Oxidants, oxidative stress and the biology of ageing. Nature, 9 November 2000. 239-247.
7. Berris, Linda. Critical Resource—Horde of the Flies. [online] http://www.ncrr.nih.gov/newspub/oct99rpt/Flies.htm,9/29/00
8. Flagg, Raymond O. (1998) Carolina Drosophila Manual. Burlington, North Carolina: Carolina Biological Supply Company.
9. Ricklefs, R. and Finch, C. (1995). Aging A Natural History. New York: Scientific American Library.