Beruflich Dokumente
Kultur Dokumente
949-954, 1984
Brian P. Moore, Philip M. Potter and R. Marian Hicks described; for example benzidine is a particularly good
substrate for prostaglandin endoperoxide synthetase, an en-
School of Pathology, The Middlesex Hospital Medical School, Riding
House Street, London W1P 7LD, UK zyme which is abundant in the renal inner medulla (13,14).
This enzyme catalyses the reduction of prostaglandin G with
The metabolism of benzidine and 2-naphthylamine, two co-oxidation of a second substrate, in this case benzidine,
aromatic amines whkh are carcinogenic for the human, was thus forming free radicals that will bind to macromolecules.
investigated in human and rat bladder organ cultures. There Prostaglandin synthetase also is involved in the metabolism
was little oxidative metabolism of either carcinogen in either of other, structurally unrelated bladder carcinogens and since
species. In particular, N-hydroxy-2-naphthylamine, a prox- it is located in the urothelium itself, could play an important
Table I. Benzidine
Rat bladder 34.0 ± 11.0(10) 0.28 ± 0.05 (9) 0.03 ± 0.02 (9) 0.3 ± 0.1 (10)
Human bladder 4 16.9 ± 6.2 (5) 0.5 ± 1.0 (4) 0.04 ± 0.02 (4) 0.19 ± 0.05 (5)
Human ureter 1 39.3 ± 3.0 (4) 0.4 ± 0.08 (4) 0.06 ± 0.003 (3) 0.38 ± 0.05 (4)
950
Metabolism of 2-aaphthytamine and benzfcttne
The two major peaks (Figure 2, peaks C and D) co-chromato- graphed well behind N-acetyl-2-naphthylamine, was variable
graphed with the starting material, 2-NA, and with N-acetyl- in size, and occasionally separated into two components. In
2-naphthylamine. The latter was not present in controls from most experiments longer than 24 h, the levels of radioactive
which tissue was absent, or in similar cultures performed at product chromatographing in this region were only slightly
4°C. When larger pieces of tissue were cultured, the higher than those occurring in parallel controls. As with the
acetylated metabolite accounted for as much as 82% of the radioactive materials associated with peak A, the products in
organo-soluble material after 24 h in human organ cultures. these peaks may have been formed metabolically and by
Its mean rate of production was 0.68 ± 0.18 and 0.88 ± naturally occurring degradation reactions. This was ex-
0.2 nmol/min/g in human bladder and human ureter emplified in 96 h human bladder cultures where a late
cultures, respectively, and 0.4 ± 0.1 nmol/min/g in rat blad- chromatographing metabolic component was found to ac-
der cultures (Table II). count for 5 1 - 5 2 % of the organo-soluble radioactive
Two of the remaining peaks of radioactivity chromato- materials whereas the equivalent controls had two peaks with
graphed near to the origin (Figure 2, peaks A and B). Peak B different retention times, accounting for 26 and 31% of the
co-chromatographed with 3-amino-2-naphthol and was pre- organo-soluble radioactivity. These experiments also sug-
sent only ever in trace amounts, < 1 % of the total organo- gested that the later chromatographing peak (E) may be a fur-
soluble radioactivity in cultures; it was undetectable in con- ther metabolite of N-acetyl-2-naphthylamine as it was found
trols. The other peak, A, which was usually slightly larger in much smaller quantities than in 24 h cultures. However,
than B, was present also in 'no tissue' controls, suggesting it this could not be confirmed in a series of rat bladder and
TaWe D. 2-NA
Rat bladder 38.5 ± 17.0 (12) 0.4 ± 0.1 (12) 0.143 ± 0.05 (11)
Human bladder 7 13.5 ± 19.2(17) 0.68 ± 0.18 (15) 0.045 ± 0.036(11)
Human ureter 4 24.4 ± 12.1 (11) 0.88 ± 0.095 (7) 0.075 ± 0.018 (9)
951
B.P.Moore, P.M.Pottcr and R.M.Hkks
J 40 80
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
Fraction No. rat bladder cultures, than in cultures of human bladder or
human ureter. Furthermore, the levels of binding to protein
Fig. 3. Co-incubation of PHJ2-NA and [l-"C]acetyl-coenzyine A with rat were slightly higher in the rat bladder cultures than in the
bladder organ cultures. (A) H.p.l.c. profile of "H-metabolites produced human cultures. The differences in species susceptibility in
from [*H]2-NA. (B) H.p.l.c. profile of "C-acetylated products from rats and humans to these two carcinogens still cannot be
[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-
readily explained (42).
bon 14 products were extracted with ethyl acetate and chromatographed as A proximate step in the bioactivation of benzidine is
described in methods. Tritium and carbon 14 were counted simultaneously N-acetyltransfer and it is possible that the high levels of
using an LKB Rackbeta liquid scintillation counter. A = N-acetyl-2-
naphthylamine and B = excess "C-acetate. 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
ducts, we concluded that either this product was not formed followed by N-hydroxylation and, although this reaction may
or was quantitatively unimportant whereas acetylation was occur with other compounds, with benzidine and 2-NA it is
quantitatively very important. In order to increase the chance quantitatively negligible. We cannot completely rule out the
of detecting any hydroxylation metabolites which might be formation of N-hydroxy-metabolites since intrinsically they
produced, various known inhibitors of acetylation enzymes are unstable and unlikely to survive the long incubation times
were co-incubated with 2-NA in rat and human bladder required for detection of the other metabolites formed in
cultures. None of the inhibitors used, namely, 130 /tM culture. However, since only trace amounts of the more
amythopterin (34), 1 mM sulphathiazole, 10 /iM melatonin stable hydroxylated metabolites were formed the same is pro-
(35) and 10 /Jvl sodium-p-hydroxymercuribenzoate (36) bably true for N-hydroxylated products. Autrup also found
significantly altered the amounts of N-acetyl-2-naphthyl- no hydroxylated metabolites of 2-naphthylamine and observ-
amine or of hydroxy metabolites produced. Acetylation en- ed more protein binding than DNA-adduct formation in the
zymes are clearly present in considerable excess of oxidative human bladder (25). However, unlike Autrup we found no
enzymes in the urothelium. evidence for formation of an N-glucuronide of 2-NA.
As with benzidine it proved impossible to measure DNA- The metabolism of 2-NA and benzidine in the bladder thus
bound 2-NA due to the small amounts of DNA in the tissue differs markedly from that of other carcinogens such as
and the low specific activity. However, values for protein benzo[a]pyrene (17 — 19). Our previous investigations
binding are recorded in Table II. demonstrated conclusively that bladder cells actively
metabolise benzo[a]pyrene, 2-acetylaminofluorene, aflatoxin
Discussion and nitrosamines, and other investigators claim that bladder
We reported previously (17) that enzymes capable of ac- tissue forms more DNA adducts with benzo[a]pyrene than do
952
Metabofigm of 2-naphtbytainine and bcmfaSne
many other tissues known to be susceptible to its carcinogenic prostaglandin endoperoxide synthetase, Cancer Res., 40, 2839-2845.
action. Bovine bladder mucosa is also known to activate cer- 14. Wise.R.W., Zenser.T.V. and Davis.B.B. (1983), Prostaglandin H synthe-
tase metabolism of the urinary bladder carcinogens benzidine and ANFT,
tain aromatic amines (21). One possible explanation is a Carcinogenesis, 4, 285-289.
restriction in the diversity of cytochrome P-450 types in the 15. Zenser.T.V., Mattammal.M.B. and Davies.B.B. (1980), Metabolism of
urothelium which, perhaps, is to be expected in view of the N-(4^5-nitro-2-furyI)-2-thiazolyDfoTmamide by prostaglandin endoperox-
small amounts of endoplasmic reticulum normally found in ide synthetase, Cancer Res., 40, 114-118.
this tissue. Nevertheless, these small amounts of activity may 16. Cohen,S.M., Zenser.T.V., Murasaki.G., Fukushima,S., Mattammal,
M.B., Rapp.N.S. and Davies.B.B. (1981), Aspirin inhibition of N-(4-(5-
be significant on exposure to carcinogens such as benzo[a]- nitro-2-furyl)-2-thiazolyl)formamide induced lesions of the urinary blad-
pyrene, a prominent contaminant of cigarette smoke. der correlated with inhibition of metabolism by bladder prostaglandin en-
In summary, our results support the suggestion that the doperoxide synthetase, Cancer Res., 41, 3355-3359.
bladder carcinogenicity of benzidine or 2-NA probably results 17. Moore.B.P., Hicks,R.M., Knowies.M.A. and Redgrave.S. (1982),
Metabolism and binding of benzo[a]pyrene and 2-acetylaminofluorene by
from liver rather than bladder epithelial metabolic activation. short-term organ cultures of human and rat bladder, Cancer Res., 42,
Oxidative metabolism in situ, when it occurs, probably 642-648.
strongly favours detoxification rather than activation of such 18. Stoner.G.D., Danid.F.B., Schenck.K.M., Schut,H.A.J., Goldblatt,P.J.
metabolites. Non-oxidative acetylation in the bladder may be and Sandwisch.D.M. (1982), Metabolism and DNA binding of benzo[aj-
pyrene in cultured human bladder and bronchus, Carcinogenesis, 3,
an important final protective mechanism against most car- 195-201.
cinogens but, in some exceptional instances, may activate 19. Autrup.H., Grafstrom.R.C, Christensen.B. and Kider.J. (1981), Metab-
urinary metabolites from the liver to more carcinogenic
953
B.P.Moore, P.M.Potter and R.M.Hkks
954