Biological role of alcohol dehydrogenase in the tolerance of Drosophila melanogaster to aliphatic alcohols. utilization of an ADH-null mutant

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Biochemical Genetics, Vol. 14, Nos. 11/12, 1976
Biological Role of Alcohol Dehydrogenase in the
Tolerance of Drosophila melanogaster to Aliphatic
Alcohols: Utilization of an ADH-Null Mutant
Jean R. David, 1 Charles Bocquet, 2 Marie-Fran~oise Arens, 1 and Pierre
Fouillet 1
Received 6 May 1976--Final 24 May 1976
The toxicity of the first eight primary alcohols and of four secondary alcohols
was compared in a wild-type strain (having active A D H ) and an ADH-negative
mutant. Differences between LCso measured in the two strains allowed an
evaluation of the biological activity of the enzyme. In vitro, A D H is mainly
active on secondary alcohols, while in vivo its main role is the detoxification
and metabolism o f ethanol. These observations suggest that originally A D H
was involved in unknown metabolic pathways and that its utilization in ethanol
metabolism could be a recent event.
KEY WORDS: Drosophila melanogaster; alcohol dehydrogenase; enzyme biological
activity; toxicity of alcohols.
INTRODUCTION
In Drosophila melanogaster, alcohol dehydrogenase (ADH) is an abundant
enzyme whose genetic variability (e.g., Ursprung and Leone, 1965; Ward and
Herbert, 1972; Birley and Barnes, 1973; Clarke, 1975; Van Delden et al.,
1975) and biochemical activity (Vigue and Johnson, 1973; Day et al., 1974)
have received much attention.
Several authors (McKenzie and Parsons, 1972, 1974; David and Becquet,
1974, 1975; Briscoe et al., 1975) have demonstrated the physiological and
t Laboratoire d'Entomologie Exp6rimentale et de G6n6tique (Associ6 au CNRS), Universit6 Claude Bernard, Villeurbanne, France.
Laboratoire de G6n6tique Evolutive du CNRS, Gif sur Yvette, France.
989
.~ 1976 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic,
mechanical, photocopying, microfilming, recording, or otherwise, without written permission o f the publisher.
990
David, Bocquet, Arens, and Fouillet
ecological importance of ethanol tolerance for this species. This tolerance
appears, at least in some cases, to be related to the presence of active ADH
(Grell et al., 1968; Clarke, 1975; David and Bocquet, 1976a; Vigue and
Sorer, 1976).
However, a contradiction exists when biochemical and biological
results are compared. In vitro, ADH is inactive on methanol, slightly active
on ethanol, and very active on secondary alcohols (Sieber et al., 1972;
Vigue and Johnson, 1973; Day et al., 1974)..Live Drosophila, on the other
hand, are highly tolerant to ethanol and very sensitive to secondary alcohols
(David and Bocquet, 1976b). It therefore appeared interesting to measure
the biological role of ADH in the tolerance to various alcohols. Such a
study is possible by comparing the sensitivities of a normal wild-type strain
and homozygous ADH-negative mutant.
MATERIAL AND METHODS
The wild-type strain used is a French strain homozygous for the Adh v
natural allele and having a high level of enzyme activity (David and Bocquet,
1976a). The mutant strain not producing ADH was homozygous for the
Adh n4 allele (O'Donnell et al., 1975). This strain was also homozygous for the
second chromosome marker black.
Larvae of these strains were grown in low density on an axenic killedyeast medium (David and Clavel, 1965) containing no alcohol. Alcohol
toxicity was measured on adults 2-3 days old by calculating the concentration
killing 50% of the flies (LCso) after 2 days of treatment. For each determination, 560 adult flies were used. Males were usually a little more sensitive
than females, but the difference between sexes was always small and so
sex was ignored. More detailed information on these bioassay techniques
can be found in previous articles (David et al., 1974; David and Bocquet,
1976b).
RESULTS
Toxicity data (LC 50) for the first eight primary alcohols and for four secondary
alcohols are given in Fig. 1.
In the ADH-negative strain, the tolerance to primary alcohols decreases
regularly with molecule length: the LCso varies from about 3 ~ for methanol
to 0.2% for octanol. With secondary alcohols, a similar observation is made
and the toxicity increases with the length of the molecule. For a given number
of carbons, the toxicities of primary and secondary alcohols are usually
similar, as shown by the ratios in Table I.
In the presence of ADH (wild-type strain), the results are greatly different
Biological Activity of ADH in Drosophila
991
®
10.
a
o
u
c
d
.A
®
e
o
~
O
o
•
number of c a r b o n s of
alcohols
Fig. 1. Variation of the tolerance to various aliphatic alcohols
(LCso, lethal concentration 50) as a function of the number of
carbons. A: Toxicity of primary alcohols. B: Secondary alcohols.
0, Wild strain with active ADH; ©, mutant strain without ADH
activity.
In most cases, the tolerance o f the wild strain is higher than that o f the null
m u t a n t and the greatest difference is observed for ethanol. A n o t h e r peculiarity o f the wild strain is that it is always more sensitive to secondary
alcohols than to primary ones (see Table I).
The differences between the wild-type and the A D H - n u l l strain are to be
attributed mainly to the presence o f the enzyme. In other words, they measure
992
David, Bocquet, Arens, and Fouillet
Table I. Comparison of the Toxicities
of Primary and Secondary Alcohols:
Ratios of the LCso of the Primary
Alcohol to the LCso of the Secondary
One
Alcohol
ADH null
Wildtype
Propanol
Butanol
Pentanol
Hexanol
0.56
2.17
1.20
0.78
2.12
4.93
2.19
3.22
the biological activity of ADH. For a better evaluation of this phenomenon,
the differences between the LCso of the two strains were calculated for each
alcohol; data are given in Fig. 2.
For primary alcohols, the difference is zero for methanol and more than
14% for ethanol, but then it decreases rapidly from propanol to octanol,
where the difference is less than 0.3%. For secondary alcohols, a positive
difference is also seen which decreases with the length of the molecule.
The biological activity is, however, much lower than for the corresponding
primary alcohols. This observation corresponds with the higher toxicity of
secondary alcohols to wild flies (Table I).
In a previous article (David and Bocquet, 1976b), it was demonstrated
that D. simulans was generally more sensitive to various alcohols than
its sibling species D. melanogaster. On the other hand, it is known that
D. simulans has a much lower A D H activity than D. melanogaster (Pipkin
and Hewitt, 1972). It seemed interesting therefore to calculate the differences
between the L¢5o values of the two species. The results are shown in Fig.
2B. There is a striking similarity between these curves and those observed
for wild and mutant strains of D. melanogaster. These observations leave
very little doubt that the physiological differences between the two species
are due to the low A D H activity level found in D. simulans.
Previous authors (Vigue and Johnson, 1973; Day et al., 1974) have
studied the biochemical substrate specificities of A D H for various alcohols.
Our results allow the estimation of the biological activity of the enzyme for
the same alcohols. Comparison of these results is made in Table II, where,
in all cases, the activity on ethanol is taken as 100.
There are some differences between authors concerning the biochemical
activity on various substrates. Such differences may be explained by the
utilization of different strains and techniques. However, results agree in
showing no activity with methanol and a higher activity on alcohols with
Biological Activity of ADH in Drosophila
993
®
~
10.
®
g
i _ .
n u m b e r of c a r b o n s
of alcohols
Fig. 2. Biological activity of A D H for the tolerance to various alcohols. A: Difference between wild-type and ADH-null mutant of
D. melanogaster. B: Difference between wild D. melanogaster and
wild D. simulans, m, Primary alcohols; ~, secondary alcohols.
David, Bocquet, Arens, and Fouillet
994
Table II. Comparison of Biological Activity and in Vitro Biochemical
Activity of ADH °
Biochemical activity
Alcohol
Methanol
Ethanol
n-Propanol
2-Propanol
n-Butanol
2-Butanol
Biological
Vigueand
activity
Johnson (1973) Day et al. (1974)
0
100
20
4
20
3
0
100
213
580
213
775
0
100
110
350
230
120
"All results are expressed relative to an activity on ethanol of 100.
three or four carbons. Moreover, A D H seems, at least for propanol, more
active on secondary than on primary alcohols. This conclusion was confirmed by Sieber et aI. (1972) and many others, and 2-propanol is now the
classical substrate for staining A D H in electrophoretic studies.
When the biochemical properties are compared with the biological
activity, a concordance is observed for methanol only. For propanol and
butanol, the biological activity is low while the biochemical activity is high.
The discordance is particularly apparent with 2-propanol.
DISCUSSION
In the absence of A D H (negative mutants), alcohols are supposedly not
metabolized so that their deleterious effects on the organism can be directly
measured. In fact, this is not absolutely true because Drosophila contains
another enzyme, octanol dehydrogenase (ODH), which is active on primary
alcohols including ethanol (Sieber et al., 1972). O D H was present in both
wild and mutant flies, but our results prove that this enzyme is not important
in alcohol tolerance.
The nature of the toxic effect of alcohols on Drosophila adults is not
known, nor is the cause of death known. Probably all cells are affected by
alcohol ingestion or by respiration of alcoholic vapors. Some cells are likely
more sensitive than others and their dysfunction is lethal. Curiously, the
toxic effect increases with length of the molecule. The shorter molecules,
which are more volatile and more water soluble and hence should penetrate
more readily into the organism, are less toxic. Penetration of the alcohols
into lipidic cell structures may be involved in the toxic effects.
The difference between the wild-type and the mutant strain may be
Biological Activity of ADH in Drosophila
995
explained by the role of ADH in the metabolic utilization of alcohols.
The inactivity of ADH on methanol explains the similar toxicity in the
two strains. For ethanol, the higher tolerance of wild flies (and also of
D. melanogaster as compared to D. simulans) is most likely due to the
presence of an active ADH which uses alcohol as a substrate. In that case,
the physiological picture becomes more complex. When a toxic product is
taken into an organism, the overall reaction and toxic effect are the result
of several interacting factors: the direct toxicity of the initial product, the
toxicity of the transformed product(s), the rate of the enzymatic transformation, and further metabolism and utilization of these products. Such interactions are summarized in Fig. 3.
In the case of ethanol, the alcohol is probably transformed by ADH
into acetaldehyde. However, this product, in a direct test, is highly toxic
(Los0 of about 0.50~). The transformation of ethanol by ADH should
therefore increase the overall toxicity. Such is obviously not the case. This
can be explained by a coupled reaction which transforms the aldehyde into
nontoxic, useful products. This transformation, probably mediated by
aldehyde oxidase (Day et al., 1974; Clarke, 1975), is demonstrated by the
"feeding value" of ethanol (Van Herrewege and David, 1974; LibionMannaert et aL, 1976). This reasoning applies also to other alcohols. In all
cases here studied, the presence of ADH decreases the toxicity of primary
alcohols. ADH most likely transforms these alcohols into very toxic aldehydes. Since the overall toxicity is decreased, further metabolism probably
transforms the long-chain aldehydes into acids of low toxicity. Such transformations may be made by aldehyde oxidase, but the activity of this enzyme
on long-chain aldehydes is apparently not known. The primary toxicity of
secondary alcohols is not higher than that of the corresponding primary
ones (see Table I). But a relative increase of toxicity is observed when ADH
is present. Such a result can be explained by the toxicity of the intermediate
products which are ketones: either such ketones are more toxic than the
corresponding aldehydes, or their further utilization is questionable.
ALCOHOL
1
primary
toxicity
metabolism
further
bY ADH I I intermediate
porduct " I utilizcti°n= I end
(energetics}
pd°rucst
1
secondary
tgxicity
Fig. 3. Relationship between alcohol metabolismand toxicityto the flies.
H
996
David, Bocquet, Arens, and Fouillet
Considering the diagram of Fig. 3, it is conceivable that, in some cases,
enzymatic action would produce a very toxic product which, if not further
transformed, would kill the flies. Such a process, in the case of ADH, has
actually been demonstrated for some unsaturated secondary alcohols (Sofer
and Hatkoff, 1972; O'Donnell et al., 1975)
Finally, the biological activity of an enzyme can either increase or
decrease the toxicity of a substrate. This explains the lack of correlation
between the biochemical efficiency and the biological utilization. A D H
is mostly active on secondary alcohols, while, in the living fly, it is mostly
useful in the detoxification of ethanol.
As pointed out previously, high ethanol concentrations are encountered
by the flies in natural conditions and high ethanol tolerance clearly has an
ecologically adaptive significance. If the normal substrate of the enzyme
were environmental ethanol, it could be assumed logically that natural
selection, acting for a long time, would have produced an enzyme with its
maximum activity on ethanol. As this is clearly not the case, it can be considered that the primary physiological role of A D H in Drosophila metabolism
was not ethanol use but some other, unknown processes involving secondary
alcohols (Pipkin et al., 1975). In that case, the clinal ethanol adaptation
observed in D. melanogaster wild populations could be a recent event, as
supposed previously (David and Bocquet, 1975). Other examples in various
organisms now exist that previously existing enzymes are often used when an
adaptation occurs to a new substrate (Bari-Kolata, 1975).
ACKNOWLEDGMENTS
We thank Professor W. Sofer for providing the Adh n* strain and Dr. R.
Grantham for help with the manuscript.
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