Cardnogausb Vol.5 no.7 pp.
949-954, 1984
Metabolism of 2-napthylamine and benzidine by rat and human
bladder organ cultures
Brian P. Moore, Philip M. Potter and R. Marian Hicks
School of Pathology, The Middlesex Hospital Medical School, Riding
House Street, London W1P 7LD, UK
The metabolism of benzidine and 2-naphthylamine, two
aromatic amines whkh are carcinogenic for the human, was
investigated in human and rat bladder organ cultures. There
was little oxidative metabolism of either carcinogen in either
species. In particular, N-hydroxy-2-naphthylamine, a prox-
imate carcinogen of 2-naphthylamine could not be detected.
In contrast, large amounts of the acetylated metabolites,
N-acetylbenzkline, N,N-diacetylbenzidine and N-acetyl-2-
naphthylamine were formed both in rat and human bladder
cultures. Theresultssuggest that metabolism of these carcino-
gens in situ in the bladder is unlikely to contribute to their car-
cinogenic effect but instead may have a positive protective
role.
Introduction
Aryiamines such as 2-naphthylamine (2-NA)* and benzidine
were amongst the first pure chemicals to be identified as
human carcinogens and this knowledge has led to legislation
restricting their manufacture and use in many countries (1).
Metabolism and binding studies with these and many other
aryiamines have shown cytochrome P-450 catalysed
N-hydroxylation to be a critical step in the activation of these
compounds leading to the formation of toxic and car-
cinogenic metabolites (2). This type of metabolic reaction,
followed by glucuronidation and excretion of the
glucuronides via the kidney can account for the ability of
2-NA to initiate bladder tumours (3,4). It is not known
whether further metabolism in situ i.e., in the bladder, con-
tributes to the carcinogenicity of these conjugates since
although it may occur, in the mildly acidic urine there will be
sufficient spontaneous binding of metabolites with DNA to
account for the formation of mutagenic DNA adducts (4—7).
This mechanism is attractively simple and may be typical for
more aryiamines that initiate bladder cancer.
Benzidine may be the exception. The metabolism of this
compound is very complex (8) and several different metabolic
pathways involving N-hydroxylation leading to DNA-binding
have been described (9,10), but that most likely to be respon-
sible for the initiation of liver tumours in rats is described by
Martin et al. (11,12). The single DNA adduct found in the
liver of treated animals in that study, N-(deoxyguanosin-
8-yl)-N'-acetylbenzidine, can only have arisen by
N-hydroxylation after acetylation of the parent compound.
However, the metabolic pathway in the bladder leading to in-
itiation is less clear. The hepatic metabolites produced are
almost certainly too unstable to reach the target DNA in the
bladder unless a further stabilising conjugation reaction oc-
curred, cf. 2-NA. Other potential mechanisms have been
* Abbreviation: 2-NA, 2-naphthylamine.
© IRL Press Ltd., Oxford, England.
described; for example benzidine is a particularly good
substrate for prostaglandin endoperoxide synthetase, an en-
zyme which is abundant in the renal inner medulla (13,14).
This enzyme catalyses the reduction of prostaglandin G with
co-oxidation of a second substrate, in this case benzidine,
thus forming free radicals that will bind to macromolecules.
Prostaglandin synthetase also is involved in the metabolism
of other, structurally unrelated bladder carcinogens and since
it is located in the urothelium itself, could play an important
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on May 13, 2016
role in bladder carcinogenesis (15,16).
The actual sequence of events leading to initiation of blad-
der cancer is further complicated by the presence of hydroxyl-
ating enzymes in the tissue itself. These enzymes are capable
of activating a wide range of carcinogens including benzo[a]-
pyrene, aromatic amines and nitrosamines (17—20) by form-
ing mutagenic products (20,21). In situ activation of car-
cinogens in a plausible proposition and may account for in-
itiation in some tissues including the bronchus by inhaled car-
cinogens (22), and gastrointestinal tract by dietary car-
cinogens (23 — 25). When assessing the importance of
metabolism in situ, it is important to consider whether the en-
zymes concerned simply perform the final step in activating
the carcinogen, or are involved in its entire metabolism. For
example, non-oxidative enzymes in the bladder wall could
reactivate urine-borne benzidine metabolites (26) and the final
stage of activation of N,N-dialkylnitrosamine bladder car-
cinogens by hydroxylation to form a-lactones, could take
place in the urothelium even though all preceeding steps occur
in the liver (27).
In the present paper we describe an attempt to assess the
extent and importance of in situ metabolism of two very im-
portant bladder carcinogens, 2-NA and benzidine, by organ
cultures of normal human bladder.
Materials and methods
Cup biopsy samples of the urotbdium plus some supporting mesenchymal
tissue were obtained from normal bladder of consenting male and female pa-
tients during routine cytoscopkal examination, and were placed immediately
in ice cold buffered medium. This tissue was cultured within three hours by a
modification (17) of the method of Knowles et al. (28). Cultures in scratched
plastic dishes (~ 3 mm square) were equilibrated for 24—96 h then rocked at
10 cycles/min with radiolabellcd aryiamines. Samples of ureter obtained from
patients undergoing renal transplant were cultured identically. For com-
parison, half bladders from Fischer F344 rats were cultured in the same
medium, (Ham's F-12 supplemented with 10% foetal calf serum, 1 /ig/ml
hydrocortisone, 1001 units penicillin, 100 /ig/ml streptomycin and 0.45/ig/ml
ferrous sulphate).
Incubations with radiolaMled aryiamines
pHJBenzidine (115 rrrCi/mmol) and PHJ2-NA (47 mCi/mmoI) were generous
gifts provided by Dr. C.N.Martin (Cancer Research Unit, University of York,
York YO1 5DD, UK) and Dr. F.A.Bdand (National Center for Toxicological
Research, Jefferson, AR 72079). ["CJBenzidine (25.7 mO/mmol) was pur-
chased from New England Nuclear. RadiotabeDed aryiamines (18—25 pan)
were added to the bladder cultures in small quantities of dimethylsulphoxide
such that the final concentration of solvent did not exceed 0.3%. Incubation,
unless otherwise stated, was for 24 ± 2 h.
949
B.P.Moore, P.M.Potter and R-M.Hlcks
Determination of metabolic products
Organo-soluble metabolites were extracted with ethyl acetate, concentrated in
vacuo and separated by h.p.Lc. using Spherisorb 5 p. octadecyl silane
(O.D.S.) or a Zorbax-C8 (Dupont Instruments Ltd.) column. Metabolites
were duted with 1 ml/min 35 or 40% methanol in 0.01 M KPO« (pH 6.0)
buffer changing to 100% methanol with a 5 min gradient after etution of the
acetylated metabolites. In a few experiments with 2-NA, N-butanol was used
for extraction in place of ethyl acetate, enabling direct h.p.l.c. of glucuronide
conjugates (6). AcetyDiydroxamic acid (0.01 M) was added as carrier for any
N-hydroxymetabolites produced in all but a few initial analyses (29).
Metabolites were identified by co-chromatography of the radioactive pro-
ducts, collected on a fraction collector at 30 s or 1 min intervals, with the
peaks of added authentic unlabdlcd metabolites. Authentic marker com-
pounds used were N-acetylbenzidine and N,N'-diacetyIbenzidine (gifts from
Dr. C.N.Martin) and benzidine for estimations of benzidine metabolism;
N-hydroxy-2-naphthylamine, 2-NA (gifts from Dr. F.F. Kadlubar), 2-amino-
1-naphthol (Pfalz and Bauer, Stamford, C D , 2-nitro-l-naphthol and
3-amino-2-naphthol (AMrich Chemical Co. Ltd.) and N-acetyl-2-
naphthylamine (synthesised by acetylation of 2-NA as described by Booth et
al. (30)) were used for 2-NA experiments. Radioactivity was quantified in
Fisofluor MPC scintillation cocktail using an LKB Rackbeta scintillation
counter.
Protein and DNA binding
Tissues were sotubuised with proteinase K (BDH Ltd., Poole, UK) and ex-
tracted with chloroform/phenol mixture as described previously (17). Pro-
teins, precipitated from the phenolic layer by addition of 2 - 3 volumes of
ethanol, were exhaustively extracted with acetone, ethyl acetate and diethyl
ether, as described by Autrup et al. (31). The protein content of the resultant
material was determined using the Hartree-Folin method (32) and the
associated radioactivity by liquid scintillation counting after redissolving in
0.1 M NaOH. Binding to DNA was estimated after purification by hydroxy-
apatite chromatography (33) as described previously (17).
Results
Benzidine
Rat and human organ cultures metabolised [14C]- and ['H]-
benzidine to small amounts of water-soluble and organo-
soluble metabolites. In human cultures, only very small
amounts of water-soluble metabolites were produced
(equivalent to 1 ± 0.6% of the total radioactivity added, 1.65
± 1.2 pmol/g/min). Rat bladder cultures also produced low
levels of water-soluble metabolites, which, as in the human
cultures, were not detectably hydrolysed by /3-glucuronidase
to ethyl acetate extractable organo-soluble material. H.p.l.c.
of the organo-soluble radioactive products showed four ma-
jor peaks of radioactivity (Figure 1). A major peak cor-
responded to the parent compound and accounted for
15 — 60% of the total organo-soluble radioactivity in a 24 h
human bladder culture. Three other peaks of radioactivity
were observed which co-chromatographed with N-acetyl-
benzidine, N,N'-diacetylbenzidine and another unknown
substance which stayed nearer the origin, possibly 3-hydroxy-
benzidine. In some human bladder cultures, N-acetyl-
benzidine accounted for as much as 64% of the total organo-
Table I. Benzidine
Number of Tissue weight Production of
human (mg) N-acetylbenzidine
patients nmol/min/g tissue
Rat bladder 34.0 ± 11.0(10) 0.28 ± 0.05 (9)
Human bladder 4 16.9 ± 6.2 (5) 0.5
Human ureter 1 39.3 ± 3.0 (4) 0.4
Figures are given as values ± S.D.
Figures in parentheses are the number of determinations.
950
soluble radioactive materials. Rates of production of the
acetylated metabolites were slightly lower in rat bladder
cultures than in human bladder or human ureter cultures
(Table I). The minor peak of radioactive material near to the
origin was very variable, due in part to co-chromatography of
another material apparently produced by auto-oxidation of
benzidine during culture; this peak was also found in 'no-
tissue' controls. Binding to DNA was not detectable either in
rat or human organ cultures due to the small amount of DNA
present and an insufficiently high specific activity of the
radiolabelled benzidine ([JH]benzidine 115 mCi/mmol). Bin-
ding to protein, however was slightly higher in rat than
human cultures (Table I).
2-NA
Rat and human bladder cultures metabolised 2-NA to
organo-soluble and water-soluble metabolites. As with
benizidine the levels of water-soluble metabolites produced
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were very low in both species. Little or no material associated
with water-soluble radioactivity could be hydrolysed to
organo-soluble material either by mild acid hydrolysis, thus
indicating the absence of N-hydroxy-2-naphthylamine
glucuronide (6)., or by enzymatic hydrolysis with /3-
glucuronidase. H.p.l.c. of the organo-soluble radioactive
material separated two major and three minor peaks of radio-
active products from both rat and human cultures (Figure 2).
4 x ID4
c
D.P.M.
2 x ID4
B
D
ft I L 50 100
F r a c t i o n No.
Fig. 1. H.p.Lc. profile of organo-soluble metabolites of ["CJbenzidine pro-
duced by human bladder organ cultures. Bladder tissue, allowed to
equilibrate for 24 h in culture, was incubated in 20 fiM ["CJbenzidine for
further 24 h. The ethyl acetate-soluble metabolites were extracted and
chromatographed as described in methods. A = unknown metabolite, B
= benzidine, C =• N-acetylbenzidine, D = N.N'-diacetylbenzidine.
Production of Protein binding
N,N'-diacetyl- nmol/mg protein
benzidine nmol/
min/g tissue
0.03 ± 0.02 (9) 0.3 ± 0.1 (10)
± 1.0 (4) 0.04 ± 0.02 (4) 0.19 ± 0.05 (5)
± 0.08 (4) 0.06 ± 0.003 (3) 0.38 ± 0.05 (4)

The two major peaks (Figure 2, peaks C and D) co-chromato-
graphed with the starting material, 2-NA, and with N-acetyl-
2-naphthylamine. The latter was not present in controls from
which tissue was absent, or in similar cultures performed at
4°C. When larger pieces of tissue were cultured, the
acetylated metabolite accounted for as much as 82% of the
organo-soluble material after 24 h in human organ cultures.
Its mean rate of production was 0.68 ± 0.18 and 0.88 ±
0.2 nmol/min/g in human bladder and human ureter
cultures, respectively, and 0.4 ± 0.1 nmol/min/g in rat blad-
der cultures (Table II).
Two of the remaining peaks of radioactivity chromato-
graphed near to the origin (Figure 2, peaks A and B). Peak B
co-chromatographed with 3-amino-2-naphthol and was pre-
sent only ever in trace amounts, < 1 % of the total organo-
soluble radioactivity in cultures; it was undetectable in con-
trols. The other peak, A, which was usually slightly larger
than B, was present also in 'no tissue' controls, suggesting it
was, in part, a natural degradation product formed in the
culture conditions. None was present in the starting material
or in controls performed at 4°C. However, when human
bladder was cultured for 96 h with [3H]2-NA, the material
chromatographing in this region increased to - 5 % of the
total organo-soluble radioactivity, i.e., approximately three
times that found in controls.
The remaining peak of radioactivity, E, which chromato-
5x10
D.P.M.
2.5xK>
A B
•n- -
40
Fraction
L No.
80
Fig. 2. H.p.l.c. profile of organo-sohible metabolites of PHJ2-NA produc-
ed by human bladder organ cultures. Bladder tissue, allowed to equilibrate
for 24 h in culture, was incubated in 20 /iM PHJ2-NA for a further 24 h.
Metabolites were extracted with ethyl acetate and chromatographed as
described in methods. A = Unknown, B = 3-amino-2-naphthol, C =
2-naphthylamine, D = N-acetyl-2-naphthylamine, E = unknown,
F = position of N-hydroxy-2-naphthylamine.
TaWe D. 2-NA
Number of Tissue
human weight (mg)
patients
Rat bladder 38.5 ± 17.0 (12)
Human bladder 7 13.5 ± 19.2(17)
Human ureter 4 24.4 ± 12.1 (11)
Figures are given as values ± S.D.
Figures in parentheses are the number of determinations.
Metabolism of 2-aaphthytamine and benzfcttne
graphed well behind N-acetyl-2-naphthylamine, was variable
in size, and occasionally separated into two components. In
most experiments longer than 24 h, the levels of radioactive
product chromatographing in this region were only slightly
higher than those occurring in parallel controls. As with the
radioactive materials associated with peak A, the products in
these peaks may have been formed metabolically and by
naturally occurring degradation reactions. This was ex-
emplified in 96 h human bladder cultures where a late
chromatographing metabolic component was found to ac-
count for 5 1 - 5 2 % of the organo-soluble radioactive
materials whereas the equivalent controls had two peaks with
different retention times, accounting for 26 and 31% of the
organo-soluble radioactivity. These experiments also sug-
gested that the later chromatographing peak (E) may be a fur-
ther metabolite of N-acetyl-2-naphthylamine as it was found
in much smaller quantities than in 24 h cultures. However,
this could not be confirmed in a series of rat bladder and
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on May 13, 2016
human ureter cultures in which labelled and unlabelled acetyl-
CoA was co-incubated with labelled and unlabelled 2-NA.
H.p.l.c. of the organo-soluble 2-NA metabolite conclusive-
ly demonstrated co-chromatography of "C deriving from
["CJacetyl-CoA with 'H from pH]2-NA in the N-acetyl-2-
naphthylamine peak (Figure 3). Apart from a very small
quantity of free ["CJacetate, which chromatographed just
after the void volume, no peaks of 14C activity other than that
co-chromatographing with N-acetyl-2-naphthylamine were
observed in the organo-soluble metabolites from these
cultures, and none at all in their controls. In these ex-
periments as much as 77% of the organo-soluble *H radio-
activity produced in human ureter and 91% of that in rat
bladder, co-chromatographed with N-aceryl-2-
naphthylamine, with little evidence for further metabolism of
the product. The added acetyl-CoA may have stimulated the
rate of production of N-acetyl-2-naphthylamine but had no
further affect on the metabolism of 2-NA.
In many experiments we attempted to detect formation of
N-hydroxy-2-naphthylamine and its glucuronide using
rigorous conditions of low yellow light, ice cold solvent ex-
traction and gassing with argon or nitrogen atmospheres to
prevent product breakdown of labile intermediates (6). In all
experiments with cultured rat and human bladder little or no
organo-soluble radioactive material co-chromatographed
with N-hydroxy-2-naphthylamine or was released by mild
acid hydrolysis to organo-soluble radioactive products co-
chromatographing with N-hydroxy-2-naphthylamine.
In our culture conditions the high oxygen tension required
to maintain the tissue is unlikely to permit large amounts of
an unstable metabolite, such as N-hydroxy-2-naphthylamine,
to survive unless trapped and stabilised by conjugation.
However, after numerous attempts to detect conjugated pro-
Production of N-acetyl Protein binding
naphthylamine nmol/ nmol/mg protein
min/g tissue
0.4 ± 0.1 (12) 0.143 ± 0.05 (11)
0.68 ± 0.18 (15) 0.045 ± 0.036(11)
0.88 ± 0.095 (7) 0.075 ± 0.018 (9)
951
B.P.Moore, P.M.Pottcr and R.M.Hkks
1.6 X ID
D.P.M.
0 . 8 X ID
i minimal in both rat and human bladder organ cultures. In
40 80
5xK)
D.P.M.
2.5 x ID
J 40 80
Fraction No.
Fig. 3. Co-incubation of PHJ2-NA and [l-"C]acetyl-coenzyine A with rat
bladder organ cultures. (A) H.p.l.c. profile of "H-metabolites produced
from [*H]2-NA. (B) H.p.l.c. profile of "C-acetylated products from
[l-"C]acetyi-CoA. Bladder tissue, allowed to equilibrate for 24 h, was co-
incubated in PHJ2-NA and [l-"C]acetyi-CoA for 24 h. Tritiated and car-
bon 14 products were extracted with ethyl acetate and chromatographed as
described in methods. Tritium and carbon 14 were counted simultaneously
using an LKB Rackbeta liquid scintillation counter. A = N-acetyl-2-
naphthylamine and B = excess "C-acetate.
ducts, we concluded that either this product was not formed
or was quantitatively unimportant whereas acetylation was
quantitatively very important. In order to increase the chance
of detecting any hydroxylation metabolites which might be
produced, various known inhibitors of acetylation enzymes
were co-incubated with 2-NA in rat and human bladder
cultures. None of the inhibitors used, namely, 130 /tM
amythopterin (34), 1 mM sulphathiazole, 10 /iM melatonin
(35) and 10 /Jvl sodium-p-hydroxymercuribenzoate (36)
significantly altered the amounts of N-acetyl-2-naphthyl-
amine or of hydroxy metabolites produced. Acetylation en-
zymes are clearly present in considerable excess of oxidative
enzymes in the urothelium.
As with benzidine it proved impossible to measure DNA-
bound 2-NA due to the small amounts of DNA in the tissue
and the low specific activity. However, values for protein
binding are recorded in Table II.
Discussion
We reported previously (17) that enzymes capable of ac-
952
tivating the polycyclic hydrocarbon, benzo[a]pyrene, and the
arylamine, 2-acetylaminofluorene are located in the rat and
human bladder. These and similar observations (18,19) sug-
gest that products excreted in human urine after smoking may
be rendered carcinogenic in the bladder (37). Metabolism in
situ thus may well contribute to the two to three fold excess of
smoking-related cancer (38).
In contrast to these earlier findings, our present observa-
tions indicate a different mechanism to be involved in the in-
itiation of bladder cancer by 2-NA and benzidine. Acetyla-
tion, normally regarded as a detoxification reaction, was very
rapid whereas hydroxylation, catalysed either by cytochrome
P450 or by prostaglandin endoperoxide synthetase, was
these experiments, acetylation of 2-NA and benzidine was
~ 100-fold faster than any other form of metabolism. Even
the addition of known inhibitors of acetylation failed to
reveal N-hydroxylation or increase aromatic oxidation of
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on May 13, 2016
these substrates.
The normal human population is divided into high and low
N-acetyltransferase phenotypes. It has been postulated that
high levels of hepatic N-acetylation activity confer natural
protection against arylamine-induced bladder carcinogenesis
(39,40). Similarly, the low levels of this enzyme normally
found in dogs, make this species particularly susceptible to
arylamine induced bladder cancer and hence a useful animal
model of the human disease (41). The high levels of N-acetyl-
transferase recorded in our human bladder cultures may also
be expected to confer protection against arylamines, though
in the small sample studied there was little evidence of
populations with high and low activity. Levels of N-acetyl-
transferase towards both carcinogens, was 40—60% lower in
rat bladder cultures, than in cultures of human bladder or
human ureter. Furthermore, the levels of binding to protein
were slightly higher in the rat bladder cultures than in the
human cultures. The differences in species susceptibility in
rats and humans to these two carcinogens still cannot be
readily explained (42).
A proximate step in the bioactivation of benzidine is
N-acetyltransfer and it is possible that the high levels of
N-acetyltransferase activity in the urothelium are important
in the initiation of bladder carcinogenesis by benzidine. How-
ever, complete activation can only occur if acetylation is
followed by N-hydroxylation and, although this reaction may
occur with other compounds, with benzidine and 2-NA it is
quantitatively negligible. We cannot completely rule out the
formation of N-hydroxy-metabolites since intrinsically they
are unstable and unlikely to survive the long incubation times
required for detection of the other metabolites formed in
culture. However, since only trace amounts of the more
stable hydroxylated metabolites were formed the same is pro-
bably true for N-hydroxylated products. Autrup also found
no hydroxylated metabolites of 2-naphthylamine and observ-
ed more protein binding than DNA-adduct formation in the
human bladder (25). However, unlike Autrup we found no
evidence for formation of an N-glucuronide of 2-NA.
The metabolism of 2-NA and benzidine in the bladder thus
differs markedly from that of other carcinogens such as
benzo[a]pyrene (17 — 19). Our previous investigations
demonstrated conclusively that bladder cells actively
metabolise benzo[a]pyrene, 2-acetylaminofluorene, aflatoxin
and nitrosamines, and other investigators claim that bladder
tissue forms more DNA adducts with benzo[a]pyrene than do

many other tissues known to be susceptible to its carcinogenic
action. Bovine bladder mucosa is also known to activate cer-
tain aromatic amines (21). One possible explanation is a
restriction in the diversity of cytochrome P-450 types in the
urothelium which, perhaps, is to be expected in view of the
small amounts of endoplasmic reticulum normally found in
this tissue. Nevertheless, these small amounts of activity may
be significant on exposure to carcinogens such as benzo[a]-
pyrene, a prominent contaminant of cigarette smoke.
In summary, our results support the suggestion that the
bladder carcinogenicity of benzidine or 2-NA probably results
from liver rather than bladder epithelial metabolic activation.
Oxidative metabolism in situ, when it occurs, probably
strongly favours detoxification rather than activation of such
metabolites. Non-oxidative acetylation in the bladder may be
an important final protective mechanism against most car-
cinogens but, in some exceptional instances, may activate
urinary metabolites from the liver to more carcinogenic
species.
Acknowledgements
This investigation was supported by PHS grant number 1 RO1 CA31082-01
from the National Cancer Institute, USA. We are indebted to Mr. E.Milroy
and other members of the Urology Department of the Middlesex Hospital for
provision of biopsy arxl tissue samples. We also thank Professor R. Berry for
his support and for providing culture facilities. Thanks are also given to Dr.
C.N.Martin for helpful discussions.
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(Received on 8 February 1984; accepted on 28 March 1984)

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