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NICOTINE AND SOME CARCINOGENS IN SPECIAL REFERENCE TO THE HEPATIC DRUG-METABOLIZING ENZYMES
NICOTINE
AND
TO
SOME
THE
CARCINOGENS
HEPATIC
IWAO
IN
SPECIAL
DRUG-METABOLIZING
YAMAMOTO,
KAZUO
NAGAI*,
AND KATSUYA
REFERENCE
ENZYMES HIROSHI
KIMURA
IWATSUBO
Departmentof Pharmacology, Osaka UniversityDental School,Kita-ku,Osaka Received for publication November 22, 1965
In (e.g.
the
past
few
years
3,4-benzpyrene
pyrine) metabolize
the
has
standpoint
of
nicotine and
in the
be
the
foreign been
relationship drug
been
reported
to
the
enzyme
(1).
be
metabolism.
the
that
of
the
effect
of
of
on
the
phenobarbital in
of
1,000
and
not
which
10
of
reports) (2)
been
carcinogen
, but
done
pharmacological
carcinogens
amino
microsomes
cigarettes
have
of
the
liver
(average of
hydrocarbons
3 ,4
studies from
the
investigations
on
nicotine
metabolism
of
metabolism
were
subjected
to
investigated. Data
presented
in
and
3-methylcholanthrene
that
nicotine
fluorene
also
and
this
paper
indicates
enhanced increased
the
3,4-benzpyrene
the
rat
Animals,
diet
Male
and
compounds
Donryu
rats
semisynthetic
diet
Sources
of
of
liver
aminofluorene 1-nicotine
Ltd),
nikethamide
Biochemical Administration BP,
of
and
山本
巌 ・永 井
Activity,
Osaka
compounds
和 男 ・木 村 : Department
were
metabolizing
enzymes
enzyme
toward
and
2-acetylamino
METHODS
used
follows
:
Co.),
all
experiments
.
They
3,4-benzpyrene
(BP)
were
and
3-methylcholanthrene
monopolycorp
.),
Industries
and
for
fed
Co.). as
Industries
(Japan
ATP
nicotine
University
g
Chemical
nikethamide
address
AND
were
Kasei
1-cotinine
and
the
and
Cotinine
* Present
used
(Takeda
MC
nicotine
microsomes.
Kokei-Shiryo
(Tokyo
and
Corp.)
60-80
compounds
(AAF)
AG.),
of
metabolizing
3,4-benzpyrene
employed weighing
(General
the
2-acetylaminofluorene,
activity
activity
in
that
MATERIALS
a
smoke
course
effect
nicotine
(e.g.
systems
carcinogens
the
the
drugs
carcinogenic
16 ƒÊg
in
and During
(3-5),
various
About
contained
nicotine
laboratory
converse,
found
and
activity
compounds
between
our
has
3-methylcholanthrene)
markedly
several
benzpyrene on
and
increase
it
Co .),
glucose-6-phosphate
(MC)
dl-ethionine NAD (Sigma
2-acetyl
(Nakarai and
Chemicals,
NADP
Chemical
(Fluka
(Nutritional
Co .).
employed were were
given injected
拓 ・岩 壺 of Medical
intraperitoneally
in
intraperitoneally
in
0 .1 0.1
or ml
0.2 of
0.9
ml per
of
corn
oil.
cent
saline
High
Nerve
克哉
Pharmacology School
and
, Kita-ku,
Biochemistry Osaka.
of
Instit
ute
for
.
Control animals received only corn oil or saline. These drugs were usually administered 24 hours prior to assay of the liver unless otherwise stated. In case of administration of AAF 0.025 per cent was added to the diet at a final concentration. In inhibition experiments, ethionine was administered in dose of 1.0 g/kg 30 minutes before the injection of nicotine, cotinine and MC. Enzymepreparation The animals were killed by a fracture of the neck and bled the carotid arteries. The livers were then removed and 20 per cent homogenate was made in cold 1.15 per cent KCl solution. Nuclear and mitochondrial fractions of the liver cells were removed by centrifugation at 9,800x g for 30 minutes, and the supernatant having microsomes and soluble fraction (MsS) was used as an enzyme sourse. Determinationof enzymeactivities Nicotine oxidase was determined by the method of Hucker et al. (6). AAF and BP hydroxylases were determined according to the method of Cramer et al. (7) and Conney et al. (8) with some modifications respectively. For the determination of nicotine oxidase activities the incubation mixture contained 3.0 ml of MsS equivalent to 600 mg of wet liver, 0.1 ml of nicotine (486 mg/ml), 0.6 ml of cofactors and 1.3 ml of 0.2 M phosphate buffer, pH 7.4. The mixtures for the determination of AAF and BP hydroxylase activities contained 0.3 ml of MsS equivalent to 60 mg of wet liver, 0.1 ml of substrate solution in ethanol (0.5 mg/ml of both substrates respectively), 0.6 ml of cofactors and 2.0 ml of 0.2 M phosphate buffer, pH 7.4. The cofactors were composed of 0.1 ml of NAD (0.25 icmole), 0.1 ml of NADP (0.25 ,umole), 0.1 ml of glucose-6-phosphate (6 pmoles), 0.1 ml of MgC12 (75,amoles), 0.1 ml of ATP (2,amoles) and 0.1 ml of nicotinamide (120 amoles) to a total volume of 0.6 ml. The incubation was carried out at 37°C in air atmosphere of oxygen for 60, 60 and 30 minutes in the experiments on nicotine-, AAF and BP-metabolizing enzymes, respec tively. In the experiment on BP hydroxylase incubation was done in the dark. RESULTS The metabolismof AAF by the liver microsomes of rats fed AAF containingdiet and of rats in jectedwith MC ' When rats were fed AAF containing diet, AAF hydroxylase activity in the liver microsomes was enhanced (Fig. 1). Maximum increase (about 80 per cent) of this enzyme activity was observed at 3 weeks after feeding the diet. When rats were injected with 20 mg/kg of MC before 6 hours to 3 days, the AAF hydroxylase activity was markedly increased. Maximum increase (about 6 times) was shown 24 hours after injection of MC, and high activity of this enzyme persisted at least until 3 days (Fig. 2).
FIG.
1.
The
metabolism
containing by
MsS
weeks
FIG.
2.
the
of
to 60
Ordinate wet
AAF
various
by
liver
of
mg
per
of
of
AAF
: pg
liver
weeks.
beginning
metabolism
MC. mg
of at
equivalent from
The
after 60
diet
of 60
wet
the
by
microsomes
Ordinate liver
AAF
rat
AAF
liver
from
: pg per
of
30
containing
fed
AAF
metabolized
minutes.
Abscissa
:
diet.
microsomes
metabolized
rats
AAF
by
at MsS
various equivalent
days to
minutes.
The metabolismof nicotineby liver microsomes of rat pretreated with AAF, MC and BP Nicotine oxidase activity was determined 3 weeks after feeding of AAF containing diet and 24 hours after injection of 20 mg/kg of MC and 20 mg/kg of BP in reference to the above results. Pretreatment with all the three carcinogenic compounds caused an increase in the nicotine metabolizing activity in rat liver microsomes. AAF enhanced the metabolizing activity toward nicotine about 50 per cent as compared to control. MC and BP enhanced this activity about 100 and 80 per cent respectively, when the metabolized nicotine was determined. The similar tendency was also shown in case that cotinine, one of the major metabolites of nicotine, was determined (Fig. 3).
FIG. 4. Time course of AAF hydro xylase activity of MsS equivalent to 60 mg of wet liver per 60 minutes after nicotine. Ordinate:
FIG. 3. The effect of AAF, MC and BP on nicotine metabolism by MsS equivalent to 600 mg of wet liver. Ordinate : pmole of nicotine metabolized and cotinine formed. Abscissa : I= control, II=pretreatment with AAF containing diet, III=pre treatment with 20 mg/kg of MC, IV=pretreatment with 20 mg/kg of BP. n : nicotine metabolized, c:cotinine formed.
g of AAF metabolized. Abscis sa : hours after 40 mg/kg of nicotine.
Effect of nicotineon AAF and BP hydroxylasein rat liver microsomes AAF hydroxylase activities of rat liver micro somes at various time intervals after injection of 40 mg/kg of nicotine are shown in Fig. 4. The AAF hydroxylase activity increased about two times 6 hours after nicotine administration and recovery of the increased activity to normal level was not found even 30 hours after nicotine administration. Fig. 5 shows the effect of the doses of pretreated nicotine on AAF hydroxylase activity. Five to 40 mg/kg and 40 mg/kg of nicotine per day for two and three days were injected 24 hours before assay of AAF hydroxylase activity. Result showed that 30 to 40 mg/kg of nicotine induced the maximum enzyme activity. On the contrary, the daily injections of nicotine for two to three days decreased the hepatic AAF hydroxylase activity to less than control.
FIG.
5.
Effect
of MsS were mg
of
various
equivalent expressed
of
wet
liver
doses to 60 mg
as
pg per
of 60
of
nicotine
of wet AAF
minutes.
on
liver
metabolized
per
AAF
hydroxylase
60 minutes. by
MsS
activities The
equivalent
activities to 60
FIG.
6.
Effect
xylase
FIG.
7.
of
Effect
activities as pg liver
of
nicotine,
rat
liver
of of
cotinine, microsomes.
nicotine, rat
liver
conitine,
nikethamide The
BP and
microsomes.
of
BP
metabolized
per
30
minutes.
by
The MsS
and
activities
MC
MC are
on
BP
activities
equivalent
on
as
60
hydro Fig.
5.
hydroxylase
were to
AAF
under
expressed mg
of
wet
When nicotine (40 mg/kg), cotinine (20 mg/kg), nikethamide (40 mg/kg) and MC (25 mg/kg) were preadministered to rats, AAF hydroxylase was enhanced by all the compounds employed (Fig. 6). The increased percentages of this enzyme activity resulted from pretreatments with nicotine, cotinine, nikethamide and MC were about 130, 50. 155 and 260, respectively. Pretreatments with nicotine (40 mg/kg), cotinine (40 mg/kg), BP (20 mg/kg) and MC (25 mg/kg) enhanced BP hydroxylase in rat liver microsomes about 90, 100, 220 and 180 per cent (Fig. 7). Effect of ethionineon the increasesin the hydroxylaseactivities In order to prove suggestions that the above increased hydroxylase activities by nicotine, cotinine and MC might be due to the increased enzyme synthesis, ethionine (1.0 g/kg) was injected 30 minutes before these drugs.
Fig. 8 shows the results ; both
FIG. 8. Effect of ethionine on the increase in AAF and BP hydroxylase activities. One gram per kg of ethionine was administered 30 minutes before injection of the inducers. stimulated and
MC
AAF were
and inhibited
BP hydroxylase respectively
activities
by pretreatment
with
nicotine,
cotinine
by ethionine.
DISCUSSION It was reported that the metabolic process of AAF by liver microsomes was hydro xylation in N-position (7) and 1-, 3-, 5 and 7-position of the phenolic ring and deacetyla tion of the N-position (8). AAF hydroxylase activity in rat liver microsomes was increased gradually as the animal fed AAF containing diet and the maximum peak was obtained after 3 weeks feeding. Miller et al. (9) had studied the similar experiment and they reported that the homogenates from the rats fed AAF generally had slightly more activity than those from rats not fed AAF for 2-13 weeks. In the present investigation, the injection of 20 mg/kg of MC enhanced markedly the rat liver AAF hydroxylase activity, although Miller et al. (9) reported that there was no difference between AAF hydroxylase activities of the liver from rat fed with and without MC (0.0036 per cent) containing diet. Administration of MC or BP alters the level of activities of a number of liver micro somal enzyme systems. In the findings presented here the enzyme system that metabo lizes nicotine has also been shown to be stimulated by preadministration of AAF, MC or BP. In this case, the amount of nicotine metabolized was considerably in parallel with that of cotinine yielded. In rats LD50 of nicotine in saline solution was 25.5 mg/kg (10), but it was found that 40 mg/kg of nicotine in corn oil solution was sometimes a lethal dose. Administra tion of 40 mg/kg of nicotine in corn oil enhanced the AAF hydroxylase activity two times as control and this activity did not restore to normal even after 30 hours. This increasing pattern of AAF hydroxylase activity by nicotine appeared to be different
from that by 25 mg/kg of MC. Nicotine in doses of 30 to 40 mg/kg showed a maximum activity of the AAF hydroxylase and 40 mg/kg of nicotine per day for two days or three conversely maintained the activity lower than that of the normal animals. As the livers from rat which received repeated doses of nicotine discolored black-brown in appearance, degeneration of the liver might occur. Administration of cotinine, niketha mide also increased the hepatic AAF hydroxylase as well as MC did. Nicotine and cotinine, moreover, increased BP hydroxylase activity. However, the stimulating activities of nicotine and cotinine for AAF or BP metabolizing system were less potent than those of carcinogenic MC and BP. The fact that the increase of these hepatic hydroxylase activities by nicotine, cotinine and MC was inhibited by preinjection of ethionine, a compound that inhibits incorporation of methionine and glycine into liver protein, may indicate that nicotine, cotinine and MC induced more enzyme protein synthesis as report
ed in the other papers about prevention of the induction of microsomalenzyme activities (1, 10, 12). Because carcinogenic BP was contained in cigarette smoke, it appears of interest to investigate the relationship between nicotine and BP in vivofrom the standpoint of the metabolism of both compounds and carcinogenesis. Phenols and fatty acids in cigarette tar would be possible as cocarcinogen which potentiates cancer producing property of the carcinogens (2), and it may be probable that nicotine and the related compounds might play a cocarcinogenic role, because Essenberg (13) reported that smoke of high nicotine content induced more lung tumors than that of low nicotine content. SUMMARY When rats were fed 2-acetylaminofluorene(AAF) containing diet (0.025per cent), AAF hydroxylase activity in the liver microsomeswas enhanced with maximum increase (80 per cent) at 3 weeks after the feeding. Pretreatment with 20 mg/kg of 3-methyl cholanthrene (MC) caused an maximum increase (55 per cent) at 24 hours after the in jection. Pretreatment with AAF (for 3 weeks feeding), MC (20 mg/kg, before 24 hours) and 3,4-benzpyrene(BP) (20 mg/kg, before 24 hours) enhanced nicotine metabolizing activity in rat liver microsomeswith about 50, 100 and 80 per cent increases, respectively. Pretreatment with nicotine also caused an increase in AAF hydroxylase activity with maximum elevation at 24 hours after the injection and in doses of 40 mg/kg. When nicotine (40 mg/kg),cotinine (20mg/kg), nikethamide (40 mg/kg) and MC (25 mg/kg)were preadministered to rats, AAF hydroxylase was enhanced about 130,50, 155and 260 per cent after 24 hours, respectively. Pretreatments with nicotine (40 mg/kg), cotinine (40 mg/kg), BP (20mg/kg) and MC (25 mg/kg) enhanced BP hydroxylase about 90, 100, 120 and 180 per cent after 24 hours, respectively. The increased AAF and BP hydroxylase activities by nicotine, cotinine and MC were inhibited by ethionine.
Acknowledgement: This investigation
was supported
in part by a Grant-in-Aid
for Fundamental
Research from the Ministry of Education. REFERENCES 1) CONNEY,A.H. AND BURNS, J.J.: Advances in Pharmacology 1, 31 (1962) 2) THE SURGEONGENERAL'SADVISORYCOMMITTEE: Smoking and Health, edited by GUTHRIE, E.H., p. 47, U.S. Department Washington (1963)
of Health, Education
and Welfare, U.S. Government
3) YAMAMOTO,I.: Seitainokagaku 6, 154 (1955) 4) TAKEUCHI,M.: Folia pharmacol. japon. 51, 62 (1955) 5) TsUJIMOTO, A.: Ibid. 53, 553 (1957) 6) HUCKER, H.B., GILLETTE,J.R. AND BRODIE,B.B. : J. Pharmacol. 129, 94 (1960) 7) CRAMER,J.W., MILLER, J.A. AND MILLER, E.C.: J. biol. Chem. 235, 885 (1960) 8) CRAMER,J.W., MILLER, J.A. AND MILLER, E.C.: Ibid. 235, 250 (1960) 9) MILLER, J.A., CRAMER,J.W. AND MILLER, E.C. : Cancer Res. 20, 950 (1960) 10) NAGAI, K. : Folia pharmacol. japon. 59, 442 (1963) 11) CONNEY,A.H., MILLER, E.C. AND MILLER,J.A.: J. biol. Chem. 228, 753 (1957) 12) CONNEY,A.H., MILLER, E.C. AND MILLER, J.A.: Cancer Res. 16, 450 (1956) 13) ESSENBERG,J.M. : West. J. Surg. Obstet. Gynecol. 65, 161 (1957)
Printing
Office,
AND
TO
SOME
THE
CARCINOGENS
HEPATIC
IWAO
IN
SPECIAL
DRUG-METABOLIZING
YAMAMOTO,
KAZUO
NAGAI*,
AND KATSUYA
REFERENCE
ENZYMES HIROSHI
KIMURA
IWATSUBO
Departmentof Pharmacology, Osaka UniversityDental School,Kita-ku,Osaka Received for publication November 22, 1965
In (e.g.
the
past
few
years
3,4-benzpyrene
pyrine) metabolize
the
has
standpoint
of
nicotine and
in the
be
the
foreign been
relationship drug
been
reported
to
the
enzyme
(1).
be
metabolism.
the
that
of
the
effect
of
of
on
the
phenobarbital in
of
1,000
and
not
which
10
of
reports) (2)
been
carcinogen
, but
done
pharmacological
carcinogens
amino
microsomes
cigarettes
have
of
the
liver
(average of
hydrocarbons
3 ,4
studies from
the
investigations
on
nicotine
metabolism
of
metabolism
were
subjected
to
investigated. Data
presented
in
and
3-methylcholanthrene
that
nicotine
fluorene
also
and
this
paper
indicates
enhanced increased
the
3,4-benzpyrene
the
rat
Animals,
diet
Male
and
compounds
Donryu
rats
semisynthetic
diet
Sources
of
of
liver
aminofluorene 1-nicotine
Ltd),
nikethamide
Biochemical Administration BP,
of
and
山本
巌 ・永 井
Activity,
Osaka
compounds
和 男 ・木 村 : Department
were
metabolizing
enzymes
enzyme
toward
and
2-acetylamino
METHODS
used
follows
:
Co.),
all
experiments
.
They
3,4-benzpyrene
(BP)
were
and
3-methylcholanthrene
monopolycorp
.),
Industries
and
for
fed
Co.). as
Industries
(Japan
ATP
nicotine
University
g
Chemical
nikethamide
address
AND
were
Kasei
1-cotinine
and
the
and
Cotinine
* Present
used
(Takeda
MC
nicotine
microsomes.
Kokei-Shiryo
(Tokyo
and
Corp.)
60-80
compounds
(AAF)
AG.),
of
metabolizing
3,4-benzpyrene
employed weighing
(General
the
2-acetylaminofluorene,
activity
activity
in
that
MATERIALS
a
smoke
course
effect
nicotine
(e.g.
systems
carcinogens
the
the
drugs
carcinogenic
16 ƒÊg
in
and During
(3-5),
various
About
contained
nicotine
laboratory
converse,
found
and
activity
compounds
between
our
has
3-methylcholanthrene)
markedly
several
benzpyrene on
and
increase
it
Co .),
glucose-6-phosphate
(MC)
dl-ethionine NAD (Sigma
2-acetyl
(Nakarai and
Chemicals,
NADP
Chemical
(Fluka
(Nutritional
Co .).
employed were were
given injected
拓 ・岩 壺 of Medical
intraperitoneally
in
intraperitoneally
in
0 .1 0.1
or ml
0.2 of
0.9
ml per
of
corn
oil.
cent
saline
High
Nerve
克哉
Pharmacology School
and
, Kita-ku,
Biochemistry Osaka.
of
Instit
ute
for
.
Control animals received only corn oil or saline. These drugs were usually administered 24 hours prior to assay of the liver unless otherwise stated. In case of administration of AAF 0.025 per cent was added to the diet at a final concentration. In inhibition experiments, ethionine was administered in dose of 1.0 g/kg 30 minutes before the injection of nicotine, cotinine and MC. Enzymepreparation The animals were killed by a fracture of the neck and bled the carotid arteries. The livers were then removed and 20 per cent homogenate was made in cold 1.15 per cent KCl solution. Nuclear and mitochondrial fractions of the liver cells were removed by centrifugation at 9,800x g for 30 minutes, and the supernatant having microsomes and soluble fraction (MsS) was used as an enzyme sourse. Determinationof enzymeactivities Nicotine oxidase was determined by the method of Hucker et al. (6). AAF and BP hydroxylases were determined according to the method of Cramer et al. (7) and Conney et al. (8) with some modifications respectively. For the determination of nicotine oxidase activities the incubation mixture contained 3.0 ml of MsS equivalent to 600 mg of wet liver, 0.1 ml of nicotine (486 mg/ml), 0.6 ml of cofactors and 1.3 ml of 0.2 M phosphate buffer, pH 7.4. The mixtures for the determination of AAF and BP hydroxylase activities contained 0.3 ml of MsS equivalent to 60 mg of wet liver, 0.1 ml of substrate solution in ethanol (0.5 mg/ml of both substrates respectively), 0.6 ml of cofactors and 2.0 ml of 0.2 M phosphate buffer, pH 7.4. The cofactors were composed of 0.1 ml of NAD (0.25 icmole), 0.1 ml of NADP (0.25 ,umole), 0.1 ml of glucose-6-phosphate (6 pmoles), 0.1 ml of MgC12 (75,amoles), 0.1 ml of ATP (2,amoles) and 0.1 ml of nicotinamide (120 amoles) to a total volume of 0.6 ml. The incubation was carried out at 37°C in air atmosphere of oxygen for 60, 60 and 30 minutes in the experiments on nicotine-, AAF and BP-metabolizing enzymes, respec tively. In the experiment on BP hydroxylase incubation was done in the dark. RESULTS The metabolismof AAF by the liver microsomes of rats fed AAF containingdiet and of rats in jectedwith MC ' When rats were fed AAF containing diet, AAF hydroxylase activity in the liver microsomes was enhanced (Fig. 1). Maximum increase (about 80 per cent) of this enzyme activity was observed at 3 weeks after feeding the diet. When rats were injected with 20 mg/kg of MC before 6 hours to 3 days, the AAF hydroxylase activity was markedly increased. Maximum increase (about 6 times) was shown 24 hours after injection of MC, and high activity of this enzyme persisted at least until 3 days (Fig. 2).
FIG.
1.
The
metabolism
containing by
MsS
weeks
FIG.
2.
the
of
to 60
Ordinate wet
AAF
various
by
liver
of
mg
per
of
of
AAF
: pg
liver
weeks.
beginning
metabolism
MC. mg
of at
equivalent from
The
after 60
diet
of 60
wet
the
by
microsomes
Ordinate liver
AAF
rat
AAF
liver
from
: pg per
of
30
containing
fed
AAF
metabolized
minutes.
Abscissa
:
diet.
microsomes
metabolized
rats
AAF
by
at MsS
various equivalent
days to
minutes.
The metabolismof nicotineby liver microsomes of rat pretreated with AAF, MC and BP Nicotine oxidase activity was determined 3 weeks after feeding of AAF containing diet and 24 hours after injection of 20 mg/kg of MC and 20 mg/kg of BP in reference to the above results. Pretreatment with all the three carcinogenic compounds caused an increase in the nicotine metabolizing activity in rat liver microsomes. AAF enhanced the metabolizing activity toward nicotine about 50 per cent as compared to control. MC and BP enhanced this activity about 100 and 80 per cent respectively, when the metabolized nicotine was determined. The similar tendency was also shown in case that cotinine, one of the major metabolites of nicotine, was determined (Fig. 3).
FIG. 4. Time course of AAF hydro xylase activity of MsS equivalent to 60 mg of wet liver per 60 minutes after nicotine. Ordinate:
FIG. 3. The effect of AAF, MC and BP on nicotine metabolism by MsS equivalent to 600 mg of wet liver. Ordinate : pmole of nicotine metabolized and cotinine formed. Abscissa : I= control, II=pretreatment with AAF containing diet, III=pre treatment with 20 mg/kg of MC, IV=pretreatment with 20 mg/kg of BP. n : nicotine metabolized, c:cotinine formed.
g of AAF metabolized. Abscis sa : hours after 40 mg/kg of nicotine.
Effect of nicotineon AAF and BP hydroxylasein rat liver microsomes AAF hydroxylase activities of rat liver micro somes at various time intervals after injection of 40 mg/kg of nicotine are shown in Fig. 4. The AAF hydroxylase activity increased about two times 6 hours after nicotine administration and recovery of the increased activity to normal level was not found even 30 hours after nicotine administration. Fig. 5 shows the effect of the doses of pretreated nicotine on AAF hydroxylase activity. Five to 40 mg/kg and 40 mg/kg of nicotine per day for two and three days were injected 24 hours before assay of AAF hydroxylase activity. Result showed that 30 to 40 mg/kg of nicotine induced the maximum enzyme activity. On the contrary, the daily injections of nicotine for two to three days decreased the hepatic AAF hydroxylase activity to less than control.
FIG.
5.
Effect
of MsS were mg
of
various
equivalent expressed
of
wet
liver
doses to 60 mg
as
pg per
of 60
of
nicotine
of wet AAF
minutes.
on
liver
metabolized
per
AAF
hydroxylase
60 minutes. by
MsS
activities The
equivalent
activities to 60
FIG.
6.
Effect
xylase
FIG.
7.
of
Effect
activities as pg liver
of
nicotine,
rat
liver
of of
cotinine, microsomes.
nicotine, rat
liver
conitine,
nikethamide The
BP and
microsomes.
of
BP
metabolized
per
30
minutes.
by
The MsS
and
activities
MC
MC are
on
BP
activities
equivalent
on
as
60
hydro Fig.
5.
hydroxylase
were to
AAF
under
expressed mg
of
wet
When nicotine (40 mg/kg), cotinine (20 mg/kg), nikethamide (40 mg/kg) and MC (25 mg/kg) were preadministered to rats, AAF hydroxylase was enhanced by all the compounds employed (Fig. 6). The increased percentages of this enzyme activity resulted from pretreatments with nicotine, cotinine, nikethamide and MC were about 130, 50. 155 and 260, respectively. Pretreatments with nicotine (40 mg/kg), cotinine (40 mg/kg), BP (20 mg/kg) and MC (25 mg/kg) enhanced BP hydroxylase in rat liver microsomes about 90, 100, 220 and 180 per cent (Fig. 7). Effect of ethionineon the increasesin the hydroxylaseactivities In order to prove suggestions that the above increased hydroxylase activities by nicotine, cotinine and MC might be due to the increased enzyme synthesis, ethionine (1.0 g/kg) was injected 30 minutes before these drugs.
Fig. 8 shows the results ; both
FIG. 8. Effect of ethionine on the increase in AAF and BP hydroxylase activities. One gram per kg of ethionine was administered 30 minutes before injection of the inducers. stimulated and
MC
AAF were
and inhibited
BP hydroxylase respectively
activities
by pretreatment
with
nicotine,
cotinine
by ethionine.
DISCUSSION It was reported that the metabolic process of AAF by liver microsomes was hydro xylation in N-position (7) and 1-, 3-, 5 and 7-position of the phenolic ring and deacetyla tion of the N-position (8). AAF hydroxylase activity in rat liver microsomes was increased gradually as the animal fed AAF containing diet and the maximum peak was obtained after 3 weeks feeding. Miller et al. (9) had studied the similar experiment and they reported that the homogenates from the rats fed AAF generally had slightly more activity than those from rats not fed AAF for 2-13 weeks. In the present investigation, the injection of 20 mg/kg of MC enhanced markedly the rat liver AAF hydroxylase activity, although Miller et al. (9) reported that there was no difference between AAF hydroxylase activities of the liver from rat fed with and without MC (0.0036 per cent) containing diet. Administration of MC or BP alters the level of activities of a number of liver micro somal enzyme systems. In the findings presented here the enzyme system that metabo lizes nicotine has also been shown to be stimulated by preadministration of AAF, MC or BP. In this case, the amount of nicotine metabolized was considerably in parallel with that of cotinine yielded. In rats LD50 of nicotine in saline solution was 25.5 mg/kg (10), but it was found that 40 mg/kg of nicotine in corn oil solution was sometimes a lethal dose. Administra tion of 40 mg/kg of nicotine in corn oil enhanced the AAF hydroxylase activity two times as control and this activity did not restore to normal even after 30 hours. This increasing pattern of AAF hydroxylase activity by nicotine appeared to be different
from that by 25 mg/kg of MC. Nicotine in doses of 30 to 40 mg/kg showed a maximum activity of the AAF hydroxylase and 40 mg/kg of nicotine per day for two days or three conversely maintained the activity lower than that of the normal animals. As the livers from rat which received repeated doses of nicotine discolored black-brown in appearance, degeneration of the liver might occur. Administration of cotinine, niketha mide also increased the hepatic AAF hydroxylase as well as MC did. Nicotine and cotinine, moreover, increased BP hydroxylase activity. However, the stimulating activities of nicotine and cotinine for AAF or BP metabolizing system were less potent than those of carcinogenic MC and BP. The fact that the increase of these hepatic hydroxylase activities by nicotine, cotinine and MC was inhibited by preinjection of ethionine, a compound that inhibits incorporation of methionine and glycine into liver protein, may indicate that nicotine, cotinine and MC induced more enzyme protein synthesis as report
ed in the other papers about prevention of the induction of microsomalenzyme activities (1, 10, 12). Because carcinogenic BP was contained in cigarette smoke, it appears of interest to investigate the relationship between nicotine and BP in vivofrom the standpoint of the metabolism of both compounds and carcinogenesis. Phenols and fatty acids in cigarette tar would be possible as cocarcinogen which potentiates cancer producing property of the carcinogens (2), and it may be probable that nicotine and the related compounds might play a cocarcinogenic role, because Essenberg (13) reported that smoke of high nicotine content induced more lung tumors than that of low nicotine content. SUMMARY When rats were fed 2-acetylaminofluorene(AAF) containing diet (0.025per cent), AAF hydroxylase activity in the liver microsomeswas enhanced with maximum increase (80 per cent) at 3 weeks after the feeding. Pretreatment with 20 mg/kg of 3-methyl cholanthrene (MC) caused an maximum increase (55 per cent) at 24 hours after the in jection. Pretreatment with AAF (for 3 weeks feeding), MC (20 mg/kg, before 24 hours) and 3,4-benzpyrene(BP) (20 mg/kg, before 24 hours) enhanced nicotine metabolizing activity in rat liver microsomeswith about 50, 100 and 80 per cent increases, respectively. Pretreatment with nicotine also caused an increase in AAF hydroxylase activity with maximum elevation at 24 hours after the injection and in doses of 40 mg/kg. When nicotine (40 mg/kg),cotinine (20mg/kg), nikethamide (40 mg/kg) and MC (25 mg/kg)were preadministered to rats, AAF hydroxylase was enhanced about 130,50, 155and 260 per cent after 24 hours, respectively. Pretreatments with nicotine (40 mg/kg), cotinine (40 mg/kg), BP (20mg/kg) and MC (25 mg/kg) enhanced BP hydroxylase about 90, 100, 120 and 180 per cent after 24 hours, respectively. The increased AAF and BP hydroxylase activities by nicotine, cotinine and MC were inhibited by ethionine.
Acknowledgement: This investigation
was supported
in part by a Grant-in-Aid
for Fundamental
Research from the Ministry of Education. REFERENCES 1) CONNEY,A.H. AND BURNS, J.J.: Advances in Pharmacology 1, 31 (1962) 2) THE SURGEONGENERAL'SADVISORYCOMMITTEE: Smoking and Health, edited by GUTHRIE, E.H., p. 47, U.S. Department Washington (1963)
of Health, Education
and Welfare, U.S. Government
3) YAMAMOTO,I.: Seitainokagaku 6, 154 (1955) 4) TAKEUCHI,M.: Folia pharmacol. japon. 51, 62 (1955) 5) TsUJIMOTO, A.: Ibid. 53, 553 (1957) 6) HUCKER, H.B., GILLETTE,J.R. AND BRODIE,B.B. : J. Pharmacol. 129, 94 (1960) 7) CRAMER,J.W., MILLER, J.A. AND MILLER, E.C.: J. biol. Chem. 235, 885 (1960) 8) CRAMER,J.W., MILLER, J.A. AND MILLER, E.C.: Ibid. 235, 250 (1960) 9) MILLER, J.A., CRAMER,J.W. AND MILLER, E.C. : Cancer Res. 20, 950 (1960) 10) NAGAI, K. : Folia pharmacol. japon. 59, 442 (1963) 11) CONNEY,A.H., MILLER, E.C. AND MILLER,J.A.: J. biol. Chem. 228, 753 (1957) 12) CONNEY,A.H., MILLER, E.C. AND MILLER, J.A.: Cancer Res. 16, 450 (1956) 13) ESSENBERG,J.M. : West. J. Surg. Obstet. Gynecol. 65, 161 (1957)
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