Carcinogenesis vol.17 no.8 pp.1769-1772, 1996
SHORT COMMUNICATION
Metabolic activation and DNA binding of food mutagens and
other environmental carcinogens in human mammary epithelial
cells
Paul L.Carmichael1, Elaine M.Stone, Philip L.Grover,
Barry A.Gusterson and David H.Phillips
The Institute of Cancer Research, Haddow Laboratories, Cotswold Road,
Belmont, Sutton, Surrey SM2 5NG, UK
'To whom requests for reprints should be addressed
Cultures of human mammary epithelial cells were treated
with one of seven heterocyclic amine food mutagens [2amiiio-3-methylimidazo[4,5-/]quinoline (IQ), 2-amino-3,4dimethylimidazo[4,5-/|quinoline (MelQ), 2-amino-3,8-dimethylimidazo[4,5-/]quinoxaline (MelQx), 2-amino-3,4,8trimethylimidazo[4,5-/|quinoxa]ine (4,8-DiMeIQx), 2-amino3,7,8-trimethylimidazo[4,5-/|quinoxaline (7,8-DiMeIQx), 2amino-3,4,7,8-tetramethyUmidazo[4^-/|quinoxaline (4,7,8TriMelQx) or 2-amino-l-methyI-6-phenyIimidazo[4,5-£]
pyridine (PhIP)], four nitropyrenes (1-nitropyrene (1-NP),
1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP)
or 1,8-dinitropyrene (1,8-DNP)] or the polycyclic aromatic
hydrocarbon dibenzo[a/]pyrene (DB[a/|P). DNA isolated
from the cultures was analysed by 32P-post-labeUing and
in each case the presence of carcinogen-DNA adducts was
detected. The patterns and numbers of adducts obtained
when human mammary cell DNA digests were separated
on polyethyleneimine-cellulose TLC were found to closely
resemble those previously demonstrated to be present in
the DNA of tissues from rodents and other primates treated
with the same agents. Up to six DNA adducts were detected
in human breast cells treated with IQ and MelQ. Fewer
adducts (1-3) were detected following treatment with
MelQx or its methylated derivatives, whilst PhIP gave rise
to at least four distinct adduct spots. Five adduct spots
were detected in breast cells treated with DB[a/\P or with
1-NP, but fewer adduct spots were formed by 1,3-, 1,6- and
1,8-DNP. These data demonstrate the ability of human
breast epithelial cells to activate to DNA binding species a
range of carcinogenic compounds known to be present in
the human diet or to which humans are known to be
exposed environmentally.
World wide, 600 000 new cases of breast cancer are diagnosed
every year. However, despite the high prevalence of breast
cancer its aetiology remains obscure. Known risk factors such
as a family history of the disease or factors which influence
lifetime exposure to endogenous oestrogens (e.g. early menarche, late menopause, age at first full-term pregnancy, etc.) may
account for only ~30% of cases (1). Indeed, although the
•Abbreviations: PAH, polycyclic aromatic hydrocarbons; IQ, 2-amino-3methylimidazo[4,5-/)quinoline; MelQ, 2-amino-3,4-dimethylimidazo[4,5/Iquinoline; MelQx, 2-amino-3,8-dimethylimidazo[4,5-/lquinoxaline; 4,8DiMelQx, 2-amino-3,4,8-trimethylimidazo[4,5-/lquinoxaline; 7,8-DiMeIQx,
2-amino-3,7,8-trimethylimidazo[4,5-/)quinoxaline; 4,7,8-TriMeIQx, 2-amino3,4,7,8-tetramethylimidazo[44-/lquinoxaline; PhIP, 2-amino-1-methyl-6phenylimidazo[4,5-i]pyridine; 1-NP, 1-nitropyrene; 1,3-DNP, 1,3-dinitropyrene; 1,6-DNP, 1,6-dinitropyrene; 1,8-DNP, 1,8-dinitropyrene; DB[a,/|P,
dibenzo[a/)pyrene; PEL polyethyleneimine.
© Oxford University Press
promoting effect of oestrogens in human breast cancer has
been widely established, a profound lack of understanding
exists concerning the initiation of this disease in the majority
of women where a familial link (such as inherited germline
mutations in BRCA genes) is not present. Interestingly, the
spectrum of p53 gene mutations observed in breast tumours
suggests the involvement of exogenous agents in a significant
proportion of cases (2). Furthermore, studies of the incidence
of breast cancer among migrant populations strongly implicate
dietary and/or environmental factors in the aetiology of the
disease (3).
A number of chemicals to which there is widespread human
exposure have been shown to cause mammary tumours in
laboratory animals. These include examples from the class
of compounds known as food mutagens, highly mutagenic
heterocyclic aromatic amines formed during the cooking of
protein-rich foods (4); environmental combustion products,
including certain polycyclic aromatic hydrocarbons (PAH*)
(5) and nitro-PAH (6). The majority of these chemical agents
appear to exert their biological effects following metabolic
activation to electrophilic species which interact covalently
with DNA. The formation of DNA-carcinogen adducts is thus
one of the critical initiating events in the multistage process
of chemical carcinogenesis. Hence, sensitive methods for the
detection and measurement of covalent DNA lesions are of
crucial importance in both the identification of potential human
carcinogens and of their mechanisms of action. In an earlier
pilot study using 32P-post-labelling we showed that in two
samples of human mammary tissue (removed at reduction
mammoplasty and cultured as epithelial cell aggregates) the
heterocyclic amines 2-amino-3-methylimidazo[4,5-/|quinoline
(IQ) and 2-amino-3,4-dimethylimidazo[4,5-/]quinoline (MelQ)
were metabolized to reactive species that gave rise to DNA
adducts (7). We now report studies in which we have investigated, in human mammary epithelial cells prepared from 10
patients, the metabolic activation to DNA binding species of the
food mutagens, IQ, MelQ, 2-amino-3,8-dimethylimidazo[4,5/Iquinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5/Iquinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-/]quinoxaline (7,8-DiMeIQx), 2-amino-3,4,7,8-tetramethylimidazo[4,5-/|quinoxaline (4,7,8-TriMeIQx) and 2amino-l-methyl-6-phenylimidazo[4,5-£]pyridine (PhIP), the
nitropyrenes 1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3DNP), 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8DNP) and the PAH dibenzo[a,/]pyrene (DB[a,/]P).
Human mammary epithelial cells were prepared as organoids
by digesting 200-400 g of breast tissue, removed from healthy
women undergoing reduction mammoplasty, as described (79). The organoids were then seeded into 175 cm2 flasks
containing 50 ml RPMI 1640 medium with 10% fetal calf
serum and supplemented with 2 mM L-glutamine, 100 |ig/ml
penicillin/streptomycin mix, 5 (ig/ml hydrocortisone, 5 Jig/ml
insulin and 5 p.g/ml cholera toxin. The flasks were gassed with
5% CO2 in air and incubated at 37°C for 48 h to allow
1769
P.L.Carmichael el al.
attachment of the organoids. The medium was then changed,
the cells maintained in this medium for 48 h and then
disaggregated, using a 0.25% trypsin/EDTA solution, to form
a single cell suspension (-1X10 6 cells/ml, >90% viable by
trypan blue exclusion) in Dulbecco's minimal essential medium
with 5% fetal calf serum. Aliquots of cell suspension (2 ml)
were treated with either IQ, MelQ, MelQx, 4,8-DiMeIQx, 7,8DiMelQx, 4,7,8-TriMeIQx, PhIP, 1-NP, 1,3-DNP. 1.6-DNP.
1,8-DNP or DB[a,/]P, added as a solution in dimethyl sulphoxide (50 JJ.1) to a final concentration of 500 |iM. Suspensions
of control cells were treated with the vehicle alone. Cell
suspensions were incubated with shaking at 37°C for 22 h,
the cells were harvested by centrifugation (200 g, 5 min) and
the cell pellets stored at -85°C. Breast epithelial cell DNA was
subsequently isolated according to the method of Gupta (10).
DNA (4 (ig/sample) from cells treated with food mutagens
was 32P-post-labelled using the limiting ATP method (60 (iCi
ATP/sample) described by Hall et al. (11). Resolution of 32Ppost-labelled DNA digests from MelQ-, IQ- and PhlP-treated
cells on polyethyleneimine (PEI)-cellulose TLC plates was
performed using the solvent system: Dl, 1.0 M sodium
phosphate, pH 6.0; D2, 2.3 M lithium formate, 5.5 M urea,
pH 3.5; D3, 0.8 M LiCl, 0.5 M Tris-HCl, 8.5 M urea, pH 8.0;
D4, 1.7 M sodium phosphate, pH 6.0. Resolution of 32Ppost-labelled DNA digests from MelQx-, 4,8-DiMeIQx-, 7,8DiMelQx- and 4,7,8-TriMeIQx-treated cells was carried out
on PEI-cellulose TLC plates using: Dl. 2.3 M sodium phosphate, pH 6.0; D2, 2.8 M lithium formate, 6.8 M urea, pH
3.5; D3, 0.7 M sodium phosphate, 0.5 M Tris-HCl, 8.5 M
urea, pH 8.0; D4, as above. DNA (4 |ig/sample) from cells
treated with a nitropyrene or DB[a,/]P was 32P-post-labelled
using the butanol extraction procedure described by Gupta
(12). Resolution of 32P-post-labelled DNA on PEI-cellulose
TLC plates was performed using the solvent system: Dl, 1.0
M sodium phosphate, pH 6.0; D2, 3.5 M lithium formate, 8.5
M urea, pH 3.5; D3, 1.4 M LiCl, 0.6 M Tris-HCl, 8.5 M urea,
pH 8.0; D4, as above.
Figure 1 shows 32P-post-labelling analyses of DNA digests
isolated from breast epithelial cells treated with each of the
food mutagens listed above. The most intense adduct patterns
were obtained with IQ and MelQ (Figure IB and C). A similar
pattern of up to six distinct adduct spots was seen with each
of these heterocyclic amines, broadly consistent with the
number and profile of adducts reported to be formed in the
tissues of treated rodents. For example Hall et al. (11) reported
the detection of a very similar profile and number of adducts
present in the liver, lung, kidney, colon and forestomach DNA
of CDF, mice that had been treated with IQ or MelQ. In
addition, Cummings and Schut (13) have described the detection of up to five DNA adducts in the white blood cells, liver,
lungs, stomach, small intestine, caecum, colon, kidneys, spleen
and heart DNA of CDF, mice treated with IQ. Furthermore,
broadly similar patterns of DNA adducts have been demonstrated to be present in the mammary glands of female Fisher
344 rats treated with MelQ and IQ (14). It should be noted at
this point that it is likely that some of the adduct spots detected
may actually result from the 32P-post-labelling of incompletely
digested DNA. due to the digestion-resistant nature of this
type of adduct (15,16).
Analysis of breast cells incubated with MelQx gave a
simpler and much less intense pattern of adducts (Figure ID).
as did the three methylated derivatives of this food mutagen
(Figure 1F-H), each giving between one and three DNA
1770
Control
MelQx
MelQ
4,8DiMeIQx i
7,8DiMelQx ••#
32
i
4,7,8TriMelQx >x
Fig. 1. Autoradiographs of P-post-labelled DNA from human breast
epithelial cells chromatographed on PEI-cellulose TLC plates following
treatment with (A) dimethyl sulphoxide, (B) IQ. (C) MelQ. (D) MelQx, (E)
PhIP, (F) 4.8-DiMeIQx. (G) 7,8-DiMeIQx or (H) 4,7,8-TriMeIQx, which
were present in the culture medium at a concentration of 500 |iM. Cultures
were incubated for 22 h at 37°C as described in the text.
adduct spots. The patterns and number of adducts seen with
breast tissue were consistent with those reported previously
for MelQx in a range of rat tissues (17) and the liver of
cynomolgus monkeys (17) and for 4,8-DiMeIQx in rat liver
(18). Incubation with PhIP gave rise to at least four distinct
adduct spots (Figure IE) with a very similar pattern to that
previously found in the female rat mammary gland (14) and
in a broad range of tissues in the cynomolgus monkey (19).
Because the limiting ATP method for 32P-post-labelling was
used in the detection of the above food mutagen DNA adducts,
only semi-quantitative data can be obtained from the analyses.
However, although the labelling efficiency is unknown and
adduct levels may actually be higher, a range of adduct levels
for the 10 cell cultures obtained from different individuals can
be calculated. For example, IQ gave a mean level of adducts
per 108 nucleotides of 12.5 (range 1.9-69.1), MelQ 8.7 (range
1.3-35.3) and PhIP 0.4 (range 0.2-0.6). Hence, although
identical patterns were obtained with cells from different
individuals for a given carcinogen, the calculated data indicate
that interindividual variations probably exist. This aspect will
require further and more detailed investigation.
Figure 2 shows the 32P-post-labelling analysis of DNA
digests obtained from human breast cells that had been treated
with the nitropyrenes or with DB[a,/]P. Up to five distinct
adduct spots were detected following treatment with 1-NP
(Figure 2A), but fewer adduct spots were seen with 1.3-. 1,6and 1,8-DNP (Figure 2B-D respectively). Similar patterns of
adducts have been detected in the mammary gland DNA of
Sprague-Dawley rats, liver DNA from CD-I mice and lung
DNA from A/J mice treated with 1-NP (20) and in the bladder
and liver DNA from male Wistar rats treated with 1.6-DNP (21).
When mammary epithelial cells were treated with DB[a,/]P
Food mutagens and other environmental carcinogens
lNPLAdfl . _
1,6DNP. ^
J
^
ll,3DNP
\mtmk S^1,8DNP
DB[a,l|P|
Fig. 2. Autoradiographs of 32P-post-labelled DNA from human breast
epithelial cells chromatographed on PEI-cellulose TLC plates following
treatment with (A) 1-NP, (B) 1,3-DNP, (C) 1,6-DNP, (D) 1,8-DNP or (E)
DB[a,/]P, which were present in the culture medium at a concentration of
500 uM. Cultures were incubated for 22 h at 37°C as described m the text.
five adduct spots that ran closer to the plate origin were
detected. Again, the patterns and number of adduct spots seen
with DB[a,/]P closely resembled those detected previously in
mouse skin DNA from topically treated animals (22). Roughly
similar levels of adducts were detected with the nitro-PAHs
and PAH as found in experiments with the food mutagens.
However, it should be noted that different methods of adduct
enhancement were used in the 32P-post-labe0ing assay, so such
comparisons should be treated with caution.
It is generally agreed that only a small percentage of the
total human cancer burden is attributable to genetic factors
alone and that many cancers could be prevented by controlling
environmental exposures to carcinogens. Indeed, it is clear
that naturally occurring and man-made chemicals play a
significant role in the aetiology of human cancer (23). With
human breast cancer, evidence from the epidemiology of this
disease in Western as compared with Far Eastern populations
strongly points to the involvement of specific components of
the Western diet and/or environment which may act as initiating
agents. Human exposure to heterocyclic amines has been
clearly established (24) and PhIP is generally considered to
be the most abundant amine of this type in the typical Western
diet of meat, fish or fowl cooked at high temperatures. PhIP,
like many of the other food mutagens, is a mammary carcinogen
in rodent models (4,25) and this fact alone suggests that PhIP
and other heterocyclic aromatic amines may be involved in
the aetiology of human breast cancer. In this report we have
shown that PhIP and six other heterocyclic amines, each known
to be present in a typical Western diet, can be metabolized by
the epithelial cells of the human breast to DNA-reactive
metabolites that give rise to detectable levels of DNAcarcinogen adducts. It would appear that two key steps are
involved in the metabolic activation of heterocyclic amines to
DNA-reactive species (26). First, the parent amine is metabolized by cytochrome P4501A2 to the N-hydroxylamine, which
is further activated to a reactive ester by phase II esterifying
enzymes. Ghoshal et al. suggest, from the results of studies
involving PhIP in rats, that the mammary gland is poor at
performing the /V-hydroxylation step and that this stage of the
activation is most likely to occur in the liver. The Nhydroxylated amine may then be transported in the bloodstream
to the mammary gland where, they suggest, O-acetyltransferase
is the primary activating phase II enzyme of this tissue. The
data presented in the present report suggests that some P450mediated 7V-hydroxylating activity appears to be present in the
first passage human breast cell cultures we employed. However,
Fan et al. (27) reported no detectable DNA adducts in human
mammary epithelial cells treated at passages 11-13 with IQ
or PhIP at 50 (iM, although detectable levels of adducts were
found with the iV-hydroxlamine metabolites at 1 JIM.
In addition to the food mutagens, we have also shown in
this paper that 1-NP, 1,3-DNP, 1,6-DNP, 1,8-DNP and DB [a,/]P,
known rodent mammary carcinogens (6,28) that are components of automotive combustion emissions and to which
humans are environmentally exposed, are also capable of
producing DNA adducts in human breast cells. These agents
are highly Hpophilic and the unique high fat environment of
mammary epithelial cells implies that chronic exposure to low
doses of these agents may have greater consequences for
the breast than for other tissues. Human exposure to such
carcinogens in food and from the environment is likely to be
to a complex mixture of heterocyclic amines, PAH and nitroPAH and hence the potential for such agents to demonstrate
synergism in terms of their carcinogenicity will also have to
be taken into consideration. A study investigating the presence
of DNA damage in situ in the breast tissue of human donors
reported the detection of unidentified DNA adducts in a small
but significant proportion of samples examined using 32P-postlabelling (29). In addition, Perera et al. (1) have reported
patterns of adducts consistent with exposure to tobacco smoke
carcinogens in breast tissue DNA from five out of 15 women
with breast cancer. An experimental approach in which work
of the type described above, whereby adduct patterns are
determined for potential breast cancer initiating agents in
otherwise untreated human breast cells, is compared with the
adduct patterns of 'endogenous' breast DNA damage, might
help in the identification of the major dietary or environmental
agents responsible for the initiation of this disease.
Acknowledgement
This work was supported by a grant from the Association for International
Cancer Research
References
l.Perera.F.P.,
EstabrookA,
HewerA.
Channing.K.,
RundleA,
Mooney.LA, Whyatt,R. and Phillips.D.H. (1995) Carcinogen-DNA
adducts in human breast tissue. Cancer Epidemiol. Biomarkers Prevent.,
4, 233-238.
2.Biggs,PJ., Warren.W., Venitt,S. and Stratton.M.R (1993) Does a genotoxic
carcinogen contribute to human breast cancer? The value of mutation
spectra in unravelling the aetiology of cancer. Mutagenesis, 8, 275-283.
3.HigginsonJ., Muir,C.S. and Munoz,N. (1992) Human Cancer:
Epidemiology and Environmental Causes. Cambridge Monographs on
Cancer Research, Cambridge, UK.
4.Nagao,M., Ushijima,T, Wakabayashi.K., Ochiai.M., Kushida,H.,
SugimuraX, Hasegowa,R., Shirai.T. and Ito.N. (1994) Dietary carcinogens
and mammary carcinogenesis. Induction of rat mammary carcinomas by
administration of heterocyclic amines. Cancer, 74 (Suppl.), 1063-1069.
5.El-Bayoumy,K. (1992) Environmental carcinogens that may be involved
in human breast cancer etiology. Chem. Res. Toxicol., 5, 585-590.
6.Beland,F.A. and Marques,M.M. (1994) DNA adducts of nitropolycyclic
aromatic hydrocarbons, hi Hemminki.K., DippleA-, Shuker.D.E.G.,
KadlubarJ.F., Segerback.D. and Bartsch,H. (eds), DNA Adducts:
Identification and Biological Significance. IARC Scientific Publication no.
125, International Agency for Research on Cancer, Lyon, France, pp.
229-244.
7.Pfau,W., O'HarcMJ., Grover,P.L. and Phillips,D.H. (1992) Metabolic
activation of the food mutagens 2-amino-3-methylimidazo [4,5-/)quinoline
(IQ) and 2-amino-3,4-dimethylimidazo[4,5-/]quinoline (MelQ) to DNA
1771
P.L.Carmichael et al.
binding species in human mammary epithelial cells. Carcinogenesis, 13,
907-909.
8.Easty,G.C, Easty.D.M., Monaghan.R, Ormerod,M.G. and Neville.A M.
(1980) Preparation and identification of human breast epithelial cells in
culture. Int. J. Cancer, 26, 577-584.
9.0'HareJvU., Ormerod,M.G., Monaghan.P, Lane,E.B. and Gusterson.B.A.
(1991) Characterization in vitro of luminal and myoepithelial cells isolated
from human mammary gland by cell sorting. Differentiation, 46, 209-221.
10.Gupta,R.C , Reddy.M.V. and Randerath.K. (1982) 32P-Postlabeling analysis
of non-radioactive aromatic carcinogen-DNA adducts. Cartinogenesis, 3,
1081-1092.
ll.Hall,M., Nf Sh6M., Wild.D., Fasshauer.I., Hewer.A. and Phillips.D.H.
(1990) Tissue distribution of DNA adducts in CDF, mice fed 2-amino-3methylimidazo[4,5-/|quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4r5/]quinoline (MelQ). Carcinogenesis, 11, 1005-1011.
12.Gupta,R.C. (1985) Enhanced sensitivity of 32P-postlabeling analysis of
aromatic carcinogen:DNA adducts. Cancer Res., 45, 5656-5662.
13.Cummings,D.A. and Schut.H.A.J. (1995) Inhibitory effect of dietary 4ipomeanol on DNA adduct formation by the food mutagen 2-amino-3methyhmidazo[4,5-/lquinoline (IQ) in male CDFi mice. Carcinogenesis,
16, 2523-2529.
14. Ghoshal.A. and Snyderwine,E.G. (1993) Excretion of food-derived
heterocyclic amine carcinogens into breast milk of lactating rats and
formation of DNA adducts in the newborn. Carcinogenesis, 14, 2199—2203.
15.Pfau,W., Brockstedt.U., Sohren.K. and Marquardt.H. (1994) 32P-Postlabelling analysis of DNA adducts formed by food-derived heterocyclic
amines: evidence for incomplete hydrolysis and a procedure for adduct
pattern simplification. Carcinogenesis, 15, 877—882.
16.Turesky,RJ. and MarkovicJ. (1994) DNA adduct formation of the food
carcinogen 2-amino-3-methylimidazo[4,5-/|quinoline at the C8 and N2
atoms of guanine. Chem Res Toxicol, 7, 752-61.
17.Davis,C.D.,
Schut.H.A.J.,
Adamson.R.H.,
Thorgeirsson.U.P,
Thorgeirsson.S.S. and Snyderwine.E.G. (1993) Mutagenic activation of
IQ, PhIP and MelQx by hepatic microsomes from rat, monkey and man:
low mutagenic activation of MelQx in cynomolgus monkeys in vitro
reflects low DNA adduct levels in vivo. Carcinogenesis, 14, 61-65.
18.Frandsen,H., Grivas.S., Turesky.RJ., Andersson.R., Dragsted.L.O. and
LarsenJ.C. (1994) Formation of DNA adducts by food mutagen 2-amino3,4,8-trimethyl-3//-imidazo[4,5-/]quinoxaline (4,8-DiMeIQx) in vitro and
in vivo. Identification of a Ar-(2'-deoxyguanosin-8-yl)-4,8-DiMeIQx
adduct. Carcinogenesis, 15, 2553-2558.
19. Snyderwine.E.G., Schut,H.AJ., Sugimura.T., Nagao,M. and Adamson.R H.
(1994) DNA adduct levels of 2-amino-l-methy!-6-phenylimidazo[4,5i>]pyridine (PhIP) in tissue of cynomolgus monkeys after single or multiple
dosing. Carcinogenesis, 15, 2757-2761.
2O.Smith,B.A., Korfmacher.W.A. and Beland.F.A. (1990) DNA adduct
formation in target tissues of Sprague-Dawley rats, CD-I mice and A/J
mice following tumorigenic doses of 1-nitropyrene. Carcinogenesis, 11,
1705-1710.
21.Wolff,T., Bogan.R , Wander.H. and Wegenke.M. (1993) Biomonitonng of
human exposure to carcinogenic nitroaromatic compounds: a pilot study
on DNA adduct formation by 1,6-dinitropyrene in rats. In Phillips.D.H.,
Castegnaro.M. and Bartsch,H. (eds), Postlabelling Methods for Detection
of DNA Adducts. IARC Scientific Publication no. 124, International
Agency for Research on Cancer, Lyon, France, pp. 195-199.
22. Hughes.N.C. and Phillips.D.H. (1990) Covalent binding of dibenzpyrenes
and benzo[a]pyrene to DNA: evidence for synergistic and inhibitory
interactions when applied in combination to mouse skin. Carcinogenesis,
11, 1611-1619.
23.Doll,R. and Peto.R. (1981) The Causes of Cancer: Quantitative Estimates
of Avoidable Risks of Cancer in the United States Today. Oxford University
Press, Oxford, UK.
24. Ushiyama,H., Wakabayashi.K., Hirose.M., Itoh.H., Sugimura.T. and
Nagao.M. (1991) Presence of carcinogenic heterocyclic amines in urine
of healthy volunteers eating normal diet, but not in patients receiving
parenteral alimentation. Carcinogenesis, 12, 1417-1422.
25.Ito,N., Hasegawa,R., Sano.M., Tanmano,S., Esumi.H., Takayama,S. and
Sugimura,T. (1991) A new colon and mammary carcinogen in cooked
food, 2-amino-l-methyl-6-phenylimidazo[4,5-fc]pyridine (PhIP). Carcinogenesis, 12, 1503-1506.
26.Ghoshal,A., Davis,C.D , Schut,H AJ. and Snyderwine.E.G. (1995) Possible
mechanisms for PhlP-DNA adduct formation in the mammary gland of
female Sprague-Dawley rats. Carcinogenesis, 16, 2725-2731.
27.Fan,L., Schut,H.AJ. and Snyderwine,E.G. (1995) Cytotoxicity, DNA
adduct formation and DNA repair induced by 2-hydroyamino-3methylimidazo[4,5-./]quinoline and 2-hydroxyamino-l-methyl-6-phenylimi-
1772
dazo[4,5-i]pyridine in cultured human mammary epithelial cells.
Carcinogenesis, 16, 775-779.
28.Cavalieri,E.L., Higginbotham.S., RamaKrishna.N.V.S., Devanesan.P.D.,
Todorovic,R., Rogan,E.G and Salmasi,S. (1991) Comparative doseresponse tumorigenicity studies of dibenzo[a,/]pyrene versus 7,12dimethylbenz[a]anthracene, benzo[a]pyrene and two dibenzo[a,/]pyrene
dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis, 12,
1939-1944.
29.Seidman,L.A., Moore.CJ. and Gould.M.N. (1988) 32P-Postlabeling
analysis of DNA adducts in human and rat mammary epithelial cells.
Carcinogensis, 9, 1071-1077.
Received on February 23, 1996; revised on April 22, 1996; accepted on May
8, 1996