Vol. 66, No. 2, 1975
BIOCHEMICAL
MUTAGENICITY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF METRONIDAZOLE: ACTIVATION BY MAMMALIAN LIVER MICROSOMES
Herbert S. Rosenkranz
and William
T. Speck
Departments of Microbiology and Pediatrics College of Physicians and Surgeons Columbia University New York, New York 10032 Received
July
16,1975
SUMMARY: The mutagenicity of metronidazole, a widely used chemotherapeutic agent, for Salmonella typhimurium was confirmed. Moreover using a mutant of of_S. typhimurium unable to activate metronidazole to a genetically active metabolite, it is shown that this activation can be carried out by a microsomal preparation derived from rat liver. Heretofore it had been postulated that this metabolic event was catalyzed solely by enzymes present in protozoa and anaerobic bacteria. The present findings which indicate that mammalian enzymes can activate metronidazole to a genetically active intermediate may have a direct relevance to the carcinogenicity of this agent.
Metronidazole
(l-(2-hydroxyethyl)-2-methyl-5-nitroimidazole)
used for the treatment
of protozoan
the therapy of infections Several recent studies several bacterial in prokaryotes
infections.
caused by anaerobic (2,3) have reported
species and ability
presumed that the genetic to be reduced enzymatically however,
(and protozoan)
and in view of the correlation
towards
bacteria
is without
convert metronidazole
has been advocated
(1).
of metronidazole
between
genetic
for
effects
to induce cancers in mammals (4, 5), the widespread
activity
has been questioned
of metronidazole
to the hydroxylamino
that this reduction
metabolism
bacteria
use of this drug for
the mutagenicity
usage of this drug to treat human infections
be argued,
Recently,
is widely
to a mutagenic
solely
the observed
It is
upon its ability
or amino derivative.
is dependent
(7-11) and therefore relevance
is dependent
(6).
It may
upon the microbial mutagenic
activity
to humans who are unable (12,13) to (and carcinogenic)
intermediate.
Vol. 66, No. 2, 1975
BIOCHEMICAL
In this study we demonstrate ically
active
intermediate
AND BIOPHYSICAL
the conversion
by mammalian
RESEARCH COMMUNICATIONS
of metronidazole
to a genet-
microsomes.
MATERIALS AND METHODS Salmonella University
of California,
nitro-reductase furazone identified Control)
td?imurium
Berkeley.
was obtained
(20 pg/ml,
by selection
from Dr. Bruce N. Ames,
of this strain deficient
(15) of bacteria
of nitrofurazone
reductase
activity
acid,
that this strain was deficient
Rat liver microsomes Mutagenicity
assays
The absence
(4% of
and 2-nitronaphthalene,thus nitro-reductase.
were prepared
as previously
described
using TAlOO and TAlOO-FRl were carried out as des-
cribed by Ames and his associates
(16) by incorporating
the indicator
the test agent and when indicated
the microsomal
fractions
co-factors)
The inoculated
plates were incubated
into an agar overlay.
37OC for 46 hours in the dark (17) and revertants were enumerated.
(15).
of other nitrofurans,
in a non-specific
(S-9 fraction)
to nitro-
calorimetrically
properties
2-nitrofluorene
in
One isolate
was confirmed
to the growth inhibitory
picrolonic
resistant
semicarbazone).
as TAlOO-FRl was used for these studies.
metronidazole,
(4).
A derivative
5-nitro-2-furaldehyde
TAlOO-FRI was resistant
indicating
TAlOO (14) was obtained
When anaerobic
conditions
placed into a Gas-Pak
jar (BBL, Cockeysville,
for 16 hours whereupon
they were incubated
(including
to histidine
at
the plates were
and incubated
an additional
required
independence
were required, Md.)
strain,
at 37’C
30 hours aerobically.
RESULTS Salmonella metronidazole pendence.
typhimurium
as evidenced
TAlOO, the parent strain, by the induction
This effect was dependent
into the plate
(Table 1, Expt.
I).
was mutagenized
of revertants
to histidine-inde-
upon the amount of drug incorporated
Contrariwise,
521
a nitro reductase
deficient
by
Aerobic Anaerobic Aerobic II ,I II ‘I
-
250 250 250 250 0 0 0 0 0 50 125 250 500 7 7 7 25 1.4 5.5 7
Metronidazole ,I I, II None $1 II I,
None Metronidazole II ,I II
Ethyl methanesulfonate II I, 1, Z-Epoxybutane Sodium Azide Propyle nimine 2-Bromoethanol Propane sultone
III
IV
+ + + +
-
Anaerobic ,I II II II
Aerobic Anaerobic Aerobic Anaerobic
Aerobic ,I Anaerobic II
Aerobic II II ,I 4,
II
-
0 25 50 125 250
Conditions
None Metronidazole I, I4 II
Microsomes
tvphimurium
I
&Plate
for Salmonella
Additions
of Metronidazole
Expt.
Mutagenicity
Table 1
5000
5000
1357 13 92 1594 1563 238 266 233 245
244 310 358 534 1171
Revertants TA 100
5000 408 2390 7000 702 1450
5000
147 892 1945 1992 1296
181 227 824 2008 155 150 163 175
159
, 161
Per Plate TAlOO-FRl
Vol. 66, No. 2, 1975
derivative
BIOCHEMICAL
of the tester
cubation
resulted
Expt II).
of mutagenic
microsomal
preparation
activity
of mutagenic
anaerobically
effectiveness
spontaneous
that microsomes
ration in converting
enhancement
background)
the bacterial
nitro reductase
dependent had no effect
enzyme is as effective
possessed
reflects
on the yield
of
It is interesting of metronidazole as the liver prepa-
When incubation
there was a slight
This presumably
(Table 1, Expt III).
effectiveness
form.
further
of the mutagenic
(Table 1, Expt II).
the drug to its activated
the
preparation
of mutants was increased
was concentration
to proceed anaerobically,
in the mutant yield.
in
(unpublished
with a microsomal
that these manipulations (i.e.
hours resulted
nitro reductase
had no effect on the mutagenic
for TAlOO; presumably
allowed
the yield
of metronidazole
mutations
(Table 1,
It appears that this reflects
This microsome-mediated
It should be stressed
activity
even in the absence of added mammalian
When the plates were supplemented
(Table 1, Expt II).
In-
of a microsomal
for several
of an oxygen-labile
anaerobically,
mutagen-
(Table 1, Expt IV).
in the presence
(Table 1, Expt II).
in these bacteria
and incubated
was not due to an in-
the nitro function
restoration
of the plates
expression
labile
lacking
in a partial
Incubation
results).
of this agent
strain to respond to mutagens as it was readily
of TAlOO-FRl with metronidazole
preparation
action
This lack of responsiveness
ized by a series of mutagens
presence
RESEARCH COMMUNICATIONS
(TAlOO-FRl) did not respond to the mutagenic
(Table 1, Expts I and II). ability
AND BIOPHYSICAL
but significant
the activity
was increase
of the oxygen-
by these bacteria.
DISCUSSION The ability nidazole an ability
of a mammalian
is surprising
microsomal
in view of the previously
unique to anaerobic
which show thatmammalian
bacteria
preparation
held assumption
and protozoa
liver enzymes are capable 523
to “activate”
(7-13).
metro-
that this was
The present data
of catalyzing
the con-
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
version of this drug to a mutagen deserves cepted view that mutagenic
activity
genic potential
(4, 5).
carcinogenicity
of metronidazole
consideration
intermediate,
controlled
of carcino-
it is perhaps germane that the
for rodents has been reported use of metronidazole
sible that the human liver is capable of activating active
because of the ac-
can be taken as an indication
In this connection
In view of the widespread
RESEARCH COMMUNICATIONS
(18).
and because it is posthis drug to a genetically
it would seem that the utilization
of this drug should be
more rigorously. ACKNOWLEDGEMENTS
The authors are grateful Berkeley for providing
to Dr. B.N. Ames, University
them with a specimen
This study was supported
by the Division
National
(NO l-CP-33395).
Cancer Institute
a Research Career Development Medical Fellow
Sciences in Clinical
of Salmonella
of California typhimurium
at
TAIOO.
of Cancer Cause and Prevention, One of the authors
Awardee of the National
Institute
(H. S.R.) is of General
(5 K3-GM 29, 024), the other (W.T.S .) is the Vivian Allen Medicine. REFERENCES
1. 2.
Tally, F.P., Sutter, V.L. and Finegold, S.M., Calif. Med. l& 22 (1972). Voogd, C.E., Van der Stel, J. J. and Jacobs, J. J. J.A.A., Mutation Res. 26, 483 (1974). 3. Legator, M . S., Connor, T. H. and Stockel, M., Science 188, 1118 (1975). 4. Ames, B.N., Durston, W.E., Yamasaki, E.and Lee, F.D.,oc. Nat. Acad. Sci. U.S. 70, 2281 (1973). 5. Miller, E.C. and Miller, J.A. inA. Hollaender (ed.), Chemical Mutaqens. Vol. 1, Plenum Press, Inc., New York, N.Y. pp. 83-119 (1971). 6. The Medical Letter on Drugs and Therapeutics & 53 (1975). 7. Edwards, D.I. and Mathison. G.E., J. Gen. Microbial. 63, 29 (1970). 8. Ings, R.M.J., McFadzean, J.A., and Ormerod, W. E., Biochem. Pharmacol . 2, 1421 (1974). 9. Coombs, G.H., _in P. de Puytorac and J. Grain (eds.), Proqress in Protozoology Proc. 4th Intern. Congr. Protozoology, p. 92 (1973). 10. Lindmark, D .G. ind Miller, M . , & P. de Puytorac and J. Grain (eds .), Progress in Protozooloqy, Proc. 4th Intern. Congr. Protozoology, p. 251 (1973). 436 (1974). 11. Lindmark, D.G. and Mbller, M., J. Protozool.21,
524
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
12, Ings, 13 . 14. 15. 16. 17. 18.
RESEARCH COMMUNICATIONS
R.M. J., Law, J.L., and Parnell, E.W ,, Biochem. Pharmacol.&, 515 (1966). Stambaugh, J . E . , Feo, L.G. and Manthei, R.W., J. Pharmacol. Exptl. Ther. 161, 373 (1968). McCann, J., Spingarn, N.E., Kobori, J. and Ames, B.N., Proc. Nat. Acad. Sci. U.S.2, 979 (1975). McCalla, D.R., Reuvers, A. and Kaiser, C., J. Bacterial. 104, 1126 (1970). Ames, B.N., Lee, F.D. and Durston, W.E., Proc. Nat. Acad. Sci. U.S. 7J, 782 (1973). Speck, W.T. and Rosenkranz, H.S., Photochem. Photobiol. a 369 (1975). Rustia, M. and Shubik, P., J. Nat. Cancer Inst. 48, 721 (1972).
525
BIOCHEMICAL
MUTAGENICITY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
OF METRONIDAZOLE: ACTIVATION BY MAMMALIAN LIVER MICROSOMES
Herbert S. Rosenkranz
and William
T. Speck
Departments of Microbiology and Pediatrics College of Physicians and Surgeons Columbia University New York, New York 10032 Received
July
16,1975
SUMMARY: The mutagenicity of metronidazole, a widely used chemotherapeutic agent, for Salmonella typhimurium was confirmed. Moreover using a mutant of of_S. typhimurium unable to activate metronidazole to a genetically active metabolite, it is shown that this activation can be carried out by a microsomal preparation derived from rat liver. Heretofore it had been postulated that this metabolic event was catalyzed solely by enzymes present in protozoa and anaerobic bacteria. The present findings which indicate that mammalian enzymes can activate metronidazole to a genetically active intermediate may have a direct relevance to the carcinogenicity of this agent.
Metronidazole
(l-(2-hydroxyethyl)-2-methyl-5-nitroimidazole)
used for the treatment
of protozoan
the therapy of infections Several recent studies several bacterial in prokaryotes
infections.
caused by anaerobic (2,3) have reported
species and ability
presumed that the genetic to be reduced enzymatically however,
(and protozoan)
and in view of the correlation
towards
bacteria
is without
convert metronidazole
has been advocated
(1).
of metronidazole
between
genetic
for
effects
to induce cancers in mammals (4, 5), the widespread
activity
has been questioned
of metronidazole
to the hydroxylamino
that this reduction
metabolism
bacteria
use of this drug for
the mutagenicity
usage of this drug to treat human infections
be argued,
Recently,
is widely
to a mutagenic
solely
the observed
It is
upon its ability
or amino derivative.
is dependent
(7-11) and therefore relevance
is dependent
(6).
It may
upon the microbial mutagenic
activity
to humans who are unable (12,13) to (and carcinogenic)
intermediate.
Vol. 66, No. 2, 1975
BIOCHEMICAL
In this study we demonstrate ically
active
intermediate
AND BIOPHYSICAL
the conversion
by mammalian
RESEARCH COMMUNICATIONS
of metronidazole
to a genet-
microsomes.
MATERIALS AND METHODS Salmonella University
of California,
nitro-reductase furazone identified Control)
td?imurium
Berkeley.
was obtained
(20 pg/ml,
by selection
from Dr. Bruce N. Ames,
of this strain deficient
(15) of bacteria
of nitrofurazone
reductase
activity
acid,
that this strain was deficient
Rat liver microsomes Mutagenicity
assays
The absence
(4% of
and 2-nitronaphthalene,thus nitro-reductase.
were prepared
as previously
described
using TAlOO and TAlOO-FRl were carried out as des-
cribed by Ames and his associates
(16) by incorporating
the indicator
the test agent and when indicated
the microsomal
fractions
co-factors)
The inoculated
plates were incubated
into an agar overlay.
37OC for 46 hours in the dark (17) and revertants were enumerated.
(15).
of other nitrofurans,
in a non-specific
(S-9 fraction)
to nitro-
calorimetrically
properties
2-nitrofluorene
in
One isolate
was confirmed
to the growth inhibitory
picrolonic
resistant
semicarbazone).
as TAlOO-FRl was used for these studies.
metronidazole,
(4).
A derivative
5-nitro-2-furaldehyde
TAlOO-FRI was resistant
indicating
TAlOO (14) was obtained
When anaerobic
conditions
placed into a Gas-Pak
jar (BBL, Cockeysville,
for 16 hours whereupon
they were incubated
(including
to histidine
at
the plates were
and incubated
an additional
required
independence
were required, Md.)
strain,
at 37’C
30 hours aerobically.
RESULTS Salmonella metronidazole pendence.
typhimurium
as evidenced
TAlOO, the parent strain, by the induction
This effect was dependent
into the plate
(Table 1, Expt.
I).
was mutagenized
of revertants
to histidine-inde-
upon the amount of drug incorporated
Contrariwise,
521
a nitro reductase
deficient
by
Aerobic Anaerobic Aerobic II ,I II ‘I
-
250 250 250 250 0 0 0 0 0 50 125 250 500 7 7 7 25 1.4 5.5 7
Metronidazole ,I I, II None $1 II I,
None Metronidazole II ,I II
Ethyl methanesulfonate II I, 1, Z-Epoxybutane Sodium Azide Propyle nimine 2-Bromoethanol Propane sultone
III
IV
+ + + +
-
Anaerobic ,I II II II
Aerobic Anaerobic Aerobic Anaerobic
Aerobic ,I Anaerobic II
Aerobic II II ,I 4,
II
-
0 25 50 125 250
Conditions
None Metronidazole I, I4 II
Microsomes
tvphimurium
I
&Plate
for Salmonella
Additions
of Metronidazole
Expt.
Mutagenicity
Table 1
5000
5000
1357 13 92 1594 1563 238 266 233 245
244 310 358 534 1171
Revertants TA 100
5000 408 2390 7000 702 1450
5000
147 892 1945 1992 1296
181 227 824 2008 155 150 163 175
159
, 161
Per Plate TAlOO-FRl
Vol. 66, No. 2, 1975
derivative
BIOCHEMICAL
of the tester
cubation
resulted
Expt II).
of mutagenic
microsomal
preparation
activity
of mutagenic
anaerobically
effectiveness
spontaneous
that microsomes
ration in converting
enhancement
background)
the bacterial
nitro reductase
dependent had no effect
enzyme is as effective
possessed
reflects
on the yield
of
It is interesting of metronidazole as the liver prepa-
When incubation
there was a slight
This presumably
(Table 1, Expt III).
effectiveness
form.
further
of the mutagenic
(Table 1, Expt II).
the drug to its activated
the
preparation
of mutants was increased
was concentration
to proceed anaerobically,
in the mutant yield.
in
(unpublished
with a microsomal
that these manipulations (i.e.
hours resulted
nitro reductase
had no effect on the mutagenic
for TAlOO; presumably
allowed
the yield
of metronidazole
mutations
(Table 1,
It appears that this reflects
This microsome-mediated
It should be stressed
activity
even in the absence of added mammalian
When the plates were supplemented
(Table 1, Expt II).
In-
of a microsomal
for several
of an oxygen-labile
anaerobically,
mutagen-
(Table 1, Expt IV).
in the presence
(Table 1, Expt II).
in these bacteria
and incubated
was not due to an in-
the nitro function
restoration
of the plates
expression
labile
lacking
in a partial
Incubation
results).
of this agent
strain to respond to mutagens as it was readily
of TAlOO-FRl with metronidazole
preparation
action
This lack of responsiveness
ized by a series of mutagens
presence
RESEARCH COMMUNICATIONS
(TAlOO-FRl) did not respond to the mutagenic
(Table 1, Expts I and II). ability
AND BIOPHYSICAL
but significant
the activity
was increase
of the oxygen-
by these bacteria.
DISCUSSION The ability nidazole an ability
of a mammalian
is surprising
microsomal
in view of the previously
unique to anaerobic
which show thatmammalian
bacteria
preparation
held assumption
and protozoa
liver enzymes are capable 523
to “activate”
(7-13).
metro-
that this was
The present data
of catalyzing
the con-
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
version of this drug to a mutagen deserves cepted view that mutagenic
activity
genic potential
(4, 5).
carcinogenicity
of metronidazole
consideration
intermediate,
controlled
of carcino-
it is perhaps germane that the
for rodents has been reported use of metronidazole
sible that the human liver is capable of activating active
because of the ac-
can be taken as an indication
In this connection
In view of the widespread
RESEARCH COMMUNICATIONS
(18).
and because it is posthis drug to a genetically
it would seem that the utilization
of this drug should be
more rigorously. ACKNOWLEDGEMENTS
The authors are grateful Berkeley for providing
to Dr. B.N. Ames, University
them with a specimen
This study was supported
by the Division
National
(NO l-CP-33395).
Cancer Institute
a Research Career Development Medical Fellow
Sciences in Clinical
of Salmonella
of California typhimurium
at
TAIOO.
of Cancer Cause and Prevention, One of the authors
Awardee of the National
Institute
(H. S.R.) is of General
(5 K3-GM 29, 024), the other (W.T.S .) is the Vivian Allen Medicine. REFERENCES
1. 2.
Tally, F.P., Sutter, V.L. and Finegold, S.M., Calif. Med. l& 22 (1972). Voogd, C.E., Van der Stel, J. J. and Jacobs, J. J. J.A.A., Mutation Res. 26, 483 (1974). 3. Legator, M . S., Connor, T. H. and Stockel, M., Science 188, 1118 (1975). 4. Ames, B.N., Durston, W.E., Yamasaki, E.and Lee, F.D.,oc. Nat. Acad. Sci. U.S. 70, 2281 (1973). 5. Miller, E.C. and Miller, J.A. inA. Hollaender (ed.), Chemical Mutaqens. Vol. 1, Plenum Press, Inc., New York, N.Y. pp. 83-119 (1971). 6. The Medical Letter on Drugs and Therapeutics & 53 (1975). 7. Edwards, D.I. and Mathison. G.E., J. Gen. Microbial. 63, 29 (1970). 8. Ings, R.M.J., McFadzean, J.A., and Ormerod, W. E., Biochem. Pharmacol . 2, 1421 (1974). 9. Coombs, G.H., _in P. de Puytorac and J. Grain (eds.), Proqress in Protozoology Proc. 4th Intern. Congr. Protozoology, p. 92 (1973). 10. Lindmark, D .G. ind Miller, M . , & P. de Puytorac and J. Grain (eds .), Progress in Protozooloqy, Proc. 4th Intern. Congr. Protozoology, p. 251 (1973). 436 (1974). 11. Lindmark, D.G. and Mbller, M., J. Protozool.21,
524
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
12, Ings, 13 . 14. 15. 16. 17. 18.
RESEARCH COMMUNICATIONS
R.M. J., Law, J.L., and Parnell, E.W ,, Biochem. Pharmacol.&, 515 (1966). Stambaugh, J . E . , Feo, L.G. and Manthei, R.W., J. Pharmacol. Exptl. Ther. 161, 373 (1968). McCann, J., Spingarn, N.E., Kobori, J. and Ames, B.N., Proc. Nat. Acad. Sci. U.S.2, 979 (1975). McCalla, D.R., Reuvers, A. and Kaiser, C., J. Bacterial. 104, 1126 (1970). Ames, B.N., Lee, F.D. and Durston, W.E., Proc. Nat. Acad. Sci. U.S. 7J, 782 (1973). Speck, W.T. and Rosenkranz, H.S., Photochem. Photobiol. a 369 (1975). Rustia, M. and Shubik, P., J. Nat. Cancer Inst. 48, 721 (1972).
525
