[CANCER RESEARCH 27 Part 1, 1931-1938,November 1967]
Stimulatory Effect of Poly cyclic Hydrocarbons
and Aromatic
Derivatives on the Metabolism of 7,12-Dimethylbenz(a)anthracene1
Azo
W. LEVIN AND A. H. CONNEY
The Wellcome Research Laboratories, Burroughs Wellcome & Co. (U.S.A.),
SUMMARY
Treatment of rats with certain polycyclic aromatic hydro
carbons and aromatic azo derivatives enhances severalfold the
hepatic metabolism of 7,12-dimethylbenz(a)anthracene
(DMBA) to fluorescent metabolites. Studies with tritiated DMBA
revealed that treatment of rats with 3-methylcholanthrene (MC)
stimulates the hepatic metabolism of DMBA to 7-hydroxymethyl-12-methylbenz(a)anthracene
(7-OHM-12-MBA),
12hydroxymethyI-7-methylbenz(a)anthracene
(12-OHM-7-MBA),
and several unidentified polar metabolites. MC treatment
also enhances the hepatic metabolism of monohydroxymethyl
DMBA to highly polar unidentified compounds. The stimula
tory effect of MC on the hepatic metabolism of DMBA and its
monohydroxymethj'l metabolites is paralleled by altered in vivo
metabolism of DMBA. Treatment of rats with MC at 24 hours
prior to DMBA-3H administration resulted in a markedly de
creased concentration of tritiated hydrocarbon in the adrenal
gland, mammary gland, and fat. More detailed examination of
the adrenal gland and fat revealed that MC pretreatment de
creased the concentrations of DMBA, 7-OHM-12-MBA, and
12-OHM-7-MBA in these tissues. The results obtained suggest
that the induction of hepatic enzymes that metabolize DMBA
and its monohydroxymethyl metabolites play an important role
in flecreasing the adrenal toxicity and carcinogenicity of DMBA
in rats pretreated with polycyclic hydrocarbons and aromatic
azo derivatives.
INTRODUCTION
DMBA2 causes mammary cancer and massive necrosis of the
adrenal gland in rats (13,15). Recently, it was found that pre
treatment with any of several polycyclic hydrocarbons or aro
matic azo derivatives protects rats from adrenal injury and
mammary cancer induced by a subsequent dose of DMBA (9,
'This study was supported by Research Contract No. PH 4365-10GGfrom the Pharmacology-Toxicology Programs, NIGMS,
NIH.
2The following abbreviations are used: DMBA, 7,12-dimethylbenz(«)anthracene; 7-OHM-12-MBA, 7-hydroxymethyl-12-methylbenz(a)anthracene;
7,12-diOHM-BA, 7,12-dihydroxymethylbenz(a)anthracene; MC, 3-methylcholanthrene; BP, benz(a)pyrene; NADPH, reduced nicotinamide adenine dinucleotide
phosphate; NADP, nicotinamide adenine dinucleotide phosphate,
NAD, nicotinamide adenine dinueleotide; ATP, adenosine triphosphate.
Received February 1, 1907; accepted June 15, 1907.
Inc., Tuckahoe, New York 10707
11, 12, 14, 16, 21). Since many of these protective aromatic
compounds rapidly induce the synthesis of enzymes in liver
microsomes that detoxify carcinogens such as benz (a) pyrene
(7, 21), aminoazo dyes (1, 6), and Ar-2-fluorenylacetamide (8,
19), the possibility was considered that the protective compounds
enhanced the in vivometabolism of DMBA by inducing the syn
thesis of hepatic enzymes that metabolize DMBA to nontoxic
products. A preliminary report from our laboratory showed that
many of the protective compounds are potent inducers of enzymes
in rat liver that metabolize DMBA (5). Shortly thereafter,
Jellinck and Goudy also reported a stimulatory effect of poly
cyclic hydrocarbons on the hepatic metabolism of DMBA (17).
The present report describes in more detail the stimulatory effect
of polycyclic hydrocarbons and aromatic azo derivatives on the
in vitro and in vivo metabolism of DMBA in the rat.
MATERIALS AND METHODS
Metabolism of DMBA to Fluorescent Metabolites by
Rat Liver Homogenate. Female Sprague-Dawley rats weighing
140-155 gm were injected intraperitoneally with enzyme indu
cers dissolved in corn oil (5). Liver homogenate was prepared
and incubated with DMBA and an NADPH-generating system
for 15 minutes at 37°Cas previously described (5). The metabo
lism of DMBA to fluorescent Metabolites A and B was measured
as previously described and was proportional to enzyme concen
tration from 0 to 10 mg, wet weight, of liver (5).
Metabolism of Tritiated DMBA by Liver Homogenate.
Tritiated DMBA (189 me or 17.7 c per mmole, generally labeled)
was obtained from Xuclear-Chicago Corporation. The hydro
carbon was purified by thin-layer chromatography on Silica Gel
G with benzene, when necessary, to give a purity greater than
98%. DMBA-3H was incubated with liver homogenate in the
presence of cofactors, and the mixture was extracted with acetone-hexane at zero time or after the incubation as described
previously (5). A 7.5-ml aliquot of the organic phase was evap
orated to dryness under a stream of nitrogen. The residue was
dissolved in 0.3 ml of acetone, and 10 microliters of this solution
were applied to Silica Gel G (Brinkman Instrument Co.) thinlayer plates and chromatographed with 5% ethanol in benzene
as described by Boyland and Simms (2). Synthetic reference
samples 7-OHM-12-MBA, 12-OHM-7-MBA, 7,12-dihydroxymethylbenz(<*)anthracene (7,12-diOHM-BA), and cts-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene
were ob
tained from Drs. E. Boyland and P. H. Jellinck and were chro
matographed with the metabolites. After development of the
chromatograms, 0.5- or 1-cm sections of the silica gel were
NOVEMBER 1967
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1931
W. Levin and A. H. Conney
TABLE 1
Induction of Hepatic DMBA Metabolism by Various Polycyclic
Aromatic Hydrocarbons and Aromatic Azo Compounds
Adult female rats were injected with the indicated amounts of
enzyme inducers at 24 hours prior to sacrifice. Liver homogenate
equivalent to 10mg (wet weight) liver was incubated for 15minutes
with 195nijumolesof 7,12-dimethylbenz(a)anthracene (DMBA) and
a system that generates reduced nicotinamide adenine dinucleotide phosphate. Formation of Metabolites A and B were measured
as described in Methods. The values given represent the average
of assays on individual rats. The number of rats used is given in
brackets.
TABLK 1—Continued
inMetabolite increase
administeredAzobenzeneNaphthalenei-StilbeneDose(mg)2040GO100100200100Percent
Compound
A
B
formation125
formation0
[2]133
[3]187
[4]200
[2]50
[2]75
[3]12
[4]Metabolite
inMetabolite increase
administered3-MethylcholanthreneSudan
Compound
Aformation113
[4]657
[7]712
0.5150.10.5150.1
[4]1148
[9]200
IIIBenz(«)pyreneChryseneBenz
Bformation75
[2]
250
[2]245
[3]309
[8]33
[6]417
[Ü]567
[6]860
[8133
[3]85
[4]110
[4]180
[6]38
[4]375
[4]50
[2]100
[2]12
[2]75
[4]200
[4]350
[6]025
[2]0
[2]40
[4]49
[4]90
[4]185
[2]62
[2]163
[4]188
[4]117
[2]112
[2]52
[2]10
[3]
scraped into scintillation vials, and the radioactivity was quan
tified in a Packard scintillation spectrometer utilizing the scin
tillation mixture described by Bray (4).
Metabolism of Tritiated DMBA in Vivo. Tritiated DMBA
(78 Amólescontaining 20-140 ¿ic)was dissolved in 0.5 ml of
dimethyl sulfoxide and was administered by stomach tube to
adult female rats. The rats were killed by decapitation and 30to 90-mg samples of various tissues were dissolved in 1.0 ml of
hydroxide of Hyamine (10 M in methanol, Packard Instrument
Co.) at 50°Cfor 4-6 hours. Radioactivity was quantified in a
liquid scintillation spectrometer after the addition of 10 ml of a
solution containing 0.5% of 2,5-diphenylo.xazole and 0.01% of
[2]
[6]
l,4-bis-2-(5-phenvloxazolyl)benzene
in toluene and 0.2 ml of
[2]212
135
[4]467
350
0.515101235100.51350.10.5150.51251010203025100Percent
glacial
acetic
acid.
The
acetic
acid
was
necessary to reduce color
[2]360
[3]912
ation and peroxides formed during tissue dissolution (10). Radio
[2]12
[4]1062
activity in each sample was quantified in the absence and in the
[4]50
presence of standard toluene-3H and radioactivity was corrected
[2]38
[2]138
for quenching and expressed as mamóle equivalents of DMBA.
[2]112
[4]450
In some experiments, the tissues were homogenized in 4 volumes
[2]300
[2]800
of ice-cold water and 2 volumes of cold acetone were added. Six
[2]50
[4]1000
volumes of hexane were then added, and the mixture was shaken
[2]87
for 20 minutes. Following centrifugation, an aliquot was removed
and the extraction procedure was repeated. A third extraction
[2]90
[4]200
anthraceneSudan
(a )
did not remove radioactivity from the tissue homogenate. Ali[2]132
[4]362
quots of the first two organic solvent extracts were pooled and
[3]207
[4]787
evaporated to dryness under nitrogen. The residue was dis
[4]15
[4]100
solved in 0.5 ml acetone and chromatographed on Silica Gel G
thin-layer Chromatographie plates with 5% ethanol in benzene.
[2]30
[4]130
IV7,12-Dimethylbenz(a)-anthraceneAnthraceneFluoreneDose(mg)0.1
Radioactivity on the plates was quantified as described above.
[2]50
[4]275
[11]61
[17]165
[20]Metabolite [14]
1932
RESULTS
Effect of Enzyme Iiidueers on the Metabolism of DMBA
to Fluorescent Metabolites by Rat Liver. The ability of
several polycyclic aromatic hydrocarbons and aromatic azo de
rivatives to enhance the in vilro metabolism of DMBA is shown
in Table 1. The intraperitoneal injection of 1 mg or less of MC,
BP, or Sudan III at 24 hours before sacrifice resulted in large
increases in the metabolism of DMBA by rat liver. Chrysene,
benz(or)anthracene, Sudan IV, and DMBA itself were also potent
inducers, but higher doses were required. Fluorene, anthracene,
and azobenzene were weakly active at very high doses whereas
naphthalene and i-stilbene had little or no activity.
Examination of the fluorescent spectra of 7-OHM-12-MBA,
CANCER RESEARCH VOL. 27
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
Polycyclic Aromatic Hydrocarbons
12-OHM-7-MBA, 7,12-diOHM-BA, and m-5,6-dihydro-5,6dihydroxy-7,12-dimethylbenz(a)anthracene
revealed that the
fluorescent metabolites, A and B, which are probably mix
tures, differed from the four reference compounds. Further
studies were initiated to determine the properties of Metab
olites A and B. Liver homogenate, equivalent to 10 mg of wet
weight liver obtained from MC-pretreated rats, was incubated
with DMBA and an NADPH-generating system. DMBA and
its metabolites were extracted into a mixture of acetone and
hexane as described in Methods, and the organic solvent extracts
from several flasks were pooled and evaporated to dryness under
nitrogen. The residue was dissolved in acetone and chromatographed on Silica Gel G with 5 % ethanol in benzene. Brief ir
radiation of the plate with ultraviolet light revealed the presence
of fluorescent compounds with the mobility of DMBA (RF 0.97),
7-OHM-12-MBA (RF 0.54), and two more polar metabolites with
RP values of 0.23 and 0.29. The fluorescent metabolite corre
sponding to 7-OHM-12-MBA was eluted from the thin-layer
plate with methanol. Examination of the ultraviolet and fluo
rescence spectra of this metabolite revealed that they were iden
tical with those obtained with an authentic reference sample of
7-OHM-12-MBA. The fluorescence spectrum of the metabolite
with an RF of 0.23 agreed with that described in Methods for
Metabolite A. Further work is required to determine whether
Metabolite A is a single compound or a mixture.
Stimulatory Effect of MC on the Metabolism of Tritiated DMBA by Rat Liver. Incubation of 195 m/jmoles of
DMBA-3H with 10 mg of liver for 15 minutes resulted in the
formation of metabolites which remained in the aqueous phase
when the incubation mixture was extracted with an acetonehexane mixture (Table 2). Considerably larger amounts of nonextractable metabolites were formed when 11.7 m/mioles of
DMBA-3H were incubated with 50 mg of liver for 60 minutes.
Treatment of rats with MC enhanced severalfold the ability of
liver to metabolize DMBA to nonextractable metabolites that
remained in the aqueous phase (Table 2). Aliquots of the aqueous
and organic phases were subjected to thin-layer chromatography
(Charts 1, 2). Incubation of 10 mg of liver with 195 mamólesof
DMBA-3H for 15 minutes resulted in the metabolism of DMBA
to 7-OHM-12-MBA, 12-OHM-7-MBA, and several unidentified
polar metabolites (Chart 1). Treatment of rats with MC increased
the hepatic metabolism of DMBA by more than 700 percent
as measured by substrate disappearance, formation of 7-OHM12-MBA, and the formation of three highly polar metabolites that
were observed near the origin of the thin-layer chromatograms
(Chart 1, Table 3). Formation of 12-OHM-7-MBA was stimu
lated to a lesser extent, and liver homogenate from control or
MC-treated rats did not metabolize DMBA to 7,12-diOHM-BA
or cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene to any appreciable extent. Interestingly, MC treatment
caused a severalfold decrease in the metabolism of DMBA to 7OHM-12-MBA and 12-OHM-7-MBA when 11.7 m/imoles of
DMBA were incubated with 50 mg of liver for 60 minutes (Chart
2, Table 3). Under these incubation conditions, MC treatment
markedly stimulated the disappearance of substrate and the
formation of metabolites more polar than 7,12-diOHM-BA.
The results obtained with a low substrate concentration and a
long incubation (Chart 2, Table 3) suggested that MC treatment
stimulated not only the metabolism of DMBA to its monohydroxymethyl metabolites, but also the further metabolism of 7OHM-12-MBA and 12-OHM-7-MBA.
Stimulatory Effect of MC on the Metabolism of Monohydroxymethyl-DMBA
by Rat Liver. Tritiated monohydroxymethyl-DMBA was prepared by incubating DMBA-3H
with liver from MC-treated rats as described in the legend to
Fig. 2. The acetone-hexane extracts from several vessels were
pooled, evaporated to a small volume and chromatographed on
Silica Gel G with benzene:ethanol (19:1, v/v). The area corre
sponding to 7-OHM-12-MBA and 12-OHM-7-MBA was scraped
from the plate and eluted with ethanol. The eluate was concen
trated and rechromatographed twice on Silica Gel G with ben
zene. This preparation contained 7-OHM-12-MBA and 12OH.M-7-MBA in a 7:1 ratio.
The intraperitoneal injection of rats with 5 mg MC at 24 hours
TABLE 2
Organic Solvent Extraction of Tritialed 7,l%-Dimethylbenz(ot)anthracene
by Liver Homogenates of Control and S-Methylcholanthrene
(DMHA-1H) Metabolites Formed
(MC)-prelreated Rats
Adult female rats were injected intraperitoneally
with 5 mg of MC at 24 hours before sacrifice. Liver
homogenate was incubated with J)MBA-*II in the presence of a system that generates reduced nicotinamide adenine dinucleotide phosphate, and the incubation mixture was extracted with acetonehexane as described in Methods. Following ceutrifugation,
aliquots of the aqueous phase and organic
phase were taken for the determination of radioactivity.
The average percent of radioactivity in the
aqueous and organic phase is given. The values in parentheses represent data from individual rats.
Experimen
tit23TreatmentCorn
oilMCCorn
oilMCCorn
oilMCCorn
oilMCLiver
NOVEMBER
inAqueous
time(minutes)154515GOPercent
(mg)10101050DMBAincubated(mamóles)19519519511.7Incubation
phase0.7
of radioactivity
phase97.6
98.2)92.9
(97.1,
(4.3,5.0)1.4
93.1)98.0
(92.8,
1.6)9.2
(1.2,
98.3)90.0
(97.8,
(8.9,9.6)0.5
90.0)98.2
(90.0,
0.5)4.5
(0.5,
(98.1,98.3)93.9
(93.8,94.1)82.2
4.8)17.0
(4.3,
17.4)70.8
(16.7,
(82.1,82.3)27.6
(69.0, 72.6)Organic
(26.6,28.6)
(0.5,0.8)4.6
1967
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1933
W. Levin and A. H. Conney
ORGANIC
PHASE
AQUEOUS
350,000250,000- .
8,200
Polor Frocfion I
Polar Fraction
PHASE
2
1,800I.40O-
TT
1,000-
800-
600-
7-OHM-/2-MBA
J.MC
400-
11 7,12-diOHM-SA
OMBA
200-
8
IO
12
14
16
024
CENTIMETERS
FROM ORIGIN
GHAUT1. Metabolism of DMBA by rat liver homogenate (high substrate and low tissue concentration). Adult female rats were in
jected intraperitoneally with corn oil or with 5 mg of MC in corn oil. The rats were killed 24 hours later. Homogenate equivalent to 10
mg of liver was incubated for 15 minutes with 195 mamólesof DMBA-'H and a system that generates reduced nicotinamide adenine
dinucleotlde phosphate in a total volume of 3.1ml. Three ml of cold acetone and 9 ml of hexane were added, and the mixture was shaken
for 20 minutes and centrifuged. A 7.5-ml aliquot of the organic phase was taken to dryness under nitrogen and redissolved in 0.3 ml of
acetone. Exactly 10 microliters of the acetone were spotted on Silica Gel G and chromâtographedin a benzene:ethanol (19:1, v/v)
mixture. Fifteen microliters of the aqueous phase were applied directly to the Silica Gel G plate and chromatographed in the
same system. cis-5,()-Dihydro-5,(i-dihydroxy-7,12-dimethylbenz(a)anthracene
was also used as a reference compound and it had the
same RF value as 7,12-diOHM-BA. DMBA, 7,12-dimethylbenz(a)anthracene; MC, 3-methylcholanthrene; 7,12-diOHM-BA, 7,12dihydroxymethylbenz(a)anthraccnc;
7-OIIM-12-MBA, 7-hydroxymethyl-12-methylbenz(«)anthracene; 12-OHM-7-MBA, 12-hydroxymethyl-7-methylbenz(a)anthracene.
TABLE 3
Stimulatory
Effect of MC" on the Metabolism of DMBA by Rat Liver
Adult female rats were injected intraperitoneally with 5 mg of MC at 24 hours before sacrifice. Ten
mg of liver were incubated with 195 mamólesof UMBA-3H for 15 minutes in Experiment 1. Fifty mg
of the same liver were incubated with 11.7 rmtmoles of DMBA for 60 minutes in Experiment 2.
The various metabolites in the acetone-hexane extract and in the aqueous phase were quantified follow
ing chromatography as described in Charts 2 and 3.
Ex
peri
ment12TreatmentCorn
metabolized
(mjimoles)2.8
Fraction 1
(mamóles)0.2
formed
Fraction
formed
(m/imoles)0.5
oil (2.9,2.7)23.4
(0.2,0.2)
(24.3, 22.6) 2.1 (2.3, 1.9) 8.1
MC
Corn oil 3.3 (3.6,3.0)
1.2)7.6
1.1 (1.1,
0.5
MCDMBA 8.6 (8.4,8.8)Polar
(7.6,7.6)Polar0.9
2
(m/imoles)1.2
formed
(mamóles)0.9
(0.5, 0.5)
(1.4,1.1)
(1.0,0.9)1.7
(8.3,7.9)
10.8 (11.5, 10.2)
(1.8, 1.6)
(0.5, 0.5)
0.9 (0.8, 1.0)
0.7 (0.7,0.7)
(0.9, 0.9)7-OHM-12-MBA
0.1 (0.1,0.0)12-OHM-7-MBAformed
0.1 (0.1, 0.0)
" MC, 3-methylcholanthrene; DMBA, 7,12-dimethylbenz(a)anthracene; 7-OHM-12-MBA, 7-hydroxymethyl-12-methylbenz(a)anthraeene; 12-OHM-7-MBA, 12-hydroxymethyl-7-methyl benz(a)anthracene.
1934
CANCER RESEARCH VOL. 27
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
Polycyclic Aromatic Hydrocarbons
ORGANIC PHASE
20,00010,000-
AQUEOUS PHASE
2,5002,400-
1,000-
800-
600-
7-OHM12-MBA
IrConfrol
400-
200-
CENTIMETERS
FROM ORIGIN
CHART 2. Metabolism of DMBA by rat liver homogenate (low substrate and high tissue concentration). Adult female rats were in
jected ¡ntraperitoneally with corn oil or with 5 mg of MC and killed 24 hours later. Homogeuate equivalent to 50 mg of liver was incu
bated for 00 minutes with 11.7 imimoles of DMBA-'H and a system that generates reduced nicotinamide adenine dinucleotide phosphate
in a total volume of 3.1 ml. Three ml of cold acetone and 9 ml of hexane were added and the mixture was shaken for 20 min and
centrifuged. A 7.5-ml aliquot of the organic phase was taken to dry ness under nitrogen and redissolved in 0.3 ml of acetone. Exactly 10 microliters of the acetone were spotted on Silica Gel G and chromâtographed in a benzene rethanol (19:l,v/v) mixture. Fifteen microliters
of the aqueous phase were chromatographed
in the same system. DMBA, 7,12-dimethylbenz(a)anthracene;
7,12-diOHM-BA. 7,12dihydroxymethylbenz(a)anthracene;
12-OHM-7-MBA, 12-hydroxymethyl-7-methylbenz(a)anthracene;
7-OHM-12-MBA, 7-hydroxymethyl-12-methylbenz(a)anthraeene.
before sacrifice markedly enhanced the hepatic metabolism of
monohydroxymethyl-DMBA to water-soluble metabolites that
were not extractable into acetone-hexane (Table 4). Chromatography of the acetone-hexane extract also revealed that MC ad
ministration enhanced severalfold the metabolism of monohy
droxymethyl-DMBA to the polar Fraction 1 shown in Charts
1 and 2.
The possibility was considered that DMBA inhibited the
metabolism of monohydroxymethyl-DMBA formed during in
cubations of DMBA with liver. The results shown in Table 5
indicate that DMBA inhibits the hepatic metabolism of its
monohydroxymethyl metabolites. The stimulatory effect of in
vivo MC treatment on the hepatic metabolism of monohydroxy
methyl-DMBA and the in vilro inhibitory effect of DMBA on
the hepatic metabolism of monohydroxymethyl-DMBA
ex
plains why treatment of rats with MC enhances the hepatic con
version of DMBA to monohydroxymethyl-DMBA when a high
substrate concentration is incubated for a short time (Chart 1)
and why MC treatment inhibits the accumulation of mono
hydroxymethyl-DMBA in the incubation mixture when a low
NOVEMBER
concentration of DMBA is incubated for a long time with large
amounts of liver (Chart 2).
Effect of MC on the Metabolism of Tritiated DMBA in
Vivo in the Rat. The intraperitoneal injection of rats with 5 mg of
MC at 24 hours prior to an oral dose of 20 mg (78 jumóles)of
DMBA-3H resulted in a 50-85% decrease in the concentration
of tritiated hydrocarbon in adrenal gland, mammary gland, and
fat at 2 and 4 hours after DM15A administration (Table 6). MC
pretreatment decreased the hepatic concentration of hydrocarbon
in some experiments, but not in others. Although MC treatment
at 24 hours prior to DMBA-3H administration markedly de
creased the concentration of tritiated hydrocarbon in the adrenal
gland and fat, no such decrease was observed when MC was given
2 hours before DMBA (Table 6).
Organic solvent extracts of adrenal gland and fat were chromat
ographed to determine whether metabolites of DMBA were
present in these tissues. It was found that most of the tritiated
hydrocarbon in both adrenal gland and fat was DMBA,
but measurable amounts of 7-OHM-12-MBA and 12-OHM-7MBA were also present (Table 7). The administration of MC at
1907
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1935
W. Levin and A. H. Conney
24 hours prior to the oral administration of DMBA-'H resulted
TABLE 6
in a 37, 71, and 35% decrease in the adrenal concentration of
DMBA, 7-OHM-12-MBA, and 12-OHM-7-MBA, respectively,
and the concentrations of these compounds in fat were decreased
73, 71, and 83%, respectively (Table 7). Pretreatment of rats
with naphthalene, a very weak stimulator of hepatic DMBA
metabolism (Table 1) did not decrease the concentration
of DMBA, 7-OHM-12-MBA, or 12-OHM-7-MBA in the adrenal
gland or fat.
Effect of3-Methylcholanlhrene (MC) Pretreatment on the Concentra
tion of 7,12-Dimethylbenz(a)anlhracene (DMBA) and Its
TABLE 4
Stimu laiory Effect of 3-Methylcholanthrene (MC) on the Metabolism
of Monoh.ydroxymelhyl-7,12-dimelhylbenz(a)anthracene
(Monohydroxymethyl-DMBA) by Rat Liver
Adult female rats were injected intraperitoneally
with 5 mg of
MC at 24 hours before sacrifice. Homogenate equivalent to 10 mg
of liver was incubated for 15 minutes with 3.7 mamóles of monohydroxymethyl-DMBA-3H
in the presence of a system that gener
Metabolites in Tissues of Rats Given DMBA-*H
In Experiments 1 and 2, rats pregnant for 15 days were injected
intraperitoneally
with 5 mg of MC at 24 hours before the oral
administration of 20 mg of DMBA-3H, and the rats were killed at
various times. These rats were heavier than those usually used and
weighed 275-325 gm. Pregnant rats were used in Experiments 1
and 2 to obtain sufficient mammary tissue for analysis. In Experi
ment 3, adult female rats (nonpregnant)
were injected intra
peritoneally with 5 mg of MC at 2 hours (acute treatment) or 24
hours prior to the oral administration of 20 mg of DMBA-'H, and
the rats were killed 2 hours later. Total radioactivity was deter
mined in the various tissues as described in Methods. Each value
represents the average and standard error from 5 rats per group.
afterHydrocarbonDMBA
Experiment123TissueAdrenalAdrenalMammaryMammaryLiverLiverAdrenalAdrenalMammaryMammaryLiverLiver
(m/imoles/gmadministration222444222tissue)51.0
oilMCCorn
ates reduced nicotinamide adenine dinucleotide phosphate. The
¡nruhalinn mixture was extracted
with acetone-hexane
as
described in the legend to Chart 2. Nonextractable
radioactivity
in the aqueous phase was quantified. Aliquota of the organic sol
vent were chromatographed
as described in the legend to Chart 2,
and radioactivity
in the various areas of the thin-layer chromatogram was quantified as described in Methods.
oilMCCorn
oilMCCorn
oilMCCorn
oilMCCorn
Experimenti23TreatmentCorn
DMBA metabolized
metabolites formed
ttremole?)0.561.820.632.320.732.39Water-soluble
(mamóles)0.401.540.582.030.652.26
oilMCCorn
oilMCCorn
oilAcute
MCMCCorn
oilMCCorn
oilMCMonohydrcxymethyl-
oilAcute
MCMCCorn
oilAcute
MCMCHours
TABLE 5
In Vitro Inhibitory Effect of 7,12-Dimethylbenz(a)anthracene
(DMBA) on the Metabolism of Monohydroxymethyl-DM HA
by Rat Liver
Adult female rats were injected intraperitoneally
with 5 mg of
3-methylcholanthrene
at 24 hours before sacrifice. Homogenate
equivalent to 10 mg of liver was incubated with 3.7 mamóles of
monohydroxymethyl-DMBA-3H
in the presence of a system that
generates reduced nicotinamide adenine dinucleotide phosphate
as described under Table 4. Various amounts of nonradioactive DMBA were-added to the incubation vessels.
3.421.1
±
1.419.5
±
2.07.3
±
0.844.7
±
5.049.7
±
3.561.4
±
5.217.8
±
1.063.4
±
±7.38.8
0.674.4
±
3.146.8
±
3.266.4
±
4.873.2
±
6.924.3
±
3.236.2
±
4.135.7
±
0.210.7
±
2.291.2
±
5.075.4
±
2.559.9
±
±4.8
DISCUSSION
The ability of several polycyclic aromatic hydrocarbons and
aromatic azo derivatives to prevent DMBA-induced adrenal
necrosis and mammary cancer (9, 11, 12, 14, 16, 21) is paralleled
by enhanced DMBA metabolism by liver homogenate under con
ditions where metabolite formation is proportional to tissue con
centration. MC, BP, Sudan III, Sudan IV, benz(a)anthracene,
soluble
chrysene, and DMBA are potent inducers of DMBA metabolism
added
DMBA metabolized
Experiment12DMBA
metabolites formed
(mamóles)039195039195780Monohydroxvmethyl(mamóles)2.321.590.772.391.781.080.80Water
and are potent inhibitors of DMBA-induced adrenal damage.
(mamóles)2.031.520.722.261.650.860.62
Azobenzene, anthracene, and fluorene are weak inducers of
DMBA metabolism at high dosage levels, and these compounds
also block DMBA-induced adrenal damage but only at
high dosages. i-Stilbene and naphthalene, which are inactive in
preventing adrenal damage by DMBA, have little or no effect
on the hepatic metabolism of DMBA even after the intraperitoneal injection of 100-200 mg of these compounds.
The use of a high substrate concentration, low tissue concen
tration, and a short incubation revealed that MC treat-
1936
CANCER
RESEARCH
VOL. 27
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
Polycyclic Aromatic Hydrocarbons
ment caused a severalfold increase in the hepatic metabolism of
DMBA as measured by substrate disappearance, formation of
7-OHM-12-MBA and the formation of three highly polar metab
olites. Formation of 12-OHM-7-MBA was stimulated to a lesser
extent. In contrast to these results, when a low substrate con
centration, high tissue concentration and a long incubation were
used, MC pretreatment increased the hepatic metabolism of
DMBA to highly polar metabolites but decreased the yield of
7-OHM-12-MBA and 12-OHM-7-MBA. These latter results
agree with recent studies by Boyland (3) and Jellinck and Goudy
TABLE 7
Effect of MC" Pretreatment on the Concentration of DMBA,
(17, 18). The effect of in vivo MC administration to stimulate
hepatic DMBA metabolism but decrease the yield of 7-OHM-127-OHM-18-MBA, and 12-OHM-7-MBA in Rats Given DMBA-3H
Adult female rats were injected intraperitoneally
with 5 mg of MBA and 12-OHM-7-MBA can be explained by a stimulatory
effect of MC on the metabolism of DMBA to 7-OHM-12-MBA
MC or 100 mg of naphthalene at 24 hours before the oral admin
istration of 20 mg of DMBA-8H (150 ¡tc).The rats were killed 2 and 12-OHM-7-MBA and also by enhanced metabolism of these
monohydroxymethyl-DMBA metabolites to highly polar com
hours later. Adrenal glands and fat from 10 rats per group were
pooled and homogenized in 4 volumes of ice-cold water. Two
pounds. The stimulatory effect of MC on DMBA metabolism is
volumes of acetone and six volumes of hexane were added, and
summarized in Chart 3.
the mixture was shaken 20 minutes and centrifuged. The or
The stimulatory effect of MC on the hepatic metabolism of
ganic phase was removed, and the aqueous phase extracted
DMBA
and its monohydroxymethyl metabolites is paralleled by
with acetone-hexane
again. A third extraction did not remove
altered metabolism of DMBA in vivo. Pretreatment of rats with
more radioactivity.
Radioactivity
in the aqueous phase and or
MC prior to DMBA-'H administration resulted in markedly
ganic solvent extract was quantified. The acetone-hexane
ex
decreased concentrations of tritiated hydrocarbon in the adrenal
tract was evaporated to dryness, redissolved in 1.0 ml of acetone,
gland, mammary gland, and fat. More detailed examination of
and exact aliquots were chromatographed
on Silica Gel G with
5% ethanol in benzene. Radioactivity
on the thin-layer plate was
the adrenal gland and fat revealed that MC pretreatment
quantified and used to calculate the amount of DMBA, 7-OHMdecreased the concentrations of DMBA, 7-OHM-12-MBA, and
12-MBA, and 12-OHM-7-MBA in adrenal gland and fat.
12-OHM-7-MBA in these tissues. The effect of MC on the con
centration of 7-OHM-12-MBA in the tissues of rats treated with
organicphase
in
DMBA is of considerable interest since 7-OHM-12-MBA is a
tissue)DMBA42.344.426.518.824.95.07-OHM(rmimoles/gm
TissueAdrenalFatTreatmentCorn (mamóles/
'
metabolite of DMBA that causes adrenal necrosis and induces
gmtissue)11.712.17.91.62.01.8Organicphase(m/imoles
gmtissue)60.161.535.325.332.68.7Hydrocarbons
mammary cancer (3). The results presented here suggest that the
7-MBA2.01.91.31.21.30.2
12-MBA8.67.82.51.61.70.312-OHMinduction of hepatic enzymes that metabolize DMBA and
its monohydroxymethyl metabolites plays an important role in
oilNaphthaleneMCCorn
decreasing the adrenal toxicity and carcinogenicity of DMBA
in rats pretreated with polycyclic hydrocarbons and aromatic azo
oilNaphthaleneMCAqueousphase
derivatives. The results of earlier but less detailed studies from
our laboratory (5) and by Jellinck and Goudy (17, 18) also sug
gest that the induction of hepatic enzymes that metabolize
DMBA plays an important role in decreasing the adrenal toxicity
°MC, 3-methylcholanthrene;
DMBA, 7,12-dimethylbenz(a)of DMBA. The ability of polycyclic hydrocarbons to stimulate
anthracene;
7-OHM-12-MBA, 7-hydroxymethyl-12-methylbenz(a)anthracene;
12-OHM-7-MBA,
12-hydroxymethyl-7-methyl
- the hepatic metabolism of DMBA and inhibit the carcinogenicity
benz(a)anthracene.
of this compound is similar to the effects of polycyclic hydrocar
CH,
I2-OHM-7-MBA
UNKNOWN
POLAR
METABOLITES
CHjOH
7-OHM-I2-MBA
CHART 3. Pathways of DMBA metabolism that are stimulated
12-OHM-7-MBA, 12-hydroxymethyl-7-methylbenz(a)anthracene;
NOVEMBER
by 3-methylcholanthrene.
DMBA, 7,12-dimethylbenz(a)anthracene;
7-OHM-12-MBA, 7-hydroxymethyl-12-methyll>enz(a)anthracene.
1907
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
1937
W. Levin and A. H. Conney
bons on the metabolism and carcinogenicity of aminoazo dyes
and Ar-2-fluorenylacetamide (1, 6, 8, 19). The inhibitory effect
of enzyme inducers on carcinogenesis by several unrelated chem
ical carcinogens suggests that this effect is of general importance
and that suitable enzyme inducers can protect animals from many
chemical carcinogens. This concept was recently discussed in
some detail by Wattenberg (20).
10.
11.
12.
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CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1967 American Association for Cancer Research.
VOL. 27
Stimulatory Effect of Polycyclic Hydrocarbons and Aromatic
Azo Derivatives on the Metabolism of 7,12-Dimethylbenz( α
)anthracene
W. Levin and A. H. Conney
Cancer Res 1967;27:1931-1938.
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