Mammary Cancer Induction by 6-NC and Metabolites
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 973
mol) and triphenylphosphine (39.3 g, 0.15 mol) in 300 mL of
benzene was heated under reflux for 3 h. The reaction mixture
was cooled, and the phosphonium salt 1 (53.1 g, 86%) was
isolated by filtration. To a stirred solution of the salt 1 (5.0 g,
8.9 mmol) and m-anisaldehyde (1.21 g, 8.9 mmol) in 50 mL of
CH2Cl2 was added a solution of NaOH (0.39 g, 9.7 mmol) in 1
mL of H2O. The mixture was stirred vigorously for 3 h, diluted
with CH2Cl2, washed with water twice, dried (MgSO4), and
concentrated. The crude product was purified by flash chroma-
tography on silica gel (eluent: hexane:CH2Cl2, 80:20) to give
olefin 2 (3.0 g, 99%) as a mixture of cis and trans isomers (37:
63): 1H NMR δ 3.47 (s, 1.89H, trans-OCH3), 3.88 (s, 1.11H, cis-
OCH3), 6.55-7.35 (m, 6H), 7.51-7.83 (m, 4H), 8.08 (d, 0.63H,
trans-olefinic, J ) 8.2 Hz), 8.19-8.22 (m, 0.37H, cis-olefinic),
8.27-8.31 (m, 1H); MS (m/e, relative intensity) 340 (M+, 85%),
338 (M+, 80), 259 (M+ - Br, 47).
chrysene isomers and benzo[a]pyrene (B[a]P) (5-9). We
demonstrated by intramammary administration that
6-NC is a powerful mammary carcinogen (10); again, it
was more potent than the ultimate carcinogen, the bay
region diol epoxide of B[a]P (11). The carcinogenic activity
of 6-NC in the mammary gland (10, 11), the colon of rats
(12), and the lung and skin of mice (5-9, 13) and its
environmental presence (14-17), as well as the ability
of human liver and lung to convert 6-NC into genotoxic
metabolites (18), suggest its potential importance with
regard to human cancer development. In fact, a report
by Zwirner-Baier and Neumann demonstrated the pres-
ence of hemoglobin adducts derived from several NO2-
PAHs, including 6-NC in humans (19).
Human exposure to NO2-PAHs is via inhalation and/
or ingestion. Therefore, in the present study, we evalu-
ated the carcinogenicity of 6-NC, upon oral administra-
tion, toward the mammary glands of rats. 2-Amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most
abundant of the mutagenic and carcinogenic heterocyclic
amines present in cooked meat and fish, served as the
positive control (20, 21).
Metabolism and DNA binding studies in mice and rats
have indicated that ring oxidation and nitroreduction are
involved in the metabolic activation of 6-NC (22). Al-
though, upon activation, 6-NC is known to covalently
bind to DNA in vivo (22), its effect on levels of 8-hydroxy-
2-deoxyguanosine (8-OHdG), an oxidative DNA damage
biomarker, in the rat mammary glands has not been
reported. The lack of effect of 6-NC on levels of 8-OHdG,
as demonstrated in this report, suggests that covalent
DNA binding of 6-NC metabolites is important in the
induction of mammary cancer in rats. Toward this end,
metabolites were synthesized as reported (23), and a new
method was developed to obtain ample materials of a
potential proximate carcinogenic metabolite of 6-NC,
namely, 1,2-DHD-6-AC, and the potency to induce mam-
mary cancer in the rat was ranked by intramammary
administration. This route has been used in our labora-
tory to induce mammary cancer in the rat and is
employed here to avoid systemic effects and to determine
the role of the mammary gland in the metabolic activa-
tion of 6-NC and its metabolites. Collectively, the results
of the bioassay indicate that metabolites derived from
ring oxidation and nitroreduction contribute to the overall
carcinogenicity of 6-NC in the rat mammary gland.
2-Meth oxy-6-br om och r ysen e (3). A solution of 2 (3.0 g,
8.85 mmol) and iodine (5.0 mg) in dry benzene (1 L) was stirred
and irradiated with a Pyrex-filtered Hanover 450 W medium-
pressure UV lamp, while air was bubbled through the solution.
The progress of the reaction was followed by TLC (hexane). After
5 h, 70-80% of the olefin was cyclized. Removal of the solvent
gave a crude mixture of 2-methoxy-6-bromochrysene (3) and
4-methoxy-6-bromochrysene (4). This mixture was separated by
chromatography on silica gel. Elution with hexane/CH2Cl2 (70:
30) gave early eluting 4-methoxy-6-bromochrysene (4) (0.63 g,
21%), mp 134-136 °C: 1H NMR δ 4.19 (s, 3H, OCH3), 7.19 (dd,
1H, H3, J 3,2 ) 7.2 Hz, J 3,1 ) 1.6 Hz), 7.55-7.62 (m, 2H, H1 and
H2), 7.71-7.76 (m, 2H, H8 and H9), 7.99 (d, 1H, H12, J 12,11
)
8.9 Hz), 8.41-8.45 (m, 1H, H7), 8.72 (d, 1H, H11, J 11,12 ) 8.9
Hz), 8.78-8.82 (m, 1H, H10), 10.17 (s, 1H, H5); MS (m/e, relative
intensity) 338 (M+, 70), 336 (M+, 74). Further elution with
hexane/CH2Cl2 (1:1) gave pure 2-methoxy 6-bromochrysene (3)
(1.4 g, 47%), mp 211-212 °C: 1H NMR δ 4.00 (s, 3H, OCH3),
7.32-7.38 (m, 2H, H1 and H3), 7.68-7.77 (m, 2H, H8 and H9),
7.94 (d, 1H, H12, J 12,11 ) 9.2 Hz), 8.42 (d, 1H, H7, J 7,8 ) 7.6
Hz), 8.59 (d, 1H, H4, J 4,3 ) 8.9 Hz), 8.65 (d, 1H, H11, J 11,12
)
9.2 Hz), 8.76 (d, 1H, H10, J 10,9 ) 7.9 Hz), 8.97 (s, 1H, H5); MS
(m/e relative intensity) 338 (M+, 92), 336 (M+, 100).
2-Hyd r oxy-6-br om och r ysen e (5). To a stirred solution of
3 (4.09 g, 12.1 mmol) in CH2Cl2 (200 mL) at 0 °C under an N2
atmosphere was added a 1 M solution of boron tribromide (35
mL, 35 mmol) in CH2Cl2 over a period of 10 min. After 12 h
stirring at room temperature, the reaction mixture was poured
into ice-cold H2O, the organic layer was washed with H2O (2 ×
50 mL) and dried (MgSO4), and the solvent was removed to yield
crude 5, which was recrystallized from CH2Cl2 (3.38 g, 85%),
mp 216-218 °C: 1H NMR δ 7.28-7.33 (m, 2H, H1 and H3),
7.68-7.77 (m, 2H, H8 and H9), 7.88 (d, 1H, H12, J 12,11 ) 9.2
Hz), 8.42 (d, 1H, H7, J 7,8 ) 7.9 Hz), 8.60 (d, 1H, H4, J 4,3 ) 9.2
Hz), 8.63 (d, 1H, H11, J 11,12 ) 9.2 Hz), 8.74 (d, 1H, H10, J 10,9
)
7.9 Hz), 8.95 (s, 1H, H5); for MS GC, TMS derivative of 5 was
prepared: MS (m/e, relative intensity) 396 (M+, 100%), 394 (M+,
96).
Ma ter ia ls a n d Meth od s
Melting points were recorded on a Fischer-J ohnson melting
point apparatus and are uncorrected. Unless stated otherwise,
proton NMR spectra were recorded using a Bruker AM 360WB
NMR spectrometer in CDCl3 with tetramethylsilane (TMS) as
internal standard. Chemical shifts were recorded in ppm
downfield from the internal standard. MS were run on a
Hewlett-Packard model 5988A instrument. Thin-layer chroma-
tography (TLC) was done on aluminum-supported precoated
silica gel plates (EM Industries, Gibbstown, NJ ). All starting
materials were obtained from Aldrich Chemical Co. (Milwaukee,
WI). 6-NC and PhIP were obtained as described previously (6,
20). 1,2-DHD-6-NC and 6-NCDE were synthesized as reported
previously (23). 1,2-DHD-6-AC can be obtained by nitroreduction
of 1,2-DHD-6-NC; however, this approach does not yield the
desired product in satisfactory yield (24). Therefore, we devel-
oped a new method (Scheme 1) for the synthesis of 1,2-DHD-
6-AC.
6-Br om och r ysen e-1,2-d ion e (6). To a stirred solution of 5
(1.60 g, 4.95 mmol) in 430 mL of C6H6/CH2Cl2/THF (16:5:1) were
added 10 drops of Adogen 464 and a solution of Fremy’s salt
(4.0 g, 14.9 mmol) in 250 mL of 0.17 M KH2PO4. Stirring was
continued for 18 h at room temperature, and the organic layer
was collected. The aqueous phase was extracted with benzene.
Combined organic extracts were washed with water, dried
(Na2SO4), and evaporated to dryness. The dark residue was
recrystallized from CH2Cl2 to yield 1.25 g (75%) of dione 6, mp
236-239 °C (dec): 1H NMR δ 6.64 (d, 1H, H3, J 3,4 ) 10.5 Hz),
7.80-7.86 (m, 2H, H8 and H9), 8.33 (d, 1H, H4, J 4,3 ) 10.5 Hz),
8.55 (d, 1H, H12, J 12,11 ) 8.5 Hz), 8.41-8.45 (m, 1H, H7), 8.52
(s, 1H, H5), 8.71-8.75 (m, 1H, H10), 8.82 (d, 1H, H11, J 11,12
)
8.5 Hz); MS (m/e, relative intensity) 338 (M+, 97), 336 (M+, 100).
(()-t r a n s-1,2-D ih y d r o x y -1,2-d ih y d r o -6-b r o m o c h r y -
sen e (7). To a stirred suspension of 6 (1.36 g, 4.04 mmol) in
absolute ethanol (500 mL) was added NaBH4 (4.1 g, 108 mmol)
in portions. The mixture was stirred for 72 h while open to the
1-(4-Br om on a p h th yl)-2-(3-m eth oxyp h en yl)eth ylen e (2).
A solution of 1-bromo-4-bromomethylnaphthalene (33.0 g, 0.11