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LACK OF INHIBITORY ACTION OF AFLATOXIN ON THE INDUCTION OF DRUG-METABOLIZING ACTIVITIES OF LIVER MICROSOMES BY PHENOBARBITAL
LACK
OF
INHIBITORY
INDUCTION OF
OF
LIVER
RYUICHI
ACTION
MICROSOMES
KATO,
OF
AFLATOXIN
DRUG-METABOLIZING
AKIRA
BY
THE
PHENOBARBITAL
TAKANAKA,
AND YOSHIHITO
ON
ACTIVITIES
KINICHI
ONODA
OMORI
Department of Pharmacology, National Institute of Hygienic Sciences,Setagaya-ku, Tokyo Received for publication
April 5, 1969
Aflatoxin is a mycotoxin and has been known as a potent hepatotoxic and carcinogenic compound (1, 2). It has been recently established that aflatoxin primarily inhibits DNA-dependent
RNA polymerase of liver
nuclei, but not primarily inhibits protein synthesis (3, 4). Recently, Wogan and Friedman have reported that aflatoxin inhibits the induction of tryptophan pyrrolase by cortisone, but does not inhibit that by tryptophan
(5).
These results suggest that aflatoxin interferes the
formation of new messenger RNA, but does not interfere the biosynthesis of the new enzyme protein (3, 5). The administration of phenobarbital somes (6).
markedly increased the drug-metabolizing activities of liver micro
The de novoenzyme protein synthesis is assumed to be involved in the phenobarbital-induced
increase in the drug-metabolizing
activities, but the detail mechanism of the induction is not yet elucidated
(6). In the present communication, the preliminary results on the effect of aflatoxin on th° induction of drug-metabolizing activities by phenobarbital are described. Male rats of Wistar strain, weighing about 90 g were used. solved in distilled water and given intraperitoneally
Phenobarbital sodium (80 mg/kg) was dis
40 hours before the experiments.
Aflatoxin B1* (3
mg/kg) was dissolved in dimethylsulfoxide (3 mg/ml) and given intraperitoneally 2 hours before phenobar bital pretreatment. The rats were sacrificed by decapitation and the liver was immediately removed and homogenized with 3 volumes of 1.15°,/° KC1 in a Teflon-glass homogenizer. at 10,000 x g for 20 minutes.
The homogemte was centrifuged
The preparation of the incubation mixture and the determinations of amino
pyrine N-demethylation and pentobarbital oxidation were essentially same as described in a previous paper (7). Pentobarbital
anesthesia was determined
by the duration of the loss of righting reflex.
As shown in Table 1, the induction of aminopyrine N-demethylating and pentobarbital oxidating activities by phenobarbital
was not prevented by aflatoxin at all.
加 藤 隆一 ・高 仲 正 ・小 野 田 欽 一 ・大 森 義 仁 ~`
Vurihed Hygienic
atlatoxin Sciences.
lil was
kindly
supplied
from
Drs . M.
Icurata
and
-ti.
1 anabe,
ivational
Institute
or
TABLE 1.
Effect
and
of
aflatoxin
pentobarbital
on
the
oxidation
induction by
of
aminopyrine
N-demethylation
phenobarbital.
Rats were intraperitoneally pretreated with phenobarbital (60 mg/kg) 40 hours before sacrifice. Aflatoxin (3 mg/kg) was given intraperitoneally 2 hours before phenobarbital pretreatment. The figures in parentheses indicate number of rats used. The results are exoressed as averave+S.E. TABLE 2.
Effect
barbital
of
aflatoxin
on
the
phenobarbital-induced
shortening
of
pento
anesthesia.
Rats were intraperitoneally pretreated with phenobarbital (60 mg/kg) 40 hours before pentobarbital administration (35 mg/kg, i.p.). Aflatoxin (3 mg/kg) was given intraperitoneally 2 hours before phenobarbital. Similarly, the decrease in pentobarbital aflatoxin (Table 2).
anesthesia by phenobarbital
treatment was not prevented by
Moreover, these results indicate that the induction of drug-metabolizing
activity by
phenobarbital may be enhanced by aflatoxin. Three mg/kg of aflatoxin is toxic in immature rats (1) and according to Wogan and Friedman this dose of aflatoxin inhibits the induction of tryptophan
pyrrolase at least for 7 days (5).
The inhibition by the inhibitors of protein synthesis on the induction of drug-metabolizing activities is well established (6, 8).
Although
the inhibition of the induction
by actinomycin D has been reported
(8), these results are assumed to be doubtful for its high toxicity (9). The results of present study, therefore, suggested that the biosynthesis of the new messenger RNA does not play an important role in the induction of drug-metabolizing
activities by phenobarbital.
According to Garren et al. actinomycin D, when given after the administration of cortisone, enhances the induction of tryptophan pyrrolase through the inhibition in formation of the repressor (10). Although these results are not fully reproduced by other investigator, therefore, the possible action of aflatoxin on
the formation of repressor for the biosynthesis of drug-metabolizing
enzymes could not be excluded.
On the other hand, Gurtoo et al. (11) have recently reported that the treatment with aflatoxin modified the apparent Michaelis constant (Km) for benzpyrien hydroxylase. the inductive effect of phenobarbital aminopyrine N-demethylation
Since the alteration of Km may nullify
in the present investigation, a possible alteration of apparent Km for
and pentobarbital
oxidation by aflatoxin should be investigated.
Further studies on the both possibilities are now under investigation for elucidating the mechanism of the induction of drug-metabolizing activities of liver microsomes by phenobarbital
and will be published in detail
in elsewhere. REFERENCES 1) WOGAN,G.N.: Bacteriol. Rev. 30, 460 (1966); 2)
BARNES,J.M. ANDBUTLER,W.H.: Nature, Lond. 202,
1016 (1964) ; 3) CLIFFORD,J.1. ANDREES, K.R.: Biochem.J. 102, 65 (1967) ; 4) CLIFFORD,J.I., REES, K.R. ANDSTEVENS,M.E.M.: Biochem.J. 103, 258 (1967) ; 5) WOGAN,G.N. ANDFRIEDMAN,M.A.: Archs Biochem.Biophys.128, 509 (1968) ;
6)
CONNEY,A.H.: Pharmac. Rev. 19, 317 (1967) ;
T. ANDTOMIZAWA, S.: Jap. J. Pharmac. 18,356 (1968) ; R.: unpublished observation;
10)
8)
7)
KATO,R., OSHIMA,
KATO, R.: Med. exp. 3, 95 (1960) ; 9)
KATO,
GARREN,L.D., HOWELL, R.R., TOMKINS,G.M. AND CRocco, R.M.:
Proc. nat. Acad. Sci., Wash. 52,1121 (1964) ;
11) GURTOO,H.L., CHAMPBELL, T.C., WEBB,R.E. ANDPLOW
MAN,K.M.: Biochem. Biophys. Res. Comm. 31, 588 (1968)
OF
INHIBITORY
INDUCTION OF
OF
LIVER
RYUICHI
ACTION
MICROSOMES
KATO,
OF
AFLATOXIN
DRUG-METABOLIZING
AKIRA
BY
THE
PHENOBARBITAL
TAKANAKA,
AND YOSHIHITO
ON
ACTIVITIES
KINICHI
ONODA
OMORI
Department of Pharmacology, National Institute of Hygienic Sciences,Setagaya-ku, Tokyo Received for publication
April 5, 1969
Aflatoxin is a mycotoxin and has been known as a potent hepatotoxic and carcinogenic compound (1, 2). It has been recently established that aflatoxin primarily inhibits DNA-dependent
RNA polymerase of liver
nuclei, but not primarily inhibits protein synthesis (3, 4). Recently, Wogan and Friedman have reported that aflatoxin inhibits the induction of tryptophan pyrrolase by cortisone, but does not inhibit that by tryptophan
(5).
These results suggest that aflatoxin interferes the
formation of new messenger RNA, but does not interfere the biosynthesis of the new enzyme protein (3, 5). The administration of phenobarbital somes (6).
markedly increased the drug-metabolizing activities of liver micro
The de novoenzyme protein synthesis is assumed to be involved in the phenobarbital-induced
increase in the drug-metabolizing
activities, but the detail mechanism of the induction is not yet elucidated
(6). In the present communication, the preliminary results on the effect of aflatoxin on th° induction of drug-metabolizing activities by phenobarbital are described. Male rats of Wistar strain, weighing about 90 g were used. solved in distilled water and given intraperitoneally
Phenobarbital sodium (80 mg/kg) was dis
40 hours before the experiments.
Aflatoxin B1* (3
mg/kg) was dissolved in dimethylsulfoxide (3 mg/ml) and given intraperitoneally 2 hours before phenobar bital pretreatment. The rats were sacrificed by decapitation and the liver was immediately removed and homogenized with 3 volumes of 1.15°,/° KC1 in a Teflon-glass homogenizer. at 10,000 x g for 20 minutes.
The homogemte was centrifuged
The preparation of the incubation mixture and the determinations of amino
pyrine N-demethylation and pentobarbital oxidation were essentially same as described in a previous paper (7). Pentobarbital
anesthesia was determined
by the duration of the loss of righting reflex.
As shown in Table 1, the induction of aminopyrine N-demethylating and pentobarbital oxidating activities by phenobarbital
was not prevented by aflatoxin at all.
加 藤 隆一 ・高 仲 正 ・小 野 田 欽 一 ・大 森 義 仁 ~`
Vurihed Hygienic
atlatoxin Sciences.
lil was
kindly
supplied
from
Drs . M.
Icurata
and
-ti.
1 anabe,
ivational
Institute
or
TABLE 1.
Effect
and
of
aflatoxin
pentobarbital
on
the
oxidation
induction by
of
aminopyrine
N-demethylation
phenobarbital.
Rats were intraperitoneally pretreated with phenobarbital (60 mg/kg) 40 hours before sacrifice. Aflatoxin (3 mg/kg) was given intraperitoneally 2 hours before phenobarbital pretreatment. The figures in parentheses indicate number of rats used. The results are exoressed as averave+S.E. TABLE 2.
Effect
barbital
of
aflatoxin
on
the
phenobarbital-induced
shortening
of
pento
anesthesia.
Rats were intraperitoneally pretreated with phenobarbital (60 mg/kg) 40 hours before pentobarbital administration (35 mg/kg, i.p.). Aflatoxin (3 mg/kg) was given intraperitoneally 2 hours before phenobarbital. Similarly, the decrease in pentobarbital aflatoxin (Table 2).
anesthesia by phenobarbital
treatment was not prevented by
Moreover, these results indicate that the induction of drug-metabolizing
activity by
phenobarbital may be enhanced by aflatoxin. Three mg/kg of aflatoxin is toxic in immature rats (1) and according to Wogan and Friedman this dose of aflatoxin inhibits the induction of tryptophan
pyrrolase at least for 7 days (5).
The inhibition by the inhibitors of protein synthesis on the induction of drug-metabolizing activities is well established (6, 8).
Although
the inhibition of the induction
by actinomycin D has been reported
(8), these results are assumed to be doubtful for its high toxicity (9). The results of present study, therefore, suggested that the biosynthesis of the new messenger RNA does not play an important role in the induction of drug-metabolizing
activities by phenobarbital.
According to Garren et al. actinomycin D, when given after the administration of cortisone, enhances the induction of tryptophan pyrrolase through the inhibition in formation of the repressor (10). Although these results are not fully reproduced by other investigator, therefore, the possible action of aflatoxin on
the formation of repressor for the biosynthesis of drug-metabolizing
enzymes could not be excluded.
On the other hand, Gurtoo et al. (11) have recently reported that the treatment with aflatoxin modified the apparent Michaelis constant (Km) for benzpyrien hydroxylase. the inductive effect of phenobarbital aminopyrine N-demethylation
Since the alteration of Km may nullify
in the present investigation, a possible alteration of apparent Km for
and pentobarbital
oxidation by aflatoxin should be investigated.
Further studies on the both possibilities are now under investigation for elucidating the mechanism of the induction of drug-metabolizing activities of liver microsomes by phenobarbital
and will be published in detail
in elsewhere. REFERENCES 1) WOGAN,G.N.: Bacteriol. Rev. 30, 460 (1966); 2)
BARNES,J.M. ANDBUTLER,W.H.: Nature, Lond. 202,
1016 (1964) ; 3) CLIFFORD,J.1. ANDREES, K.R.: Biochem.J. 102, 65 (1967) ; 4) CLIFFORD,J.I., REES, K.R. ANDSTEVENS,M.E.M.: Biochem.J. 103, 258 (1967) ; 5) WOGAN,G.N. ANDFRIEDMAN,M.A.: Archs Biochem.Biophys.128, 509 (1968) ;
6)
CONNEY,A.H.: Pharmac. Rev. 19, 317 (1967) ;
T. ANDTOMIZAWA, S.: Jap. J. Pharmac. 18,356 (1968) ; R.: unpublished observation;
10)
8)
7)
KATO,R., OSHIMA,
KATO, R.: Med. exp. 3, 95 (1960) ; 9)
KATO,
GARREN,L.D., HOWELL, R.R., TOMKINS,G.M. AND CRocco, R.M.:
Proc. nat. Acad. Sci., Wash. 52,1121 (1964) ;
11) GURTOO,H.L., CHAMPBELL, T.C., WEBB,R.E. ANDPLOW
MAN,K.M.: Biochem. Biophys. Res. Comm. 31, 588 (1968)