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.
Conclusion
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.
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The
Merck Index: Twelfth Edition. New
Jersey: Merck & Co., Inc, 1996.
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Drinking: Risk Factors and Consequences. (1997, July). Alcohol Alert, No 37
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The
Genetic of Alcoholism. (1992 October). Alcohol
Alert, No 18
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Haney,
Daniel Q. Mutant fruit flies open new understanding about effects of alcohol
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Medina,
J. (1996). The Clock of Ages.
Cambridge: Cambridge University Press.
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Finkel,
Toren and Holbrook, Nikki. Oxidants, oxidative stress and the biology of
ageing. Nature, 9 November 2000. 239-247.
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Linda. Critical Resource—Horde of the Flies. [online] http://www.ncrr.nih.gov/newspub/oct99rpt/Flies.htm,9/29/00
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Flagg,
Raymond O. (1998) Carolina Drosophila
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