Microsomal activation of dibenzo[def,mno]chrysene (anthanthrene), a hexacyclic aromatic hydrocarbon without a bay-region, to mutagenic metabolites

Article abstract of DOI:10.1021/tx010131t

Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydrocarbons are thought to be responsible for the genotoxic properties of these environmental pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene), although lacking this structural feature, was found to exhibit considerable bacterial mutagenicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in the range of 18-40 his⁺-revertant colonies/nmol after metabolic activation with the hepatic postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic effect amounted to 44-84% of the values determined with benzo[a]pyrene under the same conditions. The specific mutagenicity of anthanthrene in strain TA100 obtained with the cell fraction of untreated animals was 6 his⁺-revertant colonies/nmol and increased 2.7-fold after treatment with phenobarbital and 4.5-fold after treatment with 3-methylcholanthrene. To elucidate the metabolic pathways leading to genotoxic metabolites, the microsomal biotransformation of anthanthrene was investigated. A combination of chromatographic, spectroscopic, and biochemical methods allowed the identification of the trans-4,5-dihydrodiol, 4,5-oxide, 4,5-, 1,6-, 3,6-, and 6,12-quinones, and 1- and 3-phenols. Furthermore, two diphenols derived from the 3-phenol, possibly the 3,6 and 3,9 positional isomers, as well as two phenol dihydrodiols were isolated. Three pathways of microsomal biotransformation of anthanthrene could be distinguished: The K-region metabolites are formed via pathway I dominated by monooxygenases of the P450 1B subfamily. On pathway II the polynuclear quinones of anthanthrene are formed. Pathway III is preferentially catalyzed by monooxygenases of the P450 1A subfamily and leads to the mono- and diphenols of anthanthrene. The K-region oxide and the 3-phenol are the only metabolites of anthanthrene with strong intrinsic mutagenicity, qualifying them as ultimate mutagens or their precursors. From the intrinsic mutagenicity of these two metabolites and their metabolic formation, the maximal mutagenic effect was calculated. This demonstrates the dominating role of pathway III in the mutagenicity of anthanthrene under conditions where it exhibits the strongest bacterial mutagenicity. platt@ mail.uni-mainz.de.

Full text of DOI:10.1021/tx010131t

332
Chem. Res. Toxicol. 2002, 15, 332-342
Micr osom a l Activa tion of Diben zo[d ef,m n o]ch r ysen e
(An th a n th r en e), a Hexa cyclic Ar om a tic Hyd r oca r bon
w ith ou t a Ba y-Region , to Mu ta gen ic Meta bolites
Karl L. Platt,* Christian Degenhardt, Stefanie Grupe, Heinz Frank,‡ and
Albrecht Seidel‡
Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67,
D-55131 Mainz, Germany
Received August 8, 2001
Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydro-
carbons are thought to be responsible for the genotoxic properties of these environmental
pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene),
although lacking this structural feature, was found to exhibit considerable bacterial mutage-
nicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in
the range of 18-40 his+-revertant colonies/nmol after metabolic activation with the hepatic
postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic
effect amounted to 44-84% of the values determined with benzo[a]pyrene under the same
conditions. The specific mutagenicity of anthanthrene in strain TA100 obtained with the cell
fraction of untreated animals was 6 his+-revertant colonies/nmol and increased 2.7-fold after
treatment with phenobarbital and 4.5-fold after treatment with 3-methylcholanthrene. To
elucidate the metabolic pathways leading to genotoxic metabolites, the microsomal biotrans-
formation of anthanthrene was investigated. A combination of chromatographic, spectroscopic,
and biochemical methods allowed the identification of the trans-4,5-dihydrodiol, 4,5-oxide, 4,5-,
1,6-, 3,6-, and 6,12-quinones, and 1- and 3-phenols. Furthermore, two diphenols derived from
the 3-phenol, possibly the 3,6 and 3,9 positional isomers, as well as two phenol dihydrodiols
were isolated. Three pathways of microsomal biotransformation of anthanthrene could be
distinguished: The K-region metabolites are formed via pathway I dominated by monooxy-
genases of the P450 1B subfamily. On pathway II the polynuclear quinones of anthanthrene
are formed. Pathway III is preferentially catalyzed by monooxygenases of the P450 1A subfamily
and leads to the mono- and diphenols of anthanthrene. The K-region oxide and the 3-phenol
are the only metabolites of anthanthrene with strong intrinsic mutagenicity, qualifying them
as ultimate mutagens or their precursors. From the intrinsic mutagenicity of these two
metabolites and their metabolic formation, the maximal mutagenic effect was calculated. This
demonstrates the dominating role of pathway III in the mutagenicity of anthanthrene under
conditions where it exhibits the strongest bacterial mutagenicity.
Sch em e 1. Str u ctu r a l F or m u la s, Molecu la r
Region s, a n d Nu m ber in g of Ben zo[a ]p yr en e a n d
Diben zo[d ef,m n o]ch r ysen e (An th a n th r en e)
In tr od u ction
The class of the polycyclic aromatic hydrocarbons
(PAH),1 ubiquitously present in the environment as the
result of incomplete combustion of organic matter, con-
tains a great number of strong carcinogens. As PAH are
not genotoxic by themselves, they need enzymatic trans-
formation to reactive metabolites. A characteristic struc-
tural feature of most carcinogenic PAH is the presence
of a sterically hindered bay-region (cf. Scheme 1) which
* To whom correspondence should be addressed. E-mail: platt@
mail.uni-mainz.de.
‡ Biochemical Institute for Environmental Carcinogens, Prof. Dr.
Gernot Grimmer-Foundation, Lurup 4, D-22927 Grosshansdorf, Ger-
many.
is involved in the metabolic activation to dihydrodiol
epoxides considered to be ultimate carcinogenic metabo-
lites of PAH (1). Other pathways of metabolic activation
include enzymatic conversion to radical cations (2),
methylated derivatives (3), o-quinones (4), or ring-opened
metabolites (5, 6), all of which could finally lead to DNA
modifications, but their relationship to the carcinogenic-
ity of PAH is less clear. These alternative activation
mechanisms can best be studied in genotoxic PAH
1
Abbreviations: anthanthrene, dibenzo[def,mno]chrysene; 4,5-di-
hydrodiol, trans-4,5-dihydroxy-4,5-dihydroanthanthrene; EtOH, etha-
nol; 4,5-oxide, 4,5-epoxy-4,5-dihydroanthanthrene; 1-phenol, 1-hy-
droxyanthanthrene (other phenols are similarly designated); MeOH,
methanol; 4,5-quinone, anthanthrene 4,5-quinone (other quinones are
similarly designated); PAH, polycyclic aromatic hydrocarbon(s); PAPS,
adenosine 3′-phosphate 5′-phosphosulfate; TCPO, 1,1,1-trichloro-2-
propene oxide; UDPAG, uridine 5′-diphospho-N-acetylglucosamine;
UDPGA, uridine 5′-diphosphoglucuronic acid.
10.1021/tx010131t CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/06/2002

Metabolism and Mutagenicity of Anthanthrene
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 333
(v/v) aqueous MeOH to 100% MeOH within 50 min; flow rate:
0.8 mL/min]. The fraction of the eluate containing anthanthrene
was collected and brought to dryness with a stream of N2,
resulting in a radiochemical purity of 98%.
lacking a bay-region: The hexacyclic aromatic hydrocar-
bon dibenzo[def,mno]chrysene (anthanthrene), where the
bay-region of benzo[a]pyrene is bridged by an additional
aromatic ring (Scheme 1), is such an example. An-
thanthrene occurs in relatively high concentration in
coal-tar and in all environmental samples containing
PAH (7). Anthanthrene was found to be mutagenic in
bacterial short-term tests (8-11). It exhibited skin
carcinogenicity after long-term topical application (12) or
by employing the initiation-promotion protocol to mice
(13) as well as lung carcinogenicity after pulmonal
implantation in rats (14), while in several other in vivo
studies anthanthrene did not show any carcinogenic
activity (7). Hence, after evaluation of the experimental
data, the International Agency for Research on Cancer
concluded that ‘there is limited evidence that anthanthrene
is carcinogenic to experimental animals’ while the evi-
dence for its mutagenicity is characterized as ‘inadequate’
(7).
Syn th esis of P olyn u clea r Qu in on es of An th a n th r en e.
The three polynuclear quinones of anthanthrene were synthe-
sized by oxidation of the parent hydrocarbon with sodium
dichromate (17). Subsequent separation of the crude mixture
was achieved on Florisil by sequential elution with toluene/ethyl
acetate (98:2, then 9:1, v/v) and chloroform/acetone (9:1, v/v).
Several fractions were obtained with enriched mixtures of
anthanthrene 6,12-, 3,6- and 1,6-quinone (eluting in this order)
representing an overall yield of 87%. The pure isomers were
obtained by several rechromatographic steps (HPLC: LiChro-
spher RP18, 5 µm, 4 × 250 mm; 100% MeOH, 1.0 mL/min).
(1) Anthanthrene 6,12-quinone: 1H NMR (THF-d8) δ 8.73 (d,
2H, H1,7, J 1,2 ) J 7,8 ) 7.4 Hz), 8.73 (d, 2H, H5,11, J 4,5 ) J 10,11
)
8.6 Hz), 8.38 (d, 2H, H3,9, J 2,3 ) J 8,9 ) 7.4 Hz), 8.25 (d, 2H, H4,10),
7.94 (pseudo-t, 2H, H2,8); UV/vis (EtOH) λmax (nm) (ꢀ [M-1 cm-1]):
225 (21 800), 274 (16 500), 354 (6600), 446 (4400), 472 (5000);
EI-MS m/z (rel intensity, %) 307 (24) [MH]+, 306 (100) [M]+,
278 (15) [M - CO]+, 250 (17) [M - 2 × CO]+.
We, therefore, wished (i) to study the bacterial mu-
tagenicity of anthanthrene in more detail and investigate
its enzymatic control as well as (ii) to elucidate its
hitherto unknown biotransformation in order to reveal
the metabolic pathway(s) leading to genotoxicity.
(2) Anthanthrene 3,6-quinone: 1H NMR (CD2Cl2) δ 8.87 (d,
1H, H5, J 4,5 ) 7.8 Hz), 8.76 (dd, 1H, H7, J 7,8 ) 6.2 Hz, J 7,9 ) 1.2
Hz), 8.69 (d, 1H, H4), 8.33 (d, 1H, H9, J 8,9 ) 8.0 Hz), 8.19 (s, 1H,
H12), 8.10 (AB-system, 2H, H10,11, J 10,11 ) 8.6 Hz), 7.91 (pseudo-
t, 1H, H8), 7.90 (d, 1H, H1, J 1,2 ) 9,9 Hz), 6.75 (d, 1H, H2); UV/
vis (MeOH) λmax (nm) (ꢀ [M-1 cm-1]): 207 (47 900), 246 (20 400),
331 (18 400), 470 (5300); EI-MS m/z (rel intensity, %) 307 (27)
[MH]+, 306 (100) [M]+, 278 (47) [M - CO]+, 250 (33) [M - 2 ×
CO]+.
Exp er im en ta l P r oced u r es
Ch em ica ls. 3-Methylcholanthrene, the methoxy tetralones,
and also reagents for the synthetic procedures were supplied
by Sigma-Aldrich (Taufkirchen, Germany). Aroclor 1254 was
obtained from Bayer (Leverkusen, Germany), sodium phenobar-
bital from Synopharm (Barsbuettel, Germany), trioctanoin from
Sigma (Taufkirchen, Germany), and 1,1,1-trichloro-2-propene
oxide (TCPO) from EGA (Steinheim, Germany). Biochemicals
were from Roche Diagnostics (Mannheim, Germany); solvents
for HPLC were from Baker (Gross-Gerau, Germany); all other
chemicals of analytical grade were purchased from Merck
(Darmstadt, Germany).
(3) Anthanthrene 1,6-quinone: 1H NMR (THF-d8) δ 9.05 (s,
1H, H12), 8.77 (d, 1H, H5, J 4,5 ) 7.6 Hz), 8.76 (dd, 1H, H7, J 7,8
)
7.3 Hz, J 7,9 ) 1.2 Hz), 8.44 (dd, 1H, H9, J 8,9 ) 7.9 Hz), 8.30 (AB-
system, 2H, H10,11, J 10,11 ) 8.8 Hz), 8.10 (d, 1H, H4), 7.98 (pseudo-
t, 1H, H8), 7.96 (d, 1H, H3, J 2,3 ) 9.8 Hz), 6.75 (d, 1H, H2); UV/
vis (MeOH) λmax (nm) (ꢀ [M-1 cm-1]): 207 (43 600), 310 (7100),
322 (7600), 434 (5700); EI-MS m/z (rel intensity) 307 (27) [MH]+,
306 (100) [M]+, 278 (33) [M - CO]+, 250 (28) [M - 2 × CO]+.
Syn th esis of th e K-Region Der ivatives of An th an th r en e.
Anthanthrene was converted to the 4,5-osmate ester, hydrolyzed
to the cis-4,5-dihydrodiol, and oxidized with 2,3-dichloro-5,6-
dicyano-p-benzoquinone (18), yielding anthanthrene 4,5-quinone
Syn th esis of An th a n th r en e. Anthanthrene was obtained
(15) by a zinc dust melt from the commercially available 6,12-
quinone (TCI, Tokyo, J apan) after purification in benzene over
Florisil (60-100 mesh, Promochem, Wesel, Germany) and twice
recrystallization from toluene in 39% yield and 99% purity
(HPLC: LiChrospher RP18, 5 µm, 4 × 250 mm; 100% MeOH,
0.8 mL/min): mp >250 °C [lit.: 264 °C (16)]; 1H NMR (CD2Cl2)
δ 8.82 (s, 2H, H6,12), 8.56 (d, 2H, H1,7, J 1,2 ) J 7,8 ) 8.1 Hz), 8.24
(d, 2H, H3,9, J 2,3 ) J 8,9 ) 6.9 Hz), 8.18 (d, 2H, H5,11, J 4,5 ) J 10,11
) 9.1 Hz), 8.16 (pseudo-t, 2H, H2,8), 8.10 (d, 2H, H4,10); UV/vis
(EtOH) λmax (nm) (ꢀ [M-1 cm-1]): 210 (36 200), 232 (82 200), 258
(33 700), 293 (36 400), 306 (69 700), 381 (10 900), 400 (25 800),
405 (28 000), 421 (35 000), 429 (48 700); EI-MS m/z (rel inten-
1
(11%): mp >250 °C; H NMR (CDCl3) δ 8.90 (s, 1H, H6), 8.61
(dd, 1H, H3, J 2,3 ) 7.2 Hz, J 1,3 )1.2 Hz), 8.42 (dd, 1H, H1, J 1,2
8.2 Hz, J 1,3 ) 0.8 Hz), 8.34 (s, 1H, H12), 8.20 (d, 1H, H7, J 7,8
)
)
7.7 Hz), 8.11 (d, 1H, H9, J 8,9 ) 7.0 Hz), 7.95 (pseudo-t, 1H, H8),
7.79-7.86 (m, 3H, H2, H10, H11); UV/vis (EtOH) λmax (nm) (ꢀ [M-1
cm-1]): 204 (37 100), 250 (54 400), 285 (42 100), 381 (14 800),
401 (15 600); EI-MS m/z (rel intensity, %) 307 (21) [MH]+, 306
(81) [M]+, 279 (24) [MH - CO]+, 278 (100) [M - CO]+, 250 (36)
[M - 2 × CO]+.
The 4,5-quinone was reduced with NaBH4 in EtOH in the
presence of oxygen (19) to trans-4,5-dihydroxy-4,5-dihydro-
anthanthrene (70%): mp 222-224 °C (dec); 1H NMR (acetone-
d6/Me2SO-d6) δ 8.67 (s, 1H, H12), 8.57 (s, 1H, H6), 8.37 (d, 1H,
H7, J 7,8 ) 7.6 Hz), 8.27 (d, 1H, H9, J 8,9 ) 8.2 Hz), 8.18 (d, 1H,
H1, J 1,2 ) 7.2 Hz), 8.08 (AB-system, 2H, H10,11, J 10,11 ) 9.2 Hz),
8.06-8.02 (m, 2H, H3,8), 7.84 (dd, 1H, H2, J 2,3 ) 8.1 Hz), 5.21
(d, 1H, H4, J 4,5 ) 9.6 Hz), 5.17 (d, 1H, H5); UV/vis (EtOH) λmax
(nm) (ꢀ [M-1 cm-1]): 212 (27 900), 232 (44 200), 266 (37 100),
291 (38 700), 303 (46 400), 349 (11 000), 367 (22 000), 388
(25 400); EI-MS m/z (rel intensity, %) 311 (27) [MH]+, 310 (100)
[M]+.
sity, %) 277 (23) [MH]+, 276 (100) [M]+, 138 (28) [M]2+
.
For the preparation of [G-3H]anthanthrene by tritium ex-
change (Amersham Buchler, Braunschweig, Germany), the
parent hydrocarbon was dissolved in dioxane and treated with
3H2O (925-1850 GBq [25-50 Ci]/mmol) in the presence of an
Al/Rh catalyst. After removal of labile tritium, the raw product
(specific activity, 174 GBq [4.7 Ci]/mmol) was dissolved in
toluene and kept at -20 °C. The tritium distribution within the
anthanthrene molecule was determined from the tritium NMR
spectrum: 3H NMR (CDCl3, 41 800 scans) δ 8.87 (s, 2H, H6,12),
8.61 (s, 2H, H1,7), 8.29 (s, 2H, H3,9), 8.23 (s, 2H, H5,11), 8.21 (s,
2H, H2,8), 8.15 (s, 2H, H4,10). Integration of the signals indicated
a rather uniform tritium distribution with 25.1% at H2,8, 19.0%
at H4,10, 15.7% at H5,11, 14.1% at H6,12, 13.4% at H1,7, and 12.7%
The trans-4,5-dihydrodiol was cyclized with the dimethyl
acetal of dimethylformamide (20), furnishing anthanthrene 4,5-
1
oxide (47%): mp 185 °C (dec); H NMR (CDCl3) δ 8.56 (s, 1H,
at H3,9
.
H6), 8.54 (s, 1H, H12), 8.30 (d, 1H, H7 or H9, J ) 8.0 Hz), 8.28
(d, 1H, H9 or H7, J ) 7.7 Hz), 8.12 (d, 1H, H1, J 1,2 ) 7.5 Hz),
8.04 (d, 1H, H3, J 2,3 ) 7.3 Hz), 8.01 (pseudo-t, 1H, H8), 7.96 (AB-
system, 2H, H10,11, J 10,11 ) 9.1 Hz), 7.80 (pseudo-t, 1H, H2), 5.02
(d, 1H, H4, J 4,5 ) 3.8 Hz), 4.94 (d, 1H, H5); UV/vis (MeOH) λmax
Prior to use as substrate in microsomal incubations, an
aliquot of the stock solution was evaporated, and the crude
product was dissolved in Me2SO (50 µL) and purified by HPLC
[stationary phase as above; mobile phase: linear gradient of 65%

334 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Platt et al.
(nm) (ꢀrel): 227 (0.81), 264 (0.76), 288 (0.82), 299 (1.00), 367
(0.46), 386 (0.53), 408 (0.05); EI-MS m/z (rel intensity, %) 293
(24) [MH]+, 292 (100) [M]+, 263 (42) [M - CHO]+.
Syn th esis of th e L-Region Der iva tives of An th a n th r en e.
Vilsmeier formylation of anthanthrene according to Buu-Hoi and
Lavit (21) yielded the 6-aldehyde (45%) which was reduced with
(rel intensity, %) 293 (24) [MH]+, 292 (100) [M]+, 263 (36) [M -
CHO]+.
3-Hydroxyanthanthrene was prepared according to the syn-
thetic pathway described above utilizing 7-methoxy-1-tetralone
as starting material (overall yield 0.5%): mp >250 °C; 1H NMR
(acetone-d6) δ 9.78 (s, 1H, OH), 8.81 (s, 1H, H6 or H12), 8.75 (s,
1H, H12 or H6), 8.53-8.48 (m, 2H, H1,7), 8.42 (d, 1H, H9, J 8,9
)
NaBH4 to 6-hydroxymethylanthanthrene (93%): mp 212 °C
1
9.3 Hz), 8.17-8.04 (m, 5H, H4,5,8,10,11), 7.88 (d, 1H, H2, J 1,2 ) 8.6
Hz); UV/vis (MeOH) λmax (nm) (ꢀrel) 212 (0.60), 234 (1.00), 312
(0.91), 389 (0.22), 410 (0.47), 433 (0.30), 461 (0.47); EI-MS m/z
(rel intensity, %) 293 (24) [MH]+, 292 (100) [M]+, 263 (51) [M -
CHO]+.
(dec); H NMR (acetone-d6/Me2SO-d6) δ 9.08 (d, 1H, H7, J 7,8
)
8.3 Hz), 9.00 (s, 1H, H12), 8.74 (d, 1H, H5, J 4,5 ) 9.5 Hz), 8.66
(d, 1H, H9, J 8,9 ) 8.0 Hz), 8.39-8.36 (m, 2H, H1,3), 8.30-8.20
(m, 5H, H2,4,8,10,11), 8.06-8.02 (m, 2H, H3,8), 7.84 (dd, 1H, H2,
J 2,3 ) 8.1 Hz), 5.21 (d, 1H, H4, J 4,5 ) 9.6 Hz), 5.81 (s, 2H, -CH2-
); UV/vis (EtOH) λmax (nm) (ꢀ [M-1 cm-1]): 211 (33 600), 234
(72 300), 258 (29 200), 296 (34 600), 308 (71 100), 388 (10 900),
412 (30 000), 424 (19 600), 436 (54 700); EI-MS m/z (rel inten-
sity, %) 307 (25) [MH]+, 306 (100) [M]+, 289 (80) [MH - H2O]+,
288 (9) [M - H2O]+, 287 (23) [M - H2O - H]+, 275 (24) [MH -
CH3OH]+, 274 (28) [M - CH3OH]+.
P r ep a r a tion of Su bcellu la r F r a ction s. Adult male Spra-
gue-Dawley rats (200-240 g; Interfauna Sueddeutsche Ver-
suchstierfarm, Tuttlingen, Germany) were treated ip either with
Aroclor 1254 (500 mg/kg body weight at day 6 before sacrifice),
with 3-methylcholanthrene (40 mg/kg body weight at days 3, 2,
and 1 before sacrifice), or with sodium phenobarbital (80 mg/
kg body weight at days 3, 2, and 1 before sacrifice), or left
untreated. Aroclor 1254 and 3-methylcholanthrene were dis-
solved in trioctanoin (2.5 mL/kg body weight), sodium phenobar-
bital in aqueous NaCl (0.9%; 2.5 mL/kg body weight). The
postmitochondrial fraction, i.e., the 9000g supernatant, as well
as the microsomal fraction of the liver was prepared as previ-
ously described (25) under sterile conditions. Protein concentra-
tions were determined by the method of Lowry et al. (26) using
bovine serum albumin for calibration. The P450 content was
measured according to the procedure of Omura and Sato (27).
Wolff-Kishner reduction of 6-formylanthanthrene afforded
6-methylanthanthrene (83%): mp 184 °C [lit.: 192 °C (21)]; 1H
NMR (CDCl3) δ 8.74 (dd, 1H, H7, J 7,8 ) 8.2 Hz, J 7,9 ) 0.9 Hz),
8.72 (s, 1H, H12), 8.48 (d, 1H, H9, J 8,9 ) 7.9 Hz), 8.45 (d, 1H, H5,
J 4,5 ) 9.5 Hz), 8.22-8.04 (m, 7H, H1,2,3,4,6,8,11), 3.35 (s, 3H, -CH3);
UV/vis (EtOH) λmax (nm) (ꢀ [M-1 cm-1]): 211 (31 200), 234
(61 900), 258 (26 800), 296 (30 000), 308 (39 300), 389 (9500),
412 (23 500), 437 (32 300); EI-MS m/z (rel intensity, %) 291 (24)
[MH]+, 290 (100) [M]+.
Syn th esis of Non -K-Region P h en ols of An th a n th r en e.
The synthesis of 1-hydroxy-, 2-hydroxy-, and 3-hydroxyan-
thanthrene (Supporting Information: Scheme S1) will be pub-
lished in full detail elsewhere. In brief, 1-hydroxyanthanthrene
was prepared from 5-methoxy-1-tetralone that was subjected
to a Reformatsky reaction with ethyl bromoacetate, followed by
dehydration, aromatization, and hydrolysis, resulting in 5-meth-
oxy-1-naphthylacetic acid. This compound was converted with
methyl 3-formylbenzoate via a Perkin reaction to R-(5-methoxy-
1-naphthyl)-3-methoxycarbonylcinnamic acid which was trans-
formed to its methyl ester. Oxidative photocyclization (22, 23)
led to a mixture of 5,8-bismethoxycarbonyl- and 5,10-bis-
methoxycarbonyl-1-methoxychrysene, the former being much
less soluble in MeOH than the latter. Thus, a considerable
enrichment of 5,10-bismethoxycarbonyl-1-methoxychrysene was
achieved by rinsing the crude mixture with MeOH. Subsequent
chromatography on silica gel eluting with CHCl3 yielded pure
5,10-bismethoxycarbonyl-1-methoxychrysene which was reduced
to the benzylic alcohol with LiAlH4. 5,10-Bishydroxymethyl-1-
methoxychrysene was converted by oxidation with pyridinium
dichromate to 5,10-diformyl-1-methoxychrysene. This aldehyde
was reacted under phase-transfer conditions with trimethyl-
sulfonium iodide to furnish 5,10-bisepoxyethyl-1-methoxychry-
sene. Treatment of this bisoxiranyl compound with boron
trifluoride etherate (24) resulted in the formation of 1-meth-
oxyanthanthrene. Cleavage of the methoxy group with boron
tribromide furnished 1-hydroxyanthanthrene (overall yield
0.5%): 1H NMR (acetone-d6) δ 9.18 (s, 1H, H12), 8.76 (s, 1H,
H6), 8.52 (d, 1H, H7, J 7,8 ) 7.8 Hz), 8.25 (d, 1H, H4, J 4,5 ) 9.3
Hz), 8.22 (d, 1H, H9, J 8,9 ) 7.2 Hz), 8.15 (d, 1H, H3, J 2,3 ) 8.0
Hz), 8.13 (pseudo-t, 1H, H8), 8.12 (d, 1H, H5), 8.01 (AB-system,
2H, H10,11, J 10,11 ) 9.2 Hz), 7.70 (d, 1H, H2, J 2,3 ) 8.0 Hz); UV/
vis (MeOH) λmax (nm) (ꢀrel): 213 (0.84), 233 (1.00), 312 (0.86),
405 (0.31), 430 (0.40), 450 (0.39).
Meta bolism Stu d ies. Microsomal incubations contained
liver microsomes equivalent to 4-5 nmol of P450, 0.6 mM
NADP+, 8 mM glucose 6-phosphate, 1.5 units of glucose-6-
phosphate dehydrogenase, and 5 mM MgCl2 in a final volume
of 2 mL of 50 mM isotonic (150 mM KCl) sodium phosphate
buffer (pH 7.4). In some cases, the incubation mixture contained
1 mM TCPO in 10 µL of acetone. This mixture was preincubated
for 5 min at 37 °C. The incubation was started by the addition
of 80 µM unlabeled or tritium-labeled anthanthrene (0.8-9.1
GBq [22-246 mCi]/mmol), 3-hydroxyanthanthrene (80 µM), or
anthanthrene 4,5-oxide (25 µM) in 50 µL of Me2SO and
continued with shaking (80 min-1) at 37 °C. The incubation was
stopped with 2 mL of ice-cold ethyl acetate. After being vortexed
for 1 min, the organic and aqueous phases were separated by
centrifugation at 1500g and 20 °C for 5 min. The extraction was
repeated twice more with 2 mL of ethyl acetate. The organic
phases were combined, dried over anhydrous magnesium sul-
fate, and brought to dryness at 30-35 °C with a stream of
nitrogen; then, the residue was stored at -70 °C until HPLC
separation was performed, for which the sample was dissolved
in 30 µL of Me2SO.
All steps were performed under subdued light. The recovered
radioactivity in the organic and aqueous phase was usually
>95% of that applied.
Isola tion of Acetyla ted Meta bolites of An th a n th r en e.
Anthanthrene (90 µM) was incubated (80 min) as above in a
final volume of 10 mL containing liver microsomes equivalent
to 29 nmol of P450 of rats pretreated with Aroclor 1254. The
incubation was stopped with 10 mL of ice-cold acetone and
extracted with dichloromethane (25 mL). The organic phase was
dried over anhydrous magnesium sulfate, concentrated to a
volume of 100 µL, treated with 1.5 mL of a mixture of acetic
anhydride/pyridine (1:2, v/v) for 16 h at room temperature, and
subsequently brought to dryness with a stream of nitrogen. The
residue was dissolved in Me2SO and separated by HPLC
[stationary phase: LiChrospher 100 RP-18, 5 µm, 250 × 4 mm;
mobile phase: linear gradient of MeOH/H2O (60:40, v/v) to 100%
MeOH in 40 min; flow rate: 0.8 mL/min; detection wave-
length: 254 nm]. Prominent peaks were collected and further
purified by TLC on silica gel (60 F254; Riedel-de-Hae¨n, Seelze,
Germany) with chloroform as the mobile phase. Bands visible
under UV light were scraped off, eluted with acetone and
dichloromethane (5 × 200 µL, each), and subjected to mass
spectrometry (see below).
2-Hydroxyanthanthrene was prepared according to the syn-
thetic pathway described above utilizing 6-methoxy-1-tetralone
as starting material (overall yield 2.0%): mp 228-230 °C (dec);
1H NMR (acetone-d6/Me2SO-d6) δ 8.92 (s, 1H, H6 or H12), 8.78
(s, 1H, H12 or H6), 8.60 (d, 1H, H7, J 7,8 ) 8.0 Hz), 8.30 (d, 1H,
H9, J 8,9 ) 7.2 Hz), 8.18 (AB-system, 2H, H10,11, J 10,11 ) 9.2 Hz),
8.16 (AB-system, 2H, H4,5, J 4,5 ) 9.2 Hz), 8.15 (pseudo-t, 1H,
H8), 7.99 (d, 1H, H1 or H3, J ) 2.1 Hz), 7.87 (d, 1H, H3 or H1, J
) 2.1 Hz); UV/vis (EtOH) λmax (nm) (ꢀ [M-1 cm-1]): 213 (35 900),
236 (49 100), 269 (29 200), 295 (29 400), 309 (23 500), 321
(22 400), 410 (19 500), 425 (21 200), 440 (15 900); EI-MS m/z

Metabolism and Mutagenicity of Anthanthrene
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 335
HP LC An a lysis of An th a n th r en e Meta bolites. Chromato-
graphic separations were performed with a system consisting
of a ternary high-pressure pump (SP 8700; Spectra-Physics,
Darmstadt, Germany), a sample injection valve (C6W; Valco,
Schenkon, Switzerland) with a 25 µL sample loop, and a diode
array detector (1100 series; Agilent Technologies, Karlsruhe,
Germany) operated with LC 3D ChemStation A.08.03 software
(Agilent Technologies) connected in line with a radioactivity
flow-through detector (Ramona-LS, Raytest, Straubenhardt,
Germany) operated with Chromasoft 0.3 software (Raytest).
For the chromatographic separation, 20 µL of the solution of
the metabolites in Me2SO was injected onto a Vertex column (4
× 250 mm; Knauer, Berlin) filled with LiChrospher 100 RP-18
(5 µm; Merck, Darmstadt, Germany) as the stationary phase.
The mobile phaseskept at 1.5 bar under an atmosphere of
heliumsconsisted of a mixture of MeOH and 10 mM aqueous
NH4HCO3 (pH 8.0), with a linear increase in MeOH content
from 65 to 100% (v/v) in 50 min followed by 100% MeOH for 20
min (separation of the metabolites of anthanthrene and of the
4,5-oxide) or of a mixture of MeOH/H2O with a linear increase
in MeOH content from 60 to 100% (v/v) in 40 min (separation
of the metabolites of 3-phenol) at a flow rate of 0.8 mL/min.
Anthanthrene-1,6- and 3,6-quinone coeluted under these condi-
tions but could be separated employing Spherisorb S 5 ODS 2
as stationary and MeOH/H2O as mobile phase with the linear
increase in MeOH content and the flow rate as above. For the
detection of radioactivity, the eluate was mixed in the radio-
activity detector with liquid scintillator (Quickszint Flow 302,
Zinsser Analytik, Frankfurt, Germany) at a ratio of 1:3.
F igu r e 1. Bacterial mutagenicity in different strains of S.
typhimurium of anthanthrene and benzo[a]pyrene metabolically
activated with the postmitochondrial fraction of rats pretreated
with Aroclor 1254. For details, see Experimental Procedures.
Each bar represents the mean ( SD of three experiments.
Qu a n tifica ton of Meta bolic Con ver sion . Total metabolic
conversion was calculated from the sum of the radioactivity
eluting upon HPLC before and after anthanthrene and of the
radioactivity remaining in the aqueous phase after ethyl acetate
extraction divided by the specific activity, S, of the substrate.
The amount of distinct metabolites, m, was determined from
the radiochromatogram by the formula: m ) (ROI‚A)/(100‚S),
where ROI (region of interest) is the percentage of radioactivity
in the peak of the metabolite as compared to the total radioac-
tivity in the radiochromatogram and A is the total radioactivity
in the organic phase.
Sp ectr a l Meth od s. UV/vis absorption spectra of the syn-
thetic derivatives of anthanthrene were obtained on a Shimadzu
MPS-2000 spectrophotometer. UV/vis spectra of metabolites and
of synthetic reference compounds were recorded on-line during
HPLC with the diode array detector (cell volume: 13 µL; path
length: 10 mm; peak width: >0.2 min; slit: 2 nm). Electron
ionization (EI) mass spectra were recorded with a Varian MAT
CH 7A spectrometer at 70 eV. Proton NMR spectra were
measured on a Bruker AM 400 spectrometer at 400 MHz.
Chemical shifts (in ppm) are relative to tetramethylsilane.
Deuterated solvents were used as indicated for each compound.
F igu r e 2. Bacterial mutagenicity in S. typhimurium TA98 and
TA100 of anthanthrene metabolically activated with the post-
mitochondrial fraction of rats pretreated with different enzyme
inducers. For details, see Experimental Procedures. Each bar
represents the mean ( SD of three experiments.
TA98, TA100, and TA104 in the range of 18-40 his+-
revertant colonies/nmol (Figure 1). This mutagenic effect
amounted to 44-84% of the values observed for benzo-
[a]pyrene under the same conditions (Figure 1).
Mu ta gen icity Stu d ies. The mutagenicity experiments were
performed as described by Ames et al. (28) with minor modifica-
tions (29). The auxotrophic strains TA97, TA98, TA100, and
TA104 of S. typhimurium were generously provided by Dr. B.
N. Ames, Berkeley, CA. The source and preparation of the
postmitochondrial liver fraction are described above. The specific
mutagenicity (his+-revertant colonies/nmol) was calculated as
the slope of the linear part of the dose-response curve.
The bacterial mutagenicity of anthanthrene is strongly
dependent on the enzymatic composition of the post-
mitochondrial fraction as demonstrated with different
enzyme inducers in strains TA98 and TA100 (Figure 2):
The postmitochondrial fraction of untreated animals
yielded just 5-6 his+-revertant colonies/nmol; treatment
of rats with phenobarbital led to an 1.4-2.7-fold increase
in mutagenicity whereas treatment with 3-methylcholan-
threne or Aroclor 1254, a mixture of polychlorinated
biphenyls, raised the specific mutagenicity 3.6-4.6-fold
to 18-27 his+-revertant colonies/nmol.
Resu lts
Ba cter ia l Mu ta gen icity of An th a n th r en e. As in the
case of benzo[a]pyrene and of other tumorigenic PAH,
anthanthrene did not revert strains TA97, TA98, TA100,
and TA104 of S. typhimurium to histidine prototrophy
without additional metabolic activation (data not shown).
However, in the presence of the hepatic postmitochon-
drial fraction of Sprague-Dawley rats treated with
Aroclor 1254 and an NADPH-generating system, an-
thanthrene was converted to mutagens for strains TA97,
Id en tifica tion of Micr osom a l Meta bolites of An -
th a n th r en e. [G-3H]Anthanthrene (80 µM) was incubated
with liver microsomes of differently treated Sprague-
Dawley rats equivalent to 2.5 nmol of P450/mL in the

336 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Platt et al.
panels G, H) could be demonstrated. The ratio of meta-
bolically formed 1,6-/3,6-quinone was determined to be
1:1.5.
An indication for the identity of A2 and A3, prominent
metabolites in the case of microsomes of 3-methylcholan-
threne-treated animals (Figure 3B), was obtained by
comparison with the microsomal metabolites P1-P10 of
the 3-phenol (Supporting Information: Figure S4). A2
and A3 coelute with P7 and P8, respectively (Table 1),
and exhibit identical UV/vis spectra (Supporting Infor-
mation: Figure S2, panels D, E). Thus, it is rather certain
that A2 and A3 contain a hydroxyl group in the 3-posi-
tion. With respect to their polarity, A2 and A3 could
represent diphenols. This was further corroborated by
acetylation of the isolated mixture of metabolites followed
by HPLC as well as TLC separation as described under
Experimental Procedures. The HPLC fraction at 33-35
min resulted in two bands with Rf ) 0.41 and Rf ) 0.27
upon TLC. The compound of the first band yielded the
mass spectrum of Figure 4, which is typical for di-
acetoxyanthanthrene as depicted on the spectrum. The
second band led to a mass spectrum with the molecular
ion at m/z ) 306 and sequential loss of two molecules of
CO, leading to the fragment ions at m/z ) 278 and m/z
) 250 typical for quinones. Finally, an HPLC peak at
43.4 min exhibited a mass spectrum with the molecular
ion at m/z ) 334, loss of ketene leading to m/z ) 292,
and further loss of the fragment CHO to m/z ) 263, a
fragmentation pattern characteristical for a phenol ac-
etate. Thus, the existence of quinones as well as phenols
as metabolites of anthanthrene was additionally con-
firmed by this mass spectroscopic investigation.
Since A1a is metabolically formed from the 4,5-oxide
(metabolite E2) as well as from the 3-phenol (metabolite
P3; cf. Table 1), its identity with 4,5,9-trihydroxy-4,5-
dihydroanthanthrene is almost certain. An additional
indication that metabolites A1a, A1b, and A1 are dihy-
drodiols whereas metabolite A7 is an epoxide stems from
their metabolic behavior in the presence of TCPO (1 mM),
a potent inhibitor of microsomal epoxide hydrolase (30):
The formation of A1a, A1b, and A1 is considerably
depressed, in contrast to A7, the amount of which more
than doubles, while the remaining metabolites are only
marginally affected (Table 1).
F igu r e 3. Radiochromatograms of the ethyl acetate-extractable
metabolites of [G-3H]anthanthrene incubated with liver mi-
crosomes of rats treated with phenobarbital (A) or 3-methyl-
cholanthrene (B). For details, see Experimental Procedures.
Roman numerals represent metabolites of anthanthrene (cf.
Table 1).
presence of an NADPH-generating system for 20 min
followed by ethyl acetate extraction of lipophilic metabo-
lites. Reverse-phase HPLC separation of the extracts
yielded the radiochromatograms of Figure 3A after
phenobarbital and of Figure 3B after 3-methylcholan-
threne treatment of the animals. In these chromato-
grams, 10 peaks, A1-A10, could be distinguished which
were formed consistently, however in different amounts
depending on the induction state. Metabolite A7 (Figure
3A) could only be detected when the aqueous portion of
the mobile phase during HPLC separation consisted of
NH4HCO3 solution (10 mM, pH 8) instead of pure water.
Two peaks, A1a and A1b, eluting in the polar region of
the chromatogram occurred somewhat erraticly often
obscured by the radioactivity of tritiated water originat-
ing from [G-3H]anthanthrene via formation of quinones.
Similarly, metabolite A4a (Figure 3A) was not consis-
tently formed.
The identification of most of the ethyl acetate-extract-
able metabolites of anthanthrene was achieved by a
combination of (i) chromatographic, (ii) spectroscopic, and
(iii) biochemical methods (Table 1). Metabolites A1, A7,
A8, A9, and A10 (Figure 3A) coeluted with the 4,5-
dihydrodiol, 4,5-oxide, 1-phenol, 3-phenol, and 6,12-
quinone, respectively; the UV/vis spectra of the coeluting
compounds were identical (Supporting Information: Fig-
ure S2, panels C, I, K, L, M). The metabolic formation of
the 2-phenol, which is not well separated from the
3-phenol, could be excluded on the basis of different UV/
vis spectra (Supporting Information: Figure S2, panel
L). Metabolic conversion of synthetic 4,5-oxide with
hepatic microsomes from phenobarbital-treated rats re-
sulted in 10 peaks (E1-E10; Supporting Information:
Figure S3) of which E2, E3, E5, and E6 coeluted with
A1a, A1b, A1, and A4a (Table 1), exhibiting the same UV/
vis spectra (Supporting Information: Figure S2, panels
A, B, C, F). Due to their polarity and their origin from
the 4,5-oxide, metabolites A1a and A1b probably contain
more than two hydroxyl groups: in addition to the 4,5-
dihydrodiol moiety, perhaps one or two phenolic hydroxyl
groups. Metabolite A4a is very likely the 4,5-quinone
since it has the same UV/vis spectrum as the synthetic
compound (Supporting Information: Figure S2, panel F).
Metabolites A5 and A6 coeluted on LiChrospher 100
RP-18 (Figure 3) but could be well separated (R ) 1.05)
by employing Spherisorb ODS 2 as the stationary phase.
Under these conditions, coelution of A5 and A6 with the
1,6- and 3,6-quinone, respectively, as well as identity of
their UV/vis spectra (Supporting Information: Figure S2,
Neither chromatographic nor UV/vis spectroscopic
evidence for the microsomal formation of 6-methyl- or
6-hydroxymethylanthanthrene could be found under the
metabolic conditions employed.
In flu en ce of En zym e In d u cer s on th e Meta bolic
Con ver sion of An th a n th r en e. A remarkable influence
of the induction state of the metabolizing system on the
bacterial mutagenicity of anthanthrene is demonstrated
in Figure 2. The significance of enzyme induction is also
reflected by the qualitatively as well as quantitatively
different formation of microsomal metabolites (Figure 3).
Hence, a quantitative assessment of the metabolic con-
version of anthanthrene by hepatic microsomes of un-
treated rats and of rats treated with phenobarbital,
3-methylcholanthrene, and Aroclor 1254 was undertaken
(Table 2). Of the amount of 160 nmol of anthanthrene
applied, only 5.7% was metabolically converted in 20 min
by microsomes of untreated rats containing 4 nmol of
P450. Petreatment of the animals with phenobarbital
raised the metabolic conversion 1.5-fold, while it is more
than doubled in the case of 3-methylcholanthrene as well
as Aroclor 1254 as compared to the untreated control.

Metabolism and Mutagenicity of Anthanthrene
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 337
Ta ble 1. Cr iter ia of Id en tity betw een Micr osom a l Meta bolites of An th a n th r en e, 3-Hyd r oxya n th a n th r en e, An th a n th r en e
4,5-Oxid e, a n d of Syn th etic Der iva tives of An th a n th r en e
microsomal metabolites of
3-hydroxy-
anthanthrenea,c,e
no.
anthanthrene
4,5-oxideb,d,e
no.
synthetic
derivatives of
anthanthrene
corrected
retention
change in
presence of
TCPOd,h (%)
anthanthrenea,c
no.
identity of
timef (min)
UV spectrag
A1a
A1b
A1
P3
E2
E3
E5
10.5
11.6
24.1
29.2
30.3
32.9
37.9
39.7
41.5
41.6
43.4
47.2
48.4
48.5
51.2
64.7
69.8
yes
yes
yes
yes
yes
16
8
trans-4,5-dihydrodiol
4
A2
P7
P8
112
69
A3
A4
A4a
E6
4,5-quinone
6-hydroxymethyl
1,6-quinone
3,6-quinone
4,5-oxide
yes
A5
A6
A7
A8
A9
yes
yes
yes
yes
yes
126
110
250
71
P10
substrate
1-phenol
substrate
3-phenol
102
2-phenol
A10
substrate
6,12-quinone
anthanthrene
6-methyl
yes
92
a
b
80 µM substrate concentration. 25 µM substrate concentration. c Pretreatment of rats for the preparation of microsomes with Aroclor
1254 (see Table 2). Pretreatment of rats for the preparation of microsomes with phenobarbital (see Table 2). e Only metabolites were
d
tabulated that coelute with metabolites or synthetic derivatives of anthanthrene. f The retention time of different chromatographic runs
was linearly adjusted using the retention time of common metabolites; for details on chromatographic conditions, see Experimental
g
h
Procedures. Supporting Information: Figure S2. Change in the formation of metabolites of anthanthrene (80 µM) incubated in the
presence or absence of 1,1,1-trichloro-2-propene oxide (TCPO; 1 mM); mean of two experiments. For details on incubation conditions, see
Experimental Procedures.
This amount declined in the case of microsomes of
3-methylcholanthrene-treated rats to 8%, whereas the
metabolites were dominated by the diphenols A2 and A3,
which summed up to 34.7% of total metabolic conversion.
The K-region metabolites could not be detected. Of these,
the 4,5-dihydrodiol was formed in a low amount (0.8%)
when microsomes after treatment with Aroclor 1254 were
employed. Again, the diphenols A2 and A3 were the
principal metabolites with 29.4% of the total metabolic
conversion.
Ba cter ia l Mu ta gen icity of Meta bolites of An -
th a n th r en e a n d Its In flu en ce by En zym e Mod u la -
tion . As mentioned above, anthanthrene did not exhibit
bacterial mutagenicity without metabolic activation by
rat liver enzymes. This was also the case with the 4,5-
dihydrodiol and the 4,5-, 1,6-, 3,6-, and 6,12-quinones
formed as microsomal metabolites of anthanthrene and
F igu r e 4. EI mass spectrum of an acetylated diphenolic
available as synthetic compounds for determination of
metabolite of anthanthrene. For details, see Experimental
their mutagenicity in strain TA100 of S. typhimurium
Procedures.
(Figure 5). In contrast, the K-region oxide as well as the
3-phenol are directly acting strong bacterial mutagens,
whereas the 1-phenol is only weakly mutagenic without
metabolic activation (Figure 5). The specific mutagenicity
of the 3-phenol calculated as the slope of the linear part
of the dose-response curve was 120 his+-revertant
colonies/nmol. In the case of the 4,5-oxide, a similar
calculation was not possible due to lack of linearity of
the dose-response curve. Therefore, an average slope of
279 his+-revertant colonies/nmol in the dose range of 0.1-
3.5 nmol/plate was determined for the K-region oxide of
anthanthrene. The bacterial mutagenicity of the 4,5-oxide
as well as the 3-phenol dropped considerably in the
presence of the postmitochondrial fraction of Sprague-
Dawley rats treated with Aroclor 1254 whereas the
remaining derivatives of anthanthrene required enzy-
matic activation to exert mutagenicity (Figure 5). The
strongest bacterial mutagens were formed from the
1-phenol with 38 his+-revertant colonies/nmol; the mu-
tagenicity of the 4,5-dihydrodiol amounted to 14 his+-
Under this condition, the amount of metabolites extracted
by ethyl acetate equaled that of free and protein-bound
metabolites remaining in the aqueous phase due to their
hydrophilicity. The portion of lipophilic metabolites after
induction with phenobarbital and 3-methylcholanthrene
was similarly raised to about 70% and declined in the
case of Aroclor 1254 to about 60%. Large qualitative as
well as quantitative differences in the formation of
individual metabolites were observed when different
inducers had been applied. Microsomes of untreated
animals converted anthanthrene mainly to quinones and
to polar as yet unidentified metabolites. The metabolites
of anthanthrene formed with microsomes of phenobar-
bital-treated animals, on the other hand, were dominated
by the 4,5-dihydrodiol (14.4% of the total metabolic
conversion) followed by diphenol A3 (12.5%). Only under
this condition was it possible to detect the K-region
epoxide in a small amount (1.5%). About one-fifth of the
total metabolic conversion consisted of polar metabolites.

338 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Platt et al.
Ta ble 2. Meta bolic Con ver sion of An th a n th r en e (80 µM) by Liver Micr osom es of Ra ts Tr ea ted w ith Differ en t En zym e
In d u cer s
metabolite formation [nmol (4 nmol of P450)-1 (20 min)-1
(% of total metabolic conversion) with enzyme inducer
]
metabolites
no.a
(no treatment)
phenobarbital
3-methylcholanthrene
Aroclor 1254
polar metabolitesb
trans-4,5-dihydrodiol
3,?-diphenol
1.59 ( 0.79 (17.5)
0.08 ( 0.02 (0.9)
0.23 ( 0.14 (2.5)
0.12 ( 0.08 (1.3)
0.07 ( 0.13 (0.8)
0.77 ( 0.30 (8.5)
2.86 ( 0.39 (20.8)
1.98 ( 0.89 (14.4)
0.77 ( 0.24 (5.6)
1.72 ( 0.47 (12.5)
0.12 ( 0.05 (0.9)
0.87 ( 0.25 (6.3)
0.21 ( 0.06 (1.5)
0.24 ( 0.14 (1.7)
0.37 ( 0.20 (2.7)
0.47 ( 0.32 (3.4)
0.62 ( 0.47 (4.5)
1.58 ( 1.09 (8.0)
<0.01
2.71 ( 0.77 (13.3)
0.17 ( 0.10 (0.8)
3.15 ( 2.32 (15.5)
2.83 ( 1.68 (13.9)
0.27 ( 0.13 (1.3)
0.94 ( 0.31 (4.6)
A1
A2
4.06 ( 1.93 (20.5)
2.80 ( 1.42 (14.2)
2.68 ( 2.08 (13.6)
1.34 ( 0.90 (6.8)
<0.01
3,?-diphenol
A3
A4
1,6- + 3,6-quinone
4,5-oxide
1-phenol
3-phenol
6,12-quinone
A5+A6
A7
<0.01
<0.01
A8
≈0.01 (0.1)
0.04 ( 0.10 (0.2)
0.20 ( 0.22 (1.0)
0.06 ( 0.13 (0.3)
A9
0.02 ( 0.01 (0.2)
1.03 ( 0.26 (11.3)
0.56 ( 0.47 (6.1)
0.17 ( 0.28 (0.8)
c
c
A10
-
-
unknown ethyl acetate-
0.71 ( 1.31 (3.6)
1.46 ( 1.87 (7.2)
extractable metabolitesd
total ethyl acetate-
extractable metabolites
water-soluble and
4.48 ( 0.99 (49)
4.63 ( 2.59 (51)
10.23 ( 2.79 (74)
3.53 ( 0.84 (26)
13.41 ( 4.26 (68)
6.35 ( 3.66 (32)
11.76 ( 3.62 (58)
8.57 ( 3.23 (42)
protein-bound metabolites
total metabolic conversion
9.11 ( 3.21 (100)
13.76 ( 3.34 (100)
19.76 ( 5.79 (100)
20.33 ( 6.48 (100)
a
b
See Table 1. Metabolites eluting before anthanthrene 4,5-dihydrodiol (A1). c Amount often lower than in incubations without
d
microsomal protein. Calculated from the total radioactivity of the chromatographic run after subtraction of the background, of the
radioactivity of the substrate, and of the distinct metabolic peaks (A1-A10) including the polar metabolites; values represent means (
SD of 3-6 incubations.
F igu r e 5. Bacterial mutagenicity in S. typhimurium TA100 of metabolically relevant synthetic derivatives of anthanthrene in the
presence (solid symbols) or absence (open symbols) of the postmitochondrial fraction of rats pretreated with Aroclor 1254. For details,
see Experimental Procedures. Each point represents the mean ( SD of three experiments.
revertant colonies/nmol, while only weakly mutagenic
metabolites were formed from the K-region quinone as
well as from the three polynuclear quinones (3-5 his+-
revertant colonies/nmol).
The inhibition of xenobiotic metabolizing enzymes in
the Ames test allows an estimation of the significance of
these enzymes in the activation of the test compound or
its metabolites to mutagenic intermediates. 7,8-Benzofla-
vone, an inhibitor of the P450 1A subfamily (31), lowered
in a concentration of 50 µM in the top agar the mutage-
nicity of anthanthrene to one-third in strains TA98 and
TA100 (Table 3). The bacterial mutagenicity of the
3-phenol and the 4,5-oxide was also depressed in the
presence of 7,8-benzoflavone, however, only to 60-70%,
while that of the 4,5-dihydrodiol dropped to an average
of 23%.
Inhibition of the epoxide hydrolase by TCPO could only
be investigated in strain TA98 since this epoxide is
mutagenic in strain TA100. When the top agar contained
1 mM TCPO, the mutagenicity of anthanthrene and of

Metabolism and Mutagenicity of Anthanthrene
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 339
Ta ble 3. In flu en ce of In h ibitor s of P h a se I a n d Cofa ctor s of P h a se II En zym es on th e Ba cter ia l Mu ta gen icity of
An th a n th r en e a n d Som e of Its Meta bolica lly Releva n t Der iva tivesa
change (%) of specific mutagenicity in the presence of
specific
bacterial
strain
mutagenicityb
UDPGA +
ATP +
compound
anthanthrene
(his+-rev./nmol) 7,8-benzoflavonec
TCPOd
139 ( 3 (286 ( 16)h 77/72
GSHe
UDPAGf
sulfateg
TA98
TA100
TA98
TA100
TA98
TA100
TA98
TA100
18 ( 3 (7 ( 1)h
27 ( 2
33/45
33 ( 6
ndi
53/64
nd
54/88
44/72
16/30
27/45
55 ( 22
nd
16/29
nd
31/25
nd
33/33
94 ( 6
103 ( 15
nd
97/74
nd
77/137
nd
144/67
nd
70 ( 22
nd
55 ( 10
nd
46/87
nd
33/22
3-hydroxyanthanthrene
anthanthrene 4,5-oxide
anthanthrene 4,5-dihydrodiol
15 ( 5
112/202
31 ( 6
nd
5/5 (7 ( 4)h
13/8
220/224 (974/604)h
nd
5 ( 1 (4 ( 1)h
9 ( 2
178/210 (140/173)h
nd
a
b
Values are means ( SD of at least three experiments or individual results of two experiments. Metabolizing system: hepatic
postmitochondrial fraction of rats pretreated with Aroclor 1254. c 50 µM. 1,1,1-Trichloro-2-propene oxide (TCPO; 1 mM). e 5 mM. f Uridine
5′-diphosphoglucuronic acid (UDPGA; 4 mM) and uridine 5′-diphospho-N-acetylglucosamine (UDPAG; 4 mM). ATP (10 mM) and sodium
sulfate (5 mM); all concentrations are related to the top agar mixture. Metabolizing system: hepatic postmitochondrial fraction of rats
pretreated with phenobarbital. nd ) not determined.
d
g
h
i
its 3-phenol increased by about 40-60% while the mu-
tagenic effect of the K-region epoxide and the dihydrodiol
was doubled (Table 3). Treatment of the animals used
for the preparation of the hepatic postmitochondrial
fraction with phenobarbital instead of Aroclor 1254 led
to an even more pronounced effect of TCPO: The mu-
tagenicity of anthanthrene was raised nearly 3-fold and
that of the K-region epoxide up to 10-fold (Table 3).
In the standard Ames test, phase II enzymes are
usually not operative since due to dilution of the post-
mitochondrial fraction the concentration of their cofactors
falls considerably below the Km values. Hence, the
influence of conjugating enzymes on the bacterial mu-
tagenicity of a compound can be studied by adding the
necessary cofactor to the top agar. In the presence of 5
mM GSH, the mutagenic effect of anthanthrene and of
its 4,5-oxide was reduced to about 70%. The mutagenicity
of the 3-phenol dropped under this condition to 55% while
the strongest influence of the GSH transferase with an
average reduction to 28% was observed in the case of the
4,5-dihydrodiol (Table 3). The UDP-glucuronosyltrans-
ferase depressed the bacterial mutagenicity of an-
thanthrene and of its metabolically relevant derivatives
more efficiently than both other phase II enzymes
investigated (Table 3). Addition of 4 mM UDPGA and of
4 mM UDPAG as activator of UDP-glucuronosyltrans-
ferase (32) to the top agar lowered the mutagenicity in
all cases down to one-third. The sulfotransferase, in
contrast, put into action by the PAPS generating system
ATP/sulfate, exhibited with 86% only a weak reducing
effect in the case of the 3-phenol while the mutagenicity
of the remaining compounds including anthanthrene
remained virtually unchanged (Table 3).
anthanthrenes like benzo[a]pyrene (34, 35) to the most
potent mutagenic metabolites, but in contrast to benzo-
[a]pyrene (34, 35), the enzymes of the P450 2B subfamily,
induced by phenobarbital, also play a role in the activa-
tion of anthanthrene to mutagens for strain TA100.
To elucidate the metabolites of anthanthrene respon-
sible for its mutagenicity, the phase I biotransformation
was investigated. By employing synthetic derivatives of
anthanthrene and combining chromatographic, spectro-
scopic, and biochemical methods, 12 microsomal metabo-
lites could be fully or at least partially identified. Among
them, three metabolites at the K-region, the 4,5-oxide
(A7), 4,5-dihydrodiol (A1), and 4,5-quinone (A4a), were
found. As in the case of benzo[a]pyrene (36) and dibenz-
[a,h]anthracene (37), the isomerization of the K-region
oxide of anthanthrene (A7) could only be prevented when
its chromatographic separation was performed under
slightly alkaline conditions. Furthermore, three poly-
nuclear quinones, the 1,6- (A5), 3,6- (A6), and 6,12-
positional (A10) isomers, as well as two phenols, the 1-
(A8) and 3-positional (A9) isomers, could be detected. Two
major metabolites derived from the 3-phenol turned out
to be diphenols (A2, A3). This corresponds to results
obtained with benzo[a]pyrene that pointed to the exist-
ence of the 3,6- (38, 39) and 3,9-diphenols (38). Hence,
A2 and A3 are tentatively identified as 3,6- and 3,9-
dihydroxyanthanthrene. One polar metabolite of an-
thanthrene could be the 3-phenol 4,5-dihydrodiol since
it is formed not only from the 4,5-oxide but also from the
3-phenol. However, since the second monooxygenase
attack occurs more likely distant and not vicinal to the
first attack, this metabolite is instead the 9-phenol 4,5-
dihydrodiol (A1a), the 9-position being identical with the
3-position due to the rotational symmetry of anthanthrene.
Another polar metabolite is probably also a phenol
dihydrodiol (A1b) arising from the 4,5-oxide. A homolo-
gous phenol oxide of benzo[a]pyrene, the 9-hydroxy 4,5-
oxide, has been postulated as an ultimate mutagen of this
PAH (40), alkylating the DNA possibly after rearrange-
ment to a quinone-methide (41).
Discu ssion
Although anthanthrene lacks a bay-region and hence
the requirement for formation of bay-region dihydrodiol
epoxides thought to be responsible for the genotoxicity
of PAH, it nevertheless exhibits remarkably strong
bacterial mutagenicity after metabolic activation com-
parable with that of benzo[a]pyrene (33). Based on the
sensitivity of the strains of S. typhimurium used, the
metabolites of anthanthrene are able to cause base-pair
substitutions (TA100), frameshifts (TA98, TA97), and
possibly oxidative damage (TA104) in the bacterial DNA.
As in the case of other PAH, the enzymatic composition
of the metabolizing system determines the extent of
mutagenicity of anthanthrene. The enzymes of the P450
1A subfamily, induced by 3-methylcholanthrene, convert
With the information on the structures of the micro-
somal metabolites of anthanthrene, the pattern of its
biotransformation exhibiting three distinct pathways can
be envisaged (Scheme 2). Pathway I starts with the
epoxidation at the K-region leading to the 4,5-oxide,
which is then hydrolyzed to the 4,5-dihydrodiol, the
oxidation of which yields the K-region quinone in traces.
Whether the two phenol dihydrodiols A1a and A1b are

340 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Platt et al.
Sch em e 2. P a th w a ys of Micr osom a l Biotr a n sfor m a tion of Diben zo[d ef,m n o]ch r ysen e (An th a n th r en e)a
a
In the case of chiral metabolites, only one arbitrary enantiomer is shown. For the meaning of the Roman numerals, see Table 1.
formed directly from the 4,5-oxide or via the 4,5-dihy-
diphenol. Enzymatic monooxygenation in this pathway
is dominated by the P450 1A subfamily whereas P450
2B plays only a minor role.
drodiol as depicted in Scheme 2 cannot be decided at
present. As observed with other PAH (42, 43), monoxy-
genation of anthanthrene at the K-region is preferentially
catalyzed by the P450 2B subfamily. The formation of
metabolites A1, A1a, and A1b is considerably depressed
by inhibition of epoxide hydrolase, proving the impor-
tance of this hydrolytic enzyme in pathway I.
With the information on the mutagenicity of the
metabolites of anthanthrene, it is now possible to evalu-
ate the contribution of the three metabolic pathways to
the genotoxicity of the parent hydrocarbon. The quinones
of anthanthrene constituting rather weak mutagens are
preferentially formed under conditions where the hydro-
carbon exhibits the lowest bacterial mutagenicity. These
observations make it less likely that pathway II contrib-
utes much to the genotoxicity of anthanthrene. Among
the identified metabolites of anthanthrene, there are only
two which exhibit strong bacterial mutagenicity without
metabolic activation: the 3-phenol and the 4,5-oxide. On
a molar basis, their specific mutagenicity is about 4.4-
10.3-fold higher than that of anthanthrene after meta-
bolic activation. Thus, ultimate mutagens of anthanthrene
are produced via pathway I as well as via pathway III.
The 6,12-quinone and possibly the other polynuclear
quinones of anthanthrene can be formed via pathway II
(Scheme 2) by one-electron oxidation as proposed for
benzo[a]pyrene (44). However, due to the connection of
pathways II and III, this statement is not unequivocal.
In contrast to the other metabolites of anthanthrene,
neither the inducible P450 1A nor 2B enzymes are
considerably involved in the formation of the polynuclear
quinones. Especially in the case of the 6,12-quinone,
autoxidation of anthanthrene seems to be more relevant
than its enzymatic conversion.
Pathway III starts with the monooxygenation at the
A-ring of anthanthrene yielding the 1- and 3-phenols.
Further oxidation of the latter is observed at the 6-posi-
tion, at the 10,11-position (K-region), and at a third yet
unknown position leading to 3,6-diphenol (A2 or A3),
3-phenol 10,11-dihydrodiol (A1a; after enzymatic hy-
drolysis), and another diphenol, possibly the 3,9-posi-
tional isomer (A2 or A3). Oxidation of the 1-phenol could
produce the 1,6-quinone, while the 3,6-quinone is either
formed directly from the 3-phenol or formed via the 3,6-
The role of monooxygenases of the P450 2B subfamily
in activation of anthanthrene to mutagenic metabolites
and their inactivation by epoxide hydrolase in pathway
I is reflected by a considerable increase of mutagenicity
upon inhibition of epoxide hydrolase by TCPO, an effect
that was especially strong when the metabolizing en-
zymes had been induced with phenobarbital. Likewise,
the significance of monooxygenases of the P450 1A
subfamily in transforming anthanthrene to mutagenic
metabolites could be demonstrated by inhibition with 7,8-

Metabolism and Mutagenicity of Anthanthrene
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 341
Ta ble 4. Sign ifica n ce of 3-Hyd r oxyla tion Com p a r ed to
4,5-Ep oxid a tion for th e Gen otoxicity of An th a n th r en e
highest under conditions where this PAH exhibits the
lowest mutagenic effect, an appreciable contribution of
one-electron oxidation to the genotoxicity of anthanthrene
is not very likely. The L-region of anthanthrene could
also be the location for enzymatic methylation or hy-
droxymethylation as described for other PAH with a
meso-anthracenic center (3). The resulting benzylic al-
cohols can be substrates of sulfotransferase(s), trans-
forming them to highly reactive esters. Remarkably, the
conversion of 6-hydroxymethylanthanthrene to strong
bacterial mutagens by sulfotransferases of the rat has
already been demonstrated (50). However, no indication
for the metabolic formation of 6-methyl- or 6-hydroxym-
ethylanthanthrene could be found under the metabolic
conditions employed in this study. This result excludes
the “bioalkylation” as playing a role in the genotoxicity
of anthanthrene. Eventually, hitherto unknown metabo-
lites of anthanthrene could have escaped detection by
reaction with nucleophilic sites in proteins. Thus, inves-
tigations on the binding of metabolites of anthanthrene
to biomolecules such as proteins or DNA and identifica-
tion of the covalent adducts could shed more light on the
enzymatic activation of this mutagenic PAH without a
bay-region to genotoxic metabolites.
Specific Mutagenicitya
his+-rev. colonies of TA100/nmol in
3-hydroxyanthanthrene
120
anthanthrene 4,5-oxide
279
Metabolic Conversion
nmol (nmol of P450)-1 (20 min)-1 in hepatic microsomes
of rats after treatment with
phenobarbital
3-hydroxylationb 4,5-epoxidationc 3-hydroxylationb 4,5-epoxidationc
0.85 0.55 1.97 <0.01
3-methylcholanthrene
Calculated Maximal Number of his+-rev. Colonies of TA100
(nmol of P450)-1 (20 min)-1
102
153
236
<3
a
b
Spontaneous mutagenicity. Sum of 3-phenol, diphenols A2
and A3, as well as 3,6-quinone ()60% of the mixture 1,6-/3,6-
quinone). c Sum of 4,5-oxide and trans-4,5-dihydrodiol.
benzoflavone. Finally, the remarkable reduction of the
mutagenicity of anthanthrene and of its 3-phenol by
UDG-glucuronosyltransferase is an indication for the
formation of phenolic metabolites on the route to ultimate
mutagens.
For the evaluation of the significance of pathways I
and III for the genotoxicity of anthanthrene, not only the
intrinsic mutagenicity of the 4,5-oxide and that of the
3-phenol but also the total metabolic formation of these
two metabolites including their secondary metabolites
has to be taken into account. Calculating the maximal
mutagenic effect from these two parameters (Table 4)
demonstrates the dominating role of the 3-hydroxylation
pathway III in the mutagenicity of anthanthrene under
conditions where this hydrocarbon exhibits the strongest
bacterial mutagenicity, that is, after induction with
3-methylcholanthrene.
Since the 3-phenol is mutagenic without enzymatic
activation, the formation of a phenol epoxide as an
ultimately mutagenic metabolite (40, 41) can be excluded.
Recently we could demonstrate the spontaneous conver-
sion of the 3-phenol to fairly stable phenoxy radicals by
ESR measurements (45). These radical species could be
responsible for the genotoxicity of anthanthrene similarly
as it has been assumed for the 6-oxobenzo[a]pyrene
radical (46). The spontaneous formation of phenoxy
radicals from 3-hydroxyanthanthrene seems to be rather
unique for this PAH since 3-hydroxybenzo[a]pyrene could
not be converted to radical species under the same
conditions (45). Therefore, although 3-hydroxybenzo[a]-
pyrene is the most prominent microsomal metabolite of
this PAH in the presence of P450 1A (47), it rather
constitutes an inactivation step by virtue of its conjuga-
tion (48, 49) whereas in the case of anthanthrene its
3-phenol is the precursor of an ultimate mutagen.
Anthanthrene has one of the lowest ionization poten-
tials of all PAH (2) and could therefore undergo one-
electron oxidation to radical cations preferentially at the
L-region (6,12-position in anthanthrene; cf. Scheme 1).
These electrophilically reactive species could covalently
bind to DNA and as a consequence cause genotoxicity (2).
The occurrence of polynuclear quinones in the biotrans-
formation of PAH is thought to be an indication for the
initial formation of radical cations (44). However, since
the amount of polynuclear quinones of anthanthrene is
Ack n ow led gm en t. We express our appreciation to
M. Tommasone and B. Beile for excellent technical
assistance. The help of M. Eider and A. Vierengel in
performing the mass and NMR spectra is gratefully
acknowledged. We thank the Deutsche Forschungsge-
meinschaft for financial support (Grants SFB 302 and
PL 115/1-1).
Su p p or tin g In for m a tion Ava ila ble: Scheme S1 showing
the synthesis of the non-K-region phenols of anthanthrene,
Figure S2 depicting UV/vis spectra of metabolites of an-
thanthrene, 3-hydroxyanthanthrene, anthanthrene 4,5-oxide,
and of synthetic derivatives of anthanthrene recorded during
HPLC separation with the diode array detector, and also Figures
S3 and S4 exhibiting HPLC separations of the metabolites of
the 4,5-oxide (E1-E10) and of the 3-phenol (P1-P10), respec-
tively. This material is available free of charge via the Internet
at http://pubs.acs.org.
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