[CANCER RESEARCH 33, 1284 1289, June 1973]
Evidence for a Glucuronic Acid Conjugate of 7V-Hydroxy-4aminobiphenyl in the Urine of Dogs Given 4-Aminobiphenyl '
Jack L Radomski, Alberto A. Rey, and Earl Brill
Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33152
SUMMARY
Evidence of the presence of substantial quantities of a
glucuronic acid conjugate of /V-hydroxy-4-aminobiphenyl
in the urine of dogs given 4-aminobiphenyl was obtained.
The conjugate is very acid labile, readily liberating free
/V-hydroxy-4-aminobiphenyl at lowered pH and in the
presence of /3-glucuronidase. It was responsible for ap
proximately 25% of the tritium-labeled excretory products
of 4-aminibiphenyl in the urine. It is apparently the source
of the /V-hydroxy compound previously observed in dog
and monkey urine and is believed to be involved in the
induction of bladder cancer from 4-aminobiphenyl.
INTRODUCTION
Since the discovery of aromatic amine-induced bladder
cancer and its urogenic nature (19), much research effort
has centered on the identification of the active urinary
metabolite. A considerable body of data has been accumu
lated implicating /V-hydroxylation as the key process in
volved (1, 6, 7, 20 22). The carcinogenic aromatic amines,
4-ABP2 and 2-NA, are /V-hydroxylated by rabbit and dog
liver microsomes to hydroxylamines (9). The relatively
noncarcinogenic 1-NA is also /V-hydroxylated, but to a
markedly lesser extent (9). There is a positive correlation
in dogs between the methemoglobin-producing ability, a
measure of the concentration of /V-hydroxy metabolites
being released into the blood, and the carcinogenic potency
of 4-ABP, 2-NA, and 1-NA (20, 21). When these 3 amines
are administered to dogs in equivalent doses, the concen
tration of the hydroxylamine metabolites found in the
urine is also correlated with the carcinogenicity of the
amine (20, 21). Studies in rhesus monkeys, which also de
velop bladder cancer when given 2-NA (13), show that this
species also excretes hydroxylamine metabolites of 2-NA
and 4-ABP in the urine (23).
Of the 2 types of /V-hydroxy metabolites that might be
involved in the carcinogenic process, the hydroxamic acid
type or the hydroxylamine type, evidence favors the hy1This investigation was supported by USPHS Research Grant 2ROI
CA-05449-11.
2The abbreviations used are: 4-ABP, 4-aminobiphenyl; 2-NA, 2-naphthylamine; 1-NA, 1-naphthylamine; NOH-4-ABP, /V-hydroxy-4-aminobiphenyl; TLC, thin-layer chromatography: 4-NOBP, 4-nitrosobiphenyl.
Received November 6. 1972: accepted March 14, 1973.
1284
droxylamine as the active bladder carcinogen. An inten
sive examination with highly sensitive gas Chromatographie
techniques of the urine of dogs given maximally toler
ated doses of 4-ABP and 2-NA failed to reveal any traces
of acetylated /V-hydroxy metabolites (hydroxamic acids)
or their conjugates (unpublished results). In addition, 2acetylaminonaphthalene, a noncarcinogen in the dog (11),
was excreted as the glucuronide of /V-hydroxy-2-acetylaminonaphthalene while no traces of 2-naphthylhydroxylamine were found (23).
Acceptance of the involvement of the hydroxylamine
metabolite in the induction of bladder cancer has been
impeded by 1 primary consideration. Aryl hydroxylamines
are water insoluble and, unlike arylhydroxamic acids,
are highly unstable in aqueous media. Obviously, it is ex
tremely unlikely for such a metabolite to be synthesized
in the liver, circulate in the blood, be filtered through the
kidney, and reach the bladder unless it was being trans
ported in some protected form. Binding to serum albumin
or conjugation in the liver are 2 possibilities. However,
studies conducted up to now have failed to reveal the bio
logical existence of a conjugate of these aryl hydroxyl
amines. The metabolism of 2-NA and 4-ABP specifically
have been studied in several laboratories over the years
without uncovering such a conjugate (8, 14). Attempts to
synthesize glucuronic acid and sulfate conjugates of 2naphthylhydroxylamine and NOH-4-ABP have failed.
Therefore, experiments were conducted to evaluate the
former possibility. Failing to find evidence of plasma pro
teins functioning as protective carriers for the hydroxyl
amine metabolites of 2-NA (unpublished results), we
focused our effort on the detection of a water-soluble con
jugate of these amines. We decided to concentrate on 4ABP rather than 2-NA because of the greater carcino
genicity of the former compound and the greater stability
of its hydroxylamine derivative (NOH-4-ABP).
This paper deals with evidence which indicates the pres
ence of a glucuronic acid conjugate of NOH-4-ABP and
the properties of this unusual compound. This discovery
removes a major obstacle to the acceptance of /V-hydroxylation as the central process in the induction of bladder can
cer and the /V-hydroxy metabolite as a urinary carcinogen.
MATERIALS
AND METHODS
Preparation and Purity of Compounds. 4-ABP was ob-
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Glucuronic
tained from Aldrich Chemical Co., Milwaukee, Wis., and
redistilled twice followed by recrystallization from aqueous
ethanol. 4-ABP-3H was prepared by catalytic exchange
of 4-acetylaminobiphenyl with tritiated water and a prereduced platinum catalyst by New England Nuclear, Bos
ton, Mass. The product obtained was hydrolyzed in hydro
chloric acid and then neutralized with sodium hydroxide
yielding 4-ABP-3H, which was recrystallized 3 times from
aqueous ethanol. The final product was chromatographed
by TLC on Silica Gel G in petroleum ether:acetone (7:3,
v/v) yielding a single radioactive spot corresponding to 4ABP. NOH-4-ABP was prepared by the method of Willstatter and Kubli (25); m.p., 161 162°.4-NOBP (m.p.,
73-74°)was prepared by the diethylazodicarboxylate oxi
dation of NOH-4-ABP.
Dosage Administration and Urine Fractionation. 4-ABP
( 10 mg/kg) was administered to dogs in dry, powdered form
by capsule. Higher doses are apt to produce fatal methemoglobinemia. Tritiated 4-ABP (12 ¿tCi)was incorporated
in each dose. Urine was collected every 4 hr by catheter.
Both male and female dogs were used. The urine was im
mediately applied to a Sephadex G-10 column which had
been packed in double-distilled, N2-treated water. Ten to
15 ml of urine were chromatographed on a 3- x 60-cm
column. Elution was with N2-treated distilled water ad
justed to pH 7.2 to 7.6. Fifteen-mi fractions were collected
through a LKB UV absorption meter (280 nm) until no
further UV absorption was noted. These columns usually
ran for about 16 hr at an elution rate of 1 ml/min. The
fractions were collected in a refrigerated fraction collector
(5°).
Column partition chromatography was also used. The
partially purified material from the Sephadex G-10
columns was dried by flash evaporation at 10°onto silica
gel (Woelm partition chromatography). After thorough
drying, this material was added to a dry column of the same
material (1.2 x 15 cm). The column was then eluted with
«-butylalcohol:benzene (4:1, v/v), saturated with water.
Chromatography was carried out at 5°in a cold room.
Eight-mi fractions were collected.
Column Chromatograph was also carried out on Sepha
dex LH-20 ( 1.2 x 55 cm). The column was packed with 95%
ethyl alcohol: water (7:3, v/v). Partially purified material
from the Sephadex G-10 column was added to the top of
the column, and elution was carried out with this solvent.
TLC. This was used to confirm the gas Chromatogra
phie identification of 4-NOBP and to identify the 0-sulfate
of 4-amino-3-hydroxybiphenyl.
In the former case,
benzenethyl alcohol (19:1, v/v), n-hexane:acetone (100:7,
v/v), chloroform:ethyl
alcohol (100:5, v/v), and pe
troleum ether 30°/60°:
acetone (7:3, v/v) were used. The
RF'S observed with 4-NOBP were 0.60, 0.45, 0.78, and 0.65,
respectively. In the latter case, identification was accom
plished with w-butyl alcohol:«-propyl alcohol:0.2 N NH3
(2:1:1, v/v), and w-butyl alcohol: water: acetic acid
(2:1:1, v/v). In these solvent systems RF's of 0.56 and 0.54,
respectively, were obtained with synthetic 4-amino-3-hydroxylbiphenylyl sulfate.
Spray reagents used were p-dimethylaminocinnamal-
Acid Conjugate
of NOH-4-ABP
in Dog Urine
dehyde (1% w/v in ethyl alcohol:3 N HC1, 50% v/v);
trisodium pentacyanoamino ferrate (5% aqueous): and
diazotized 4-nitro-o-toluidine and 5-nitro-o-toluidine. This
latter reagent was prepared from a stock solution of 400
mg of the mixed amine in boiling 0.5 \ HC1 (200 ml). To
20 ml of this stock solution were added 20 mg of anhydrous
sodium nitrite. After 5 min, an excess of urea was added
and then a 20-min wait was allowed to permit decomposi
tion of the excess nitrite. The mixture was then filtered
and diluted with 4 volumes of water.
UV (both 254 and 365 nm) was also used to identify these
metabolites.
Isotope Experiments. Radioactivity in the fractions was
determined by counting in dioxane: scintillation mixture in
a Nuclear-Chicago liquid scintillation counter.
The Quantitative Determination of NOH-4-ABP. Ini
tially, the quantitative determination of NOH-4-ABP in
these fractions was carried out by our previously pub
lished procedure (21). One ml of the fraction was diluted
to 10 ml with water. HC1 (1 N) was added to pH 3: then
0.2 ml of 10% K3Fe(CN)6 solution was added. This solu
tion was transferred to a separatory funnel and shaken with
5 ml of gas chromatographically pure petroleum ether for
5 min. The petroleum ether extract was transferred to a
15-ml centrifuge tube and a small amount of anhydrous
sodium sulfate added. The volume was adjusted to 5 ml
with petroleum ether, and 5 n\ were injected on the gas
Chromatograph. In later experiments, the use of K3Fe(CN)6
was found to be superfluous and was eliminated.
Gas Chromatographie analyses of the nitroso and /V-hydroxy compounds were achieved with a Tracor MT 220 gas
Chromatograph equipped with a 63Ni high-temperature
electron capture detector. The columns utilized were 5% SE
30 and 5% OV 210, 6 ft x 0.25 inch in glass columns on
Chromosorb W (HP). On-column injection was used.
Temperatures: inlet, 200; column, 140; detector, 365°.
Inlet pressure: 40 psi. Flow rates 40 to 60 ml/min; pulse
mode, 3 /usée
on, 270 ¿isecoff. Carrier gas: prepurified
nitrogen.
Nitroso and /V-hydroxy compounds give exactly the
same quantitative response and retention time on the gas
Chromatograph. Therefore, all analyses are for nitroso
and/or /V-hydroxy compound present frequently referred
to as total /V-oxidation products.
Determination of Glucuronic Acid. Glucuronic acid con
centrations were measured by the method of Bitter and
Muir, without modification (2). Measurement of the color
developed in the carbazole reaction was made on a Beckmann Model DBG spectrophotometer.
Incubation with /i-Glucuronidase. Incubation of the puri
fied conjugate was carried out at room temperature to
minimize the decomposition of the liberated /V-hydroxy
compound. To 10 ml of solution at pH 6 containing 3.2 ¿ig
of the conjugate were added 20 mg of bacterial /3-glucuronidase Type 1 (Sigma Chemical Co., St. Louis, Mo.) and 3
drops of chloroform. Incubation times were 5 and 30
min. The incubate was extracted with 5 ml of gas chromatographic grade petroleum ether. Five ^1 of this ex
tract were injected on the gas Chromatographie column.
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1285
Jack L. Radomski, Alberto A. Rev, and Earl Brill
RESULTS
Detection of a Water-soluble Conjugate of NOH-4-ABP.
In an effort to detect a water-soluble conjugate of NOH-4ABP, tritium-labeled 4-ABP was administered to dogs.
The urine, obtained by catheter, was chromatographed
on a Sephadex G-10 column with double-distilled water
as an eluent. The UV scan (280 nm) of the eluate from the
column indicated the presence of 5 major UV-absorbing
peaks (Chart 1). An aliquot of each tube was assayed for
tritium activity. The presence of 3 major radioactive com
ponents was observed. Each fraction was assayed for the
presence of NOH-4-ABP. This analysis involved acidifi
cation and the addition of ferricyanide. In acid solution,
ferricyanide converts /V-hydroxy compounds present to
nitroso compounds. It was found that the 2nd major ra
dioactive peak comprised a compound which gave a gas
Chromatographie response for 4-NOBP on both the SE
30 column and the OV 210 column. A close correlation
between the concentration of the nitroso compound and
the radioactivity in each fraction was observed. The frac
tions giving this positive response were also tested by ex
traction without acidification and ferricyanide addition fol
lowed by gas chromatography of the extract. No gas chromatographic response for 4-NOBP was observed. Extracts
giving positive results for 4-NOBP on the gas chromatograph were pooled and spotted on a TLC plate next to
standard 4-NOBP. The plate was developed in benzene:
ethyl alcohol (19:1 v/v) and sprayed with trisodium pentacyanoamino ferrate (5% aqueous). The extract from
dog urine gave the same blue-gray spot at the same RF
(0.60) as the standard. The extract and standard gave
identical RK's and spray reactions in 3 additional solvent
systems (see "Materials and Methods"). As would be
expected because of the addition of ferricyanide, no
NOH-4-ABP was observed on this plate. At this point, it
was observed that an equal gas Chromatographie response
for 4-NOBP was observed with or without the presence
of ferricyanide, as long as the sample was acidified to pH
3, and the use of ferricyanide was abandoned in future
analyses. Extracts of fractions hydrolyzed at pH 3 without
ferricyanide were shown by TLC to contain 4-NOBP, not
NOH-4-ABP. However, when the experimental proce
dure was repeated with standard NOH-4-ABP, only 4NOBP was found on TLC chromatography. Thus, NOH4-ABP is rapidly converted to 4-NOBP at pH 3 in
aqueous solution.
These results demonstrate the presence of a water-solu
ble form of a metabolite of 4-ABP which yields, under the
conditions of the analysis, 4-NOBP. This metabolite ap
peared to be a conjugate of NOH-4-ABP that hydrolyzes
at pH 3. By computation of the area under the curve of
tritium activity due to the conjugate (Chart 1), it was
estimated that it comprises approximately 25% of the
urinary excretory products of 4-ABP in the early period
after dosage administration.
Experiments on the Identity of the Conjugate. In view of
the tendency of the dog to excrete exogenous aromatic
amines as sulfate conjugates, it was suspected that the
1286
II
15
21
25
HIE M
35
41
Chart 1. Fractionation of urine of dog given 4-ABP-3H. Units on or
dinale: UV scan (
), 100 to 0% transmission; 3H activity (O), O to
1000 ppm; /V-hydroxy analysis (•),0 to 5 pg/m\. The dose administered
to the dog was 10 mg/kg containing 10 ¿jCiof 4-ABP-3H. Ten ml of
urine collected during the 4- to 6-hr period after dosage administration
were chromatographed on Sephadex G-10.
conjugate observed in the preceding experiments might be
a sulfate. Because of our inability to synthesize a syn
thetic standard for the purposes of identifying this con
jugate, we decided to attempt to use a radioisotope labeling
technique to determine whether the conjugate contained sul
fur. A dog was therefore given an s.c. injection of 2 mCi of
Na35SO4. The following morning the dog was given
unlabeled 4-ABP p.o., and the urine was collected at 4-hr
intervals. Chromatography of this urine on Sephadex
G-10 carried out in the same manner as previously de
scribed revealed 4 major peaks of radioactivity when the
samples were assayed in a liquid scintillation counter.
These fractions were also assayed for NOH-4-ABP and/or
4-NOBP. A sharp peak of 35S activity was obtained in
the region of the water-soluble conjugate of NOH-4-ABP.
However, it did not coincide with the peak obtained from
the analysis of the samples for 4-NOBP (Chart 2). The
same results were obtained by repeating this experiment
several times with different urine specimens and the urines
from different dogs. The 6th peak in the UV scan was,
however, definitely rendered radioactive. This peak ap
peared by TLC in 2 solvent systems (see "Materials and
Methods") to be due to 4-amino-3-hydroxybiphenylyl sul
fate, a well-known metabolite of 4-ABP in dogs. There
fore the technique used successfully achieved the labeling
of exogenous sulfate conjugates, and it could be concluded
that the conjugate of NOH-4-ABP in question is not an
organic sulfate.
Fractions yielding a positive test for NOH-4-ABP were
combined and rechromatographed on a similar Sephadex
G-10 column. In this experiment a single major peak was
observed in the UV scan which corresponded to the peak
of tritium activity and the peak of /V-hydroxy concen
tration (Chart 3). These fractions were analyzed for the
presence of glucuronic acid. A peak of glucuronic acid
concentration was found which corresponded precisely
to the peak of radioactivity, the UV peak at 280 nm and
the peak representing the concentration of nitroso com
pound. In addition, the ratio of glucuronic acid to NOH-4ABP observed in the 2 fractions containing substantial
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Glucuronic Acid Conjugate of NOH-4-ABP
in Dog Urine
sub-milligram amounts sufficient for the determination
of its Chromatographie and spectrophotometric charac
teristics were obtained.
Fractions containing the conjugate obtained from 3 to 5
runs of the initial fractionation step utilizing Sephadex
G-10 were concentrated and chromatographed in 95%
ethyl alcohol:water (7:3, v/v) on Sephadex LH-20. The
fractions containing the conjugate were then pooled and
evaporated to dryness. The solid was dissolved in water
and streaked on a thin-layer plate coated (0.5 mm) with
Silica G. The plate was developed first in «-butyl
alcohol :«-propyl alcohol:water (2:1:1, v/v) and then
redeveloped in «-butylalcohol:benzene (4:1, v/v) saturated
2l
25
with water. The streak containing the conjugate was eluted
TUENI
with water and reduced to dryness by lyophilization. This
Chart 2. Fractionation of urine of dog given 4-ABP and Na235SO4.
material was used to determine the properties of the
Units on ordinate: UV scan (
). 100 to 0% transmission: S5S activity
conjugate.
(O), O to 2000 cpm; A'-hydroxy analysis (•),0 to 2 //g/ml. The 4-ABP
Properties of this conjugate on TLC with Silica Gel G
dose was 10 mg/kg. The Naz35SO4 dose was 2 mCi. Ten ml of urine col
are as follows. In «-butylalcohol:benzene (4:1 v/v) sat
lected during the 4- to 8-hr period after dosage administration were chro
urated with water, RF is 0.06. In «-butyl alcohol :«matographed on Sephadex G-10.
propyl alcohol:water (2:1:1, v/v), RK is 0.40. Under
long-wave-length UV (365 nm) it showed a dark gray
and under short-wave-length UV (254 nm), it showed a
dark purple fluorescence. It gave a deep, plum-red
reaction with p-dimethylaminocinnamaldehyde
and a
brown color with iodine vapor. No reaction was ob
tained with trisodium pentacyanoamine ferrate spray of
Folin's phenol reagent. The dark fluorescence observed
with both long- (365 nm) and short- (254 nm) wave
length UV is consistent with the proposed structure of
the metabolite, having no free, ring-activating func
tional group on the aromatic rings. Failure to give a color
test with the trisodium pentacyanoamine ferrate spray
reagent also indicates that the A'-hydroxy group is blocked.
Chromatographie solvent systems containing any acid
i
21
25
35
4l
45
II
or base including acetic acid and ammonia could not be
THENO
utilized because of the destruction of the conjugate.
Chart 3. Rechromatography of pooled fractions containing the con
The UV absorption spectrum of the conjugate was de
jugate uronic acid analyses. Units on ordinate: UV scan (
), 100 to 0%
transmission; 3H activity (O), O to 4000 cpm/ml; A'-hydroxy analysis termined (Chart 4). Good correspondence to the spec
trum of NOH-4-ABP was observed indicating the pres
(•).0 to 20 ppm. Uronic acid (A), 0 to 15 jig/ml. Fractions from 3 prior
ence of this moiety within the conjugate molecule. Glucu
runs were chromatographed on Sephadex G-10.
ronic acid does not absorb in the UV.
An experiment was performed to determine the sen
amounts of the conjugate (Samples 18 and 19) was sitivity
of the conjugate to hydrolysis with ß-glucuroniconstant and stoichiometric for the glucuronide of this
dase.
The
conjugate was incubated with and without this
substance (Table 1). Fraction 20 contained very little of
enzyme
for
5 and 30 min. The results indicate that the
the A-hydroxy compound and a disproportionate amount
conjugate is rapidly hydrolyzed by 0-glucuronidase
of glucuronic acid due undoubtedly to contamination
with another glucuronic acid conjugate. These results (Table 2). Slightly over 50% hydrolysis was observed
indicate the presence of the glucuronic acid conjugate
Table 1
of NOH-4-ABP.
Quantitative analyses for NOH-4-ABP
and glucuronic acid in fractions
Purification and Properties of the Conjugate. Repeated
from Sephadex G-10 column
attempts were made to isolate the conjugate in pure
acid"M/ml13.1
crystalline form from dog urine. These attempts were de
Fraction18
feated by the extreme sensitivity of this substance to
hydrolysis which continued to occur at a slow rate re
gardless of all attempts at preservation, i.e., dark, low
6.61.2(imole/ml0.076
0.035
6.0
0.034
1.03
1920NOH-4-ABPnl/ml14.1
temperature, freezing, and strict maintenance of neutral
0.007D-Glucuronic
3.4^mole/ml0.074
0.019Moleratio1.03
0.47
pH. Considerable purification was finally achieved by a
1Calculated Us n-glucuronolactone.
succession of Chromatographie fractionations, but only
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1973
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1287
Jack L. Radomski, Alberto A. Rev, and Earl Brill
»
«unit
i immurimi
211
250
311
350 400200 250 300 350 4M
»
»«HEIST»
»
Chart 4. The UV absorption curves of the conjugate and of NOH-4ABP. The concentration of NOH-4-ABP was 0.3 mg/ml in 95% ethanol.
The conjugate was in aqueous solution.
after 30 min. Previous experiments indicated that longer
incubation times yield less /V-hydroxy due to decompo
sition of the liberated aglycone.
An attempt was made to prepare the triacetyl methyl
ester of the glucuronide in hopes that the metabolite could
be stabilized in this way. This attempt was not successful
due possibly to hydrolysis of the conjugate during the
acetylation step.
DISCUSSION
While NOH-4-ABP has not been thoroughly tested,
such experiments as have been performed indicate that
it is carcinogenic. Gorrod et al. (15) found this substance
to enhance the incidence of hepatomas in newborn mice,
and an experiment in our laboratory not yet complete
shows an increase in peritoneal tumors following i.p.
injection in rats. In addition, the closely related compound
/V-hydroxy-2-naphthylamine
produced bladder tumors
after prolonged instillation in dogs (20, 22).
The discovery of the presence of substantial quantities
of a glucuronic acid conjugate of NOH-4-ABP in the
urine of dogs given 4-ABP removes the main remaining
stumbling block to the acceptance of an /V-oxidized me
tabolite of 4-ABP as a urinary carcinogen and provides
a ready explanation for the transport of this metabolite
from its site of formation in the liver to its site of action in
the bladder. Of course, further activation steps may be
necessary before the "final" carcinogenic substance that at
tacks the transitional cells of the bladder is formed. The
proposed hypothesis of the urogenesis of bladder cancer
is that 4-ABP is /V-hydroxylated and conjugated in the
liver and then transported in this inactive form to the
bladder where the free NOH-4-ABP is liberated by de
creased pH and/or /3-glucuronidase. This hypothesis is
consistent with all previously recorded observations con
cerning the behavior and nature of the urinary carcinogen,
including the failure of aromatic amines to induce tumors
in the dog in tissues other than the urinary bladder. Its
1288
extreme sensitivity to hydrolysis at any pH below 7 is suf
ficient to explain the liberation of the /V-hydroxy metab
olite in the bladder.
Sensitivity to lowered pH probably explains the failure
of previous investigators to observe the presence of this
conjugate. All of the customary methods of studying the
urinary excretion of exogenous compounds have involved
acidification. Methods most commonly used include ex
traction into solvents such as diethyl ether, n-butyl
alcohol, or tert-buly\ alcohol following acidification to
render the acidic conjugates soluble in organic solvents.
Other methods include the lead precipitation procedure
for the isolation of glucuronides and the charcoal adsorp
tion technique. Both procedures involve acidification.
Fractionation of urine is also commonly carried out on
ion-exchange cellulose columns, utilizing buffers of acidic
pH. The finding of this conjugate is undoubtedly largely
due to our use of a Chromatographie column that frac
tionated urine with only distilled water as the eluent. This
procedure has the additional advantage of yielding frac
tions of the urinary metabolites that are free of salt.
While glucuronides of the hydroxamic acid type of
/V-hydroxy compound have been frequently observed, few
instances of the biological existence of glucuronic acid
conjugates of the aryl hydroxylamines have been re
ported. A glucuronide of /V-hydroxy-2-aminofluorene
was reported in the urine of rats and guinea pigs given
2-aminofluorene (18, 24). This conjugate was also synthe
sized from glucuronide of /V-hydroxy-2-acetylaminofluorene by hydrolytic removal of the acetyl group (17).
However, these authors found this conjugate to be ex
tremely unstable in the presence of water, making its
presence in excreted urine improbable. The whole matter
has been thoroughly reviewed by Irving (16). The pro
posed glucuronide of 4-ABP here reported has consid
erably greater aqueous stability than this at neutral pH.
Hydrolysis of this conjugate is catalyzed by 0-glucuronidase. Whether or not ß-glucuronidaseplays an active role
in the liberation of the /V-hydroxy compound in vivo is
not certain, for the normally acidic nature of the urine
seems sufficient. That the liberated /V-hydroxy compound
is spontaneously converted to the nitroso derivative in
Table 2
Incubation of the purified conjugate with ß-glucuronidase
ronidase"
ExperimentlaIb2a2b3a3b4a4b0-Glucuperiod(min)555530303030Conjugate"
(Mg)3.23.23.23.23.23.23.23.2NOH-4-ABPreco
(mg)002020002020Incubation
"The total incubation volume was 10 ml.
" The amount of conjugate added is expressed as /V-hydroxy com
pound.
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Glucuronic Acid Conjugate of NOH-4-ABP
aqueous solutions suggests that the latter substance may be
more important to the carcinogenic process than the Nhydroxy compound. However, this is not necessarily true,
for it may well be that the liberation of the A'-hydroxy
compound from the conjugate by ß-glucuronidase in
close proximity to the bladder mucosa is the significant
event.
One of the most compelling virtues of the former ohydroxyamine theory of bladder carcinogenesis (3-5) was
that it provided a theoretical explanation for the lack of
carcinogenicity of 1-NA. The theory was that aromatic
amines have a strong tendency to be p-hydroxylated, as in
the case of I-NA. Only when the p-position is blocked, as
in the case of 2-NA and 4-ABP, did o-hydroxylation occur
to yield the metabolite then believed to be the urinary car
cinogen. Subsequent results showed that, while p-hydroxylation occurred to the greatest extent, significant
quantities of o-hydroxylation also occurred with 1-NA.
However, adopting a dynamic view of the metabolism of
compounds by liver, one can apply the same reasoning to
the AMiydroxylation theory of bladder carcinogensis,
namely, that p-hydroxylation competes with /V-hydroxylation as well as o-hydroxylation. In other words, compared
to 2-NA and 4-ABP, the strong tendency of 1-NA to be
p-hydroxylated would markedly reduce the amount of
amine available for /V-hydroxylation.
We are continuing to attempt to isolate significant quan
tities of crystalline conjugate and to prepare the conjugate
synthetically. Availability of this substance will be a con
siderable asset to in vitro and in vivo studies of the effects
of this carcinogen on transitional epithelial cells. Such
experiments conducted by adding water-insoluble car
cinogens such as these A'-hydroxy and nitroso compounds
to biological tissues and preparations in some organic sol
vent are always open to justifiable criticism.
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JUNE 1973
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1973 American Association for Cancer Research.
1289
Evidence for a Glucuronic Acid Conjugate of N
-Hydroxy-4-aminobiphenyl in the Urine of Dogs Given
4-Aminobiphenyl
Jack L. Radomski, Alberto A. Rey and Earl Brill
Cancer Res 1973;33:1284-1289.
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