Synthesis and quantification of DNA adducts of 4,4'-methylenedianiline

Article abstract of DOI:10.1021/tx950194+

4,4'-Methylenedianiline (MDA) is used as a hardener in the manufacture of plastics and polyurethanes. MDA has been classified as a carcinogen in animals and is a suspected human carcinogen. Assuming that MDA would yield similar DNA adducts to other arylamines, we synthesized the following C-8 guanine adducts: N'-acetyl-N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8- yl)-MDA, N-(deoxyguanosin-8-yl)-4MA, and their corresponding 3'-monophosphate derivatives. We developed methods to identify these adducts of MDA in liver DNA using ³²P-postlabeling, HPLC, and GC-MS techniques. Liver DNA was obtained from rats treated with radiolabeled MDA (1.11 and 116.5 μmol/kg body weight). The total radioactivity bound to the DNA corresponded to 0.06 and 2.7 adducts per 10⁷ nucleotides [covalent binding index (CBI = (μmol of adduct per mol of nucleotide)/(mmol of compound per kg body weight)) of 1.05 and 2.3]. This DNA-binding potency is in the range of weakly genotoxic compounds. The liver DNA was analyzed for the presence of the synthesized adducts by the following methods: (I) HPLC analysis of nucleotides and purines after enzymatic and acid hydrolysis, and (II) ³²P-postlabeling after enzymatic hydrolysis. The major adducts found in vivo did not correspond to the synthesized standards. Further work was carried out to determine the structure of the unidentified adducts. It was possible to release MDA and MDA-d₄ from DNA of rats dosed with MDA and/or MDA-d₄ and from the synthesized adducts using strong base hydrolysis. Liver of two female Wistar rats given 500 μmol/kg MDA · 2HCl was hydrolyzed in 0.1 M NaOH overnight at 110 °C. GC-MS analysis of the heptafluorobutyric anhydride derivatized dichloromethane extracts detected 428 ± 40 fmol of MDA/mg of DNA. In the control animals no MDA was found. The experiment was repeated with livers from animals dosed 500 μmol/kg MDA-d₄ · 2DCl. In these rats 488 ± 19 final MDA-d₄ was found to be bound at liver DNA. Taking into account a 68% yield of the method, the CBI found in these cases was 0.82 and 1.0, respectively.

Full text of DOI:10.1021/tx950194+

Chem. Res. Toxicol. 1996, 9, 1103-1112
1103
Syn th esis a n d Qu a n tifica tion of DNA Ad d u cts of
4,4′-Meth ylen ed ia n ilin e
Dietrich Schu¨tze,† Peter Sagelsdorff,‡ Ovnair Sepai,§ and Gabriele Sabbioni*,†
Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Wu¨rzburg, Versbacherstrasse 9,
D-97078 Wu¨rzburg, Germany, Ciba-Geigy AG, Cell Biology Unit, CH-4001 Basel, Switzerland, and
Department of Environmental and Occupational Medicine, The Medical School,
University of Newcastle upon Tyne, Newcastle NE2 4HH, U.K.
Received November 20, 1995X
4,4′-Methylenedianiline (MDA) is used as a hardener in the manufacture of plastics and
polyurethanes. MDA has been classified as a carcinogen in animals and is a suspected human
carcinogen. Assuming that MDA would yield similar DNA adducts to other arylamines, we
synthesized the following C-8 guanine adducts: N′-acetyl-N-(deoxyguanosin-8-yl)-MDA,
N-(deoxyguanosin-8-yl)-MDA, N-(deoxyguanosin-8-yl)-4MA, and their corresponding 3′-mono-
phosphate derivatives. We developed methods to identify these adducts of MDA in liver DNA
using 32P-postlabeling, HPLC, and GC-MS techniques. Liver DNA was obtained from rats
treated with radiolabeled MDA (1.11 and 116.5 µmol/kg body weight). The total radioactivity
bound to the DNA corresponded to 0.06 and 2.7 adducts per 107 nucleotides [covalent binding
index (CBI ) (µmol of adduct per mol of nucleotide)/(mmol of compound per kg body weight))
of 1.05 and 2.3]. This DNA-binding potency is in the range of weakly genotoxic compounds.
The liver DNA was analyzed for the presence of the synthesized adducts by the following
methods: (I) HPLC analysis of nucleotides and purines after enzymatic and acid hydrolysis,
and (II) 32P-postlabeling after enzymatic hydrolysis. The major adducts found in vivo did not
correspond to the synthesized standards. Further work was carried out to determine the
structure of the unidentified adducts. It was possible to release MDA and MDA-d4 from DNA
of rats dosed with MDA and/or MDA-d4 and from the synthesized adducts using strong base
hydrolysis. Liver of two female Wistar rats given 500 µmol/kg MDA‚2HCl was hydrolyzed in
0.1 M NaOH overnight at 110 °C. GC-MS analysis of the heptafluorobutyric anhydride
derivatized dichloromethane extracts detected 428 ( 40 fmol of MDA/mg of DNA. In the control
animals no MDA was found. The experiment was repeated with livers from animals dosed
500 µmol/kg MDA-d4‚2DCl. In these rats 488 ( 19 fmol MDA-d4 was found to be bound at
liver DNA. Taking into account a 68% yield of the method, the CBI found in these cases was
0.82 and 1.0, respectively.
rabbits which range between 200 and 800 mg/kg (1). It
has been reported to be hepatotoxic in rats (2), dogs (3),
and man (4). It was mutagenic in the Ames test (5) in
the presence of an S9 metabolizing system and carcino-
genic in rats and mice (2). MDA has been classified as a
carcinogen in animals and as a suspected carcinogen in
humans (6).
In tr od u ction
4,4′-Methylenedianiline (MDA)1 is used as a hardener
in the manufacture of plastics and the production of
polyurethanes. The compound has a low mammalian
toxicity with an oral LD50 in mice, rats, guinea pigs, and
* Corresponding author. Tel/FAX: (0931) 201 2281; email: Gabriele.
Sabbioni@t-online.de.
Exposure to MDA has been monitored by several
researchers by measuring MDA or N′-acetyl-4,4′-meth-
ylenedianiline (AcMDA) in urine (7-9) and as hemoglo-
bin adducts (8, 10). Most arylamines react with C-8 of
guanine (11, 12). In rats dosed with 4,4′-methylenebis-
(2-chloroaniline) (MOCA), two major adducts of mono-
cyclic arylamines with adenine have been isolated:
N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol
and N-(deoxyadenosin-8-yl)-4-amino-3-chlorotoluene (13,
14). No adducts with guanine were found in this case.
The authors postulate that the selective reaction of
N-hydroxy-MOCA with DNA-adenine and the formation
of the single arylamine ring adducts is a consequence of
the strong inductive effect of the ortho-chlorine (13).
Assuming that MDA would yield similar DNA adducts
like most arylamines (11, 12), we synthesized the follow-
ing C-8 guanine adducts of MDA: N′-acetyl-N-(deoxygua-
nosin-8-yl)-MDA (dG-AcMDA), N-(deoxyguanosin-8-yl)-
MDA (dG-MDA), and their corresponding 3′-mono-
phosphate derivatives. The C-8 adduct of 4-methyl-
† Universita¨t Wu¨rzburg.
‡ Ciba-Geigy AG.
§ University of Newcastle upon Tyne.
X Abstract published in Advance ACS Abstracts, August 15, 1996.
1
Abbreviations: 4,4′-methylenedianiline (MDA), covalent binding
index (CBI), N′-acetyl-4,4′-methylenedianiline (AcMDA), 4,4′-methyl-
enebis(2-chloroaniline) (MOCA), 4-methylaniline (4MA), N′-acetyl-N-
(deoxyguanosin-8-yl)-MDA (dG-AcMDA), N-(deoxyguanosin-8-yl)-MDA
(dG-MDA), N-(deoxyguanosin-8-yl)-4MA (dG-4MA), 2′-deoxyguanosine
monohydrate (dG), 2′-deoxyguanosine 3′-monophosphate (dGp), T4
polynucleotide kinase (PNK), direct insertion probe (DIP), desorption
chemical ionization (DCI), field desorption (FD), electrospray ionization
mass spectrometry (ESI-MS), N′-acetyl-4′-amino-4-nitrodiphenyl-
methane (nitro-AcMDA), N-acetoxy-N′-acetyl-MDA (OAc-AcMDA), am-
monium formate (AF), 3-chloroperbenzoic acid (mCPBA), N′-acetyl-N-
(3′-monophospho-2′-deoxyguanosin-8-yl)-MDA (dGp-AcMDA), N-(3′-
monophosphate-2′-deoxyguanosin-8-yl)-MDA (dGp-MDA), N-(3′-mono-
phosphate-2′-deoxyguanosin-8-yl)-4MA (dGp-4MA), 250 mM mannitol,
70 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid, pH 7.4 (HM buffer), heptafluorobutyric anhydride (HFBA), 2′-
deoxyadenosine (dA), N2-(2′-deoxyguanosin-8-yl)-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (dG-PhiP), 2-amino-1-methyl-6-phenylim-
idazo[4,5-b]pyridine (PhIP), nuclease P1 (NP1), tetrabutylammonium
chloride (TBA), units (U), 2′-deoxycytidine 3′-phosphate (dCp), 2′-
deoxythymidine 3′-phosphate (dTp), and 2′-deoxyadenosine 3′-phos-
phate (dAp).
S0893-228x(95)00194-9 CCC: $12.00 © 1996 American Chemical Society

1104 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Schu¨tze et al.
150 mL). The organic phase was dried with magnesium sulfate,
and the solvent was evaporated in vacuo. The yellow solid (6.2
g) obtained was purified on a silica gel column with ethyl acetate
as solvent. The collected fraction was evaporated in vacuo,
yielding 5.2 g (80%) of yellowish needles of nitro-AcMDA, which
melt at 164-165 °C (lit. (17) 160-161 °C). 1H NMR (250 MHz,
DMSO-d6): δ ) 2.02 (s, 3 H), 4.02 (s, 2 H), 7.17 (d, J ) 8.5 Hz,
2 H), 7.47 (d, J ) 8.8 Hz, 2 H), 7.51 (d, J ) 8.5 Hz, 2 H), 8.14
(d, J ) 8.8 Hz, 2 H), 9.90 (s, 1 H). MS (70eV): m/ z (%) ) 271
(9), 270 (46) [M+], 229 (15), 228 (100), 227 (34), 182 (35), 181
(17), 180 (11), 107 (10), 106 (66).
aniline (4MA) was synthesized in case the N-(deoxygua-
nosin-8-yl)-4MA (dG-4MA) would be formed from an
unstable dG-MDA adduct as seen for the adenine adducts
of MOCA. These DNA adducts were used to develop
methods of adduct quantification by the 32P-postlabeling
technique and by GC-MS following the procedure recently
published by Lin et al. (15) and Friesen et al. (16).
Exp er im en ta l P r oced u r es
Ch em ica ls. Nuclease P1 (NP1) (from Penicillium citrinium,
236225), RNase A (from bovine pancreas, 109142), and protein-
ase K (from Tritirachium album, 745723) were from Boehringer
(Mannheim, Germany); 2′-deoxyguanosine monohydrate (dG),
pyruvonitrile, heptafluorobutyric anhydride (HFBA), and MDA
were purchased from Fluka (Neu-Ulm, Germany); PEI-cellulose
thin-layer plates were from Macherey-Nagel (Du¨ren, Germany);
water (chromatography grade, 15333) and anhydrous sodium
sulfate (p.a. 6639) were from Merck (Darmstadt, Germany); T4
polynucleotide kinase (PNK) was from Pharmacia (Freiburg,
Germany); ethanol (Rotipuran >99.8%, 9062.2) and Roti-Phenol
()redistilled phenol, equilibrated in and covered with 10 mM
Tris, 1 mM EDTA-Na2 (pH 7.5-8.0)) were from Roth (Karlsruhe,
Germany); 2′-deoxyguanosine 3′-monophosphate (dGp) and
dichloromethane (Pestanal) were from Riedel de Haen (Seelze,
Germany); alkaline phosphatase from calf intestine (P-4252),
apyrase (A6132), calf thymus DNA, micrococcal endonuclease
(N-3755), spleen phosphodiesterase (P-6897), and pig liver
esterase (E-3128) were acquired from Sigma (Deisenhofen,
Germany); MDA-d4 was synthesized as described by Schu¨tze et
al. (8). All the other chemicals were from Aldrich (Steinheim,
N,N′-Dia cetyl-N-h yd r oxy-MDA. Hydrazine hydrate (206
mg, 4.12 mmol) was added to nitro-AcMDA (270 mg, 1 mmol)
and 5% palladium on charcoal (Pd/C) (50 mg) in THF (25 mL),
keeping the temperature below 5 °C. After 1 h, further
hydrazine hydrate (103 mg, 2.06 mmol) was added. After a total
of 2 h, no starting material was detectable by TLC (silica gel,
acetone/hexane 1:1). Triethylamine (219 mg, 2.16 mmol) was
added. Acetyl chloride (1.65 mg, 21.0 mmol) in THF (5 mL) was
added very slowly, keeping the reaction temperature below 10
°C, and the reaction mixture was kept stirring at room tem-
perature for a further 30 min. The solution was filtered, and
the solid residue was washed with diethyl ether. The combined
organic phases were vigorously stirred with saturated sodium
bicarbonate solution and then washed with 1 M NaOH (6 × 25
mL). The pH of the aqueous solution was adjusted to between
pH 1 and 2 with HCl and then extracted with ethyl acetate (5
× 25 mL). After the organic phases were dried with magnesium
sulfate and evaporated down in vacuo, the orange brown oil was
then taken up in 0.1 M NaOH (30 mL) and stored overnight
after addition of 37% HCl (15 mL). The precipitate was filtered,
washed with water, and dried in a desiccator. Colorless short
needles of N,N′-diacetyl-N-hydroxy-MDA (202 mg, 68%) with a
melting point of 75-77 °C (lit. (18) 138-139 °C) were obtained.
1H NMR (250 MHz, DMSO-d6): δ ) 2.02 (s, 3 H), 2.19 (s, 3 H),
3.86 (s, 2 H), 7.13 (d, J ) 8.3 Hz, 2 H), 7.19 (d, J ) 8.4 Hz, 2 H),
7.50 (d, J ) 8.3 Hz, 2 H), 7.52 (d, J ) 8.4 Hz, 2 H), 9.89 (s, 1 H),
10.56 (br s, 1 H). 13C NMR (63 MHz, DMSO-d6): δ ) 22.7 (q),
the other signals are listed in Table 2. MS (70eV): m/ z (%) )
299 (3), 298 (12) [M+], 283 (15), 282 (78), 256 (47), 241 (11), 240
(59), 239 (26), 199 (10), 198 (65), 197 (100), 195 (11), 183 (12),
182 (41), 181 (19), 180 (24), 148 (10), 136 (19), 121 (29), 106
(76), 93 (15).
Germany).
[
14C]MDA (6.84 mCi/mmol) was obtained from
Amersham (Little Chalfont, U.K.). The radioactive purity
(>98%) was checked by TLC (toluene/acetone ) 1:1).
Syn th eses. Ca u tion : N-Arylhydroxylamines and their O-
acyl derivatives are potentially carcinogenic. They should be
handled with protective clothing in a well-ventilated fume hood.
In str u m en ta tion . HPLC was performed with a quaternary
HPLC pump of the 1050 series by Hewlett Packard (Waldbronn,
Germany) equipped with a photodiode array detector LKB 2140
(Pharmacia, Freiburg, Germany). The following HPLC columns
were used: LiChrospher RP 18 (250 × 4 mm, 5 µm) and
LiChrospher RP Select B (250 × 4 mm, 5 µm) from Merck
(Darmstadt, Germany); Nucleosil C18 (250 × 12.5 mm, 7 µm)
and Nucleosil C18 (250 × 4.6 mm, 10 µm) from Macherey-Nagel
(Du¨ren, Germany). NMR spectra were recorded on a Bruker
AC 250 and Bruker WM 400 spectrometer. The 13C signals were
distinguished by a DEPT (distortionless enhancement by po-
larization transfer) experiment. Internal standard was tetra-
methylsilane or DMSO. Mass spectra of volatile compounds
were measured on a mass spectrometer 5989A with a GC-MS
interface or with a direct insertion probe (DIP). Mass spectra
were measured on a Finnigan MAT 90 using the field desorption
(FD) technique or on a Finnigan MAT 8200 with isobutane (0.3
mbar) or ammonia (0.3 mbar) as reactant gas using the
desorption chemical ionization (DCI) technique (Dr. Gerda
Lange, Department of Organic Chemistry of the University of
Wu¨rzburg). The electrospray ionization mass spectra (ESI-MS)
were provided by Dr. Peter Farmer (MRC Toxicology, Leicester).
The experiments were conducted on a Fisons-Quattro mass
spectrometer using cone-induced fragmentation. Samples were
introduced via loop injection in water/acetonitrile. UV spectra
were recorded on a Kontron spectrophotometer Uvikon 860. IR
spectra were registered on a Perkin-Elmer 1420 recording
spectrophotometer. TLC was carried out with TLC-plates
Alugram SIL G/UV254 with fluorescence indicator (Macherey-
Nagel, Du¨ren, Germany).
N′-Acetyl-N-h yd r oxy-MDA. Hydrazine hydrate (5.16 g, 103
mmol) was added over 15 min at 5 °C to a suspension of nitro-
AcMDA (6.76 g, 25 mmol) in THF (400 mL) and 5% Pd/C (1 g).
After 1.5 h no more starting material was present according to
TLC analysis (silica gel, acetone/hexane ) 1:1). The reaction
mixture was dried over anhydrous sodium sulfate and filtered
through Celite. The solid on the filter was washed with THF.
The combined organic phases were evaporated in vacuo to about
50 mL, covered with hexane (400 mL), and stored overnight at
-20 °C. After filtration and drying with a vacuum pump,
yellowish crystals were obtained with a yield of 95% (6.09 g).
AcMDA (15%) was present as a byproduct. For further purifica-
tion, the hydroxylamine was dissolved in a minimum quantity
of dichloromethane/THF 2:3, filtered, and covered with a 4-5-
fold volume of diethyl ether and stored at -20 °C. The
precipitate was filtered and washed twice with diethyl ether
and dried at the vacuum pump. The slightly yellow needles
decompose at 115 °C (lit. (18) ca. 110 °C). 1H NMR (250 MHz,
DMSO-d6): δ ) 2.02 (s, 3 H), 3.76 (s, 2 H), 6.77 (d, J ) 8.4 Hz,
2 H), 7.00 (d, J ) 8.4 Hz, 2 H), 7.10 (d, J ) 8.4 Hz, 2 H), 7.48
(d, J ) 8.4 Hz, 2 H), 8.13 (br s, 1 H), 9.86 (s, 1 H). MS (70eV):
m/ z (%) ) 256 (3) [M+], 255 (7), 254 (38), 241 (18), 240 (100),
212 (21), 198 (45), 197 (95), 183 (19), 182 (64), 181 (22), 180
(31), 165 (16), 106 (97), 104 (10), 93 (14), 89 (12), 77 (13).
N′-Acetyl-4′-a m in o-4-n itr od ip h en ylm eth a n e (Nitr o-Ac-
MDA). AcMDA (5.8 g, 24.1 mmol) in dichloromethane (200 mL)
was added dropwise over 2 h to 50% 3-chloroperbenzoic acid
(mCPBA) (25 g, 72.4 mmol) in dichloromethane (300 mL). After
a further 1 h stirring at room temperature, the reaction solution
was washed with 1 M NaOH (5 × 100 mL) and 1 M HCl (1 x
N-Acetoxy-N′-a cetyl-MDA (OAc-AcMDA). Pyruvonitrile
(1.07 mL, 15 mmol) in THF (25 mL) was added slowly to N′-
acetyl-N-hydroxy-MDA (3.84 g, 15.0 mmol) and triethylamine
(2.36 mL, 17 mmol) in THF at -50 to -40 °C over an hour.
After stirring the solution for 1.5 h at -20 °C, the solvents were
evaporated in vacuo at 0 °C. The yellow-orange oil was

DNA Adducts of 4,4′-Methylenedianiline
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1105
dissolved in THF and used without further purification for the
reactions with dG and dGp.
M ammonium sulfate, the reaction mixture was kept at 37 °C.
After 36 h, the pH was adjusted to 7.7 with 1 M NaOH, and
further carboxylesterase (100 µL, 230 U) was added. HPLC
analysis [250 × 4 mm, RP18 column, methanol gradient in AF
buffer (pH 5.1): 30-60% in 15 min, 60-80% in 1 min, flow 1
mL/min] showed that all dG-AcMDA was used up after further
12 h reaction time. The reaction mixture was then evaporated
in vacuo and taken up in water (1 mL) and purified by HPLC
with the same conditions described above. The peak eluting at
15.3 min was collected, evaporated, taken up in a minimum
quantity of DMSO, and rechromatographed [250 × 4 mm, RP
select B column, THF gradient in water: 10-40% in 15 min,
40-80% in 1 min, flow 1 mL/min]. The peak eluting at 14.6
min was collected. THF was evaporated in vacuo. Fine colorless
needles (3.4 mg, 7.34 µmol, 74%) were obtained after freeze-
drying.
1H NMR (250 MHz, DMSO-d6): δ ) 1.99 (dd, J ) 13.2 Hz, J
) 5.6 Hz, 2 H), 3.68 (s, 2 H), 3.73 (m, 2 H), 3.91 (d, J ) 1.8 Hz,
1 H), 4.40 (m, 1 H), 4.84 (br s, 2 H), 5.32 (d, J ) 3.2 Hz, 1H),
5.87 (t, J ) 4.5 Hz, 1 H), 6.31 (dd, J ) 5.6 Hz, J ) 9.6 Hz, 1 H),
6.34 (s, 2 H), 6.47 (d, J ) 8.4 Hz, 2 H), 6.84 (d, J ) 8.4 Hz, 2 H),
7.05 (d, J ) 8.6 Hz, 2 H), 7.60 (d, J ) 8.6 Hz, 2 H), 8.54 (s, 1 H),
10.50 (br s, 1 H). The signal for 2′-Ha[dG] is covered by the
DMSO-d6 signal. UV (methanol): λmax (log ꢀ) ) 204 nm (4.734),
236 (4.208), 285 (4.472). MS (DCI/isobutane, 70 eV): m/ z (%)
) 349 (2), 348 (10) [M+ - D-ribose], 197 (9), 169 (12), 142 (19),
141 (100), 140 (24), 129 (13), 127 (13), 125 (11), 118 (23), 117
(100), 116 (29), 113 (16).
N′-Acetyl-N-(2′-d eoxygu a n osin -8-yl)-MDA (d G-AcMDA).
A cold solution (0 °C) of OAc-AcMDA (ca. 5 mmol) in THF (8
mL) was added to dG (200 mg, 701 µmol) and triethylamine
(607 mg, 6 mmol) in water (8 mL) at 37 °C. After 1.5 h at room
temperature, water (400 mL) was added and the pH was
adjusted to 5 with formic acid. Extraction with diethyl ether
was followed by evaporation of the water phase in vacuo. The
residue was taken up in water/methanol 8:2 (10 mL) and
purified by HPLC on a 250 × 12.5 mm RP18 column with 50
mM ammonium formate (AF) buffer, pH 7.4/methanol 50:50 and
a flow of 3 mL/min. The fraction at 17 min was collected,
evaporated, and repurified by HPLC with the same conditions
but with water instead of the AF buffer. After evaporation, the
residue was taken up in DMSO (1.5 mL) and purified by HPLC
(250 × 4 mm, RP select B column, THF gradient in water: 10-
40% in 15 min, then 5 min at 80%, flow of 1 mL/min). The peak
at 14.1 min was collected (the main byproduct, N,N′-diacetyl-
N-hydroxy-MDA eluted at 15.7 min). Evaporation in vacuo of
THF was followed by freeze-drying. Colorless, fine needles of
dG-AcMDA were obtained. 1H NMR (400 MHz, DMSO-d6): δ
) 1.99 (dd, J ) 6.6 Hz, 2 H), 2.00 (s, 3 H), 3.73 (m, 2 H), 3.81
(s, 2 H), 3.91 (d, J ) 1.9 Hz, 1 H), 4.40 (m, 1 H), 5.32 (d, J ) 3.6
Hz, 1 H), 5.87 (t, J ) 4.8 Hz, 1 H), 6.31 (dd, J ) 5.8 Hz, J ) 9.8
Hz, 1 H), 6.33 (s, 2 H), 7.09 (d, J ) 8.6 Hz, 2 H), 7.11 (d, J ) 8.5
Hz, 2 H), 7.46 (d, J ) 8.5 Hz, 2 H), 7.61 (d, J ) 8.6 Hz, 2 H),
8.56 (s, 1 H), 9.84 (s, 1 H), 10.51 (br s, 1 H). The signal for
2′-Ha[dG] is covered by the DMSO-d6-signal. UV (methanol):
N-(3′-Mon oph osph ate-2′-deoxygu an osin -8-yl)-MDA (dGp-
MDA). dGp-AcMDA solution in water/methanol 4:1 (500 µL,
1.01 µmol) was evaporated to dryness with a stream of nitrogen.
The residue was taken up in 10 mL of 50 mM phosphate buffer
and incubated with a carboxylesterase suspension (500 µL, ca.
1150 U) in 3.2 M ammonium sulfate solution (pH 8) at 37 °C
under nitrogen. An HPLC run of the reaction mixture after 24
h [250 × 4 mm, RP18 column, methanol gradient in AF buffer
(pH 5.1): 10-80% in 30 min, flow 1 mL/min, tR (dGp-MDA) )
20.2 min, tR (dGp-AcMDA) ) 21.5 min] showed that dGp-
AcMDA was still present. The pH of the reaction solution was
readjusted to 7.7 with NaOH, and further carboxylesterase (125
µL, ca. 300 U) was added. After a total of 40 h reaction time,
dGp-MDA was obtained after HPLC purification [250 × 4 mm,
RP18 column, THF gradient in AF buffer (pH 5.1): 10-30% in
15 min and 30-80% in 1 min, flow 1 mL/min, tR ) 10.1 min].
Evaporation of THF in vacuo was followed by freeze-drying. The
colorless crystals of dGp-MDA were transferred to a 1 mL
volumetric flask and dissolved in water/methanol 4:1 for further
analysis. Structural elucidation and quantitation of dGp-
MDA: dGp-MDA solution (10 µL) in 30 mM sodium acetate
buffer, pH 7.2 (500 µL), and 10 mM zinc sulfate (20 µL) was
incubated at 37 °C with a solution of NP1 (20 µL, ca. 20 U) in
30 mM sodium acetate buffer (pH 5.3) for 36 h. The whole
reaction solution was analyzed with the same HPLC conditions
used for the control of the reaction after 24 h described above.
The peak area at 27.6 min was quantified against a calibration
curve obtained with dG-AcMDA (5.9-23.5 nmol) at three
wavelengths: 230, 280, and 310 nm. The concentration of the
dGp-MDA solution was 710 nmol/mL. The yield of the deacety-
lation was therefore 70%. The collected peak was then reana-
lyzed by HPLC [250 × 4 mm, RP select B column, THF gradient
in AF buffer (pH 5.1): 0-45% in 20 min, then 5 min at 80%,
flow 1 mL/min]. The retention time (23.7 min) and the UV
spectra were the same as the standard solution of dG-MDA.
λ
max (log ꢀ) ) 204 nm (4.787), 246 (4.386), 284 (4.533). MS (DCI/
isobutane, 70 eV): m/ z (%) ) 432 (2), 428 (1), 391 (8), 390 (31)
[M+ - D-ribose], 117 (100), 116 (16), 113 (16). MS (FD): m/ z
(%) ) 528 (77) [M+ + Na], 507 (58), 506 (41), 505 (100) [M+].
N′-Acetyl-N-(3′-m on op h osp h a te-2′-d eoxygu a n osin -8-yl)-
MDA (d Gp -AcMDA). The cold solution (0 °C) of OAc-AcMDA
(ca. 0.7 mmol) in THF (1.8 mL) was added to dGp (22.5 mg,
60.4 µmol) and triethylamine (63 µL, 454 µmol) in water (8 mL)
at 0 °C under nitrogen. After 1.5 h at 37 °C the THF was
evaporated with a stream of nitrogen and AF buffer, pH 5.1 (10
mL), was added. dGp-AcMDA was purified by HPLC [250 × 4
mm, RP18 column, methanol gradient in AF buffer (pH 5.1):
5-65% in 20 min, then 65-85% in 1 min, flow 1 mL/min]. The
peak at 20 min was collected, evaporated in vacuo, taken up in
2 mL of AF buffer (pH 5.1)/methanol 4:1, and rechromato-
graphed [250 × 4 mm, RP select B column, THF gradient in
AF buffer (pH 5.1): 0-30% in 15 min, then 5 min 80%, flow 1
mL/min]. The peak at 13.5 min was collected. After evapora-
tion of the organic solvents in vacuo and freeze-drying, colorless
crystals (2.02 µmol, 3.3%, see below) of dGp-AcMDA were
obtained. For further analysis, the crystals were dissolved in
water/methanol 1:4 and transferred to a 1 mL volumetric flask.
The purity determined by HPLC at the wavelengths 245 and
280 nm was 99% and 99.5% [250 × 4 mm, RP18 column,
methanol gradient in AF buffer (pH 5.1): 10-80% in 30 min,
flow 1 mL/min, tR ) 21.6 min]. Structural elucidation and
quantitation of dGp-AcMDA: dGp-AcMDA solution (10 µL) in
30 mM sodium acetate buffer, pH 7.2 (500 µL), and 10 mM zinc
sulfate (20 µL) was incubated at 37 °C with a solution of NP1
(20 µL, ca. 20 U) in 30 mM sodium acetate buffer (pH 5.3). After
20 h the whole reaction mixture was analyzed by HPLC with
the same conditions described above. The peak areas at 245
and 305 nm were quantified against a calibration curve obtained
under the same HPLC conditions with 12.1 -36.2 nmol of dG-
AcMDA. A concentration of 2.02 nmol/µL was calculated for
dGp-AcMDA. The collected peak was then reanalyzed by HPLC
[250 × 4 mm, RP select B column, THF gradient in AF buffer
(pH 5.1): 0-30% in 20 min, then 5 min at 80%, flow of 1 mL/
min]. The retention time (19.7 min) and the UV spectra were
the same as the standard solution of dG-AcMDA.
N-Hyd r oxy-4MA. Hydrazine hydrate (20.6 g, 412 mmol)
was added slowly to 4-nitrotoluene (13.7 g, 100 mmol) and 5%
Pd/C (1 g) in THF (500 mL) at 0-5 °C. After 2.5 and 3.5 h, a
further 5% Pd/C (500 mg) was added. The reaction was stopped
after 5 h. The reaction suspension was dried over sodium
sulfate and filtered through Celite. The filtrate was evaporated
to 50 mL, then poured to cold hexane (400 mL), and stored
overnight at -20 °C. The precipitate was filtered, washed with
cold hexane, and dried at a vacuum pump. Slightly yellow and
shiny leaves of N-hydroxy-4MA (8.95 g, 73%) were obtained,
N-(2′-Deoxygu a n osin -8-yl)-MDA (d G-MDA). dG-AcMDA
(5.00 mg, 9.90 µmol) was dissolved in DMSO (1 mL) and diluted
with 50 mM sodium phosphate buffer, pH 7.7 (20 mL). After
addition of carboxylesterase suspension (500 µL, 1150 U) in 3.2

1106 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Schu¨tze et al.
which melt at 92-93 °C (lit. (19) 93-94 °C). 1H NMR (250 MHz,
DMSO-d6): δ ) 2.20 (s, 3 H), 6.76 (d, J ) 8.4 Hz, 2 H), 6.98 (d,
J ) 8.4 Hz, 2 H), 8.05 (br s, 1 H). 13C-NMR (63 MHz, DMSO-
d6): δ ) 20.6 (q, -CH3), 113.7 (d, C-2,6), 128.2 (s, C-4), 129.2 (d,
C-3,5), 150.1 (s, C-1). MS (70eV): m/ z (%) ) 124 (5), 123 (56)
[M+], 121 (10), 107 (38), 106 (100), 91 (22), 79 (43), 78 (19), 77
(48), 65 (20), 63 (13), 61 (13), 53 (12), 52 (24), 51 (29).
min). The peak areas at 240 and 310 nm were quantified
against a calibration curve obtained with the same HPLC
conditions with 29.3-58.5 nmol of dG-4MA. A concentration
of 6.43 nmol/µL was calculated for dGp-4MA. This corresponds
to a total yield of 15% (6.43 µmol). The collected peak was then
reanalyzed by HPLC [250 × 4 mm, RP select B column, THF
gradient in AF buffer (pH 5.1): 0-30% in 20 min, then 5 min
at 80%, flow 1 mL/min]. The retention time (17.3 min) and the
UV spectra were the same as the standard solution of dG-4MA.
N-(2′-Deoxygu a n osin -8-yl)-4-m et h yla n ilin e (d G-4MA).
Pyruvonitrile (1.73 g, 25.0 mmol) dissolved in diethyl ether (20
mL) was added to N-hydroxy-4MA (3.08 g, 25.0 mmol) and
triethylamine (2.73 g , 27.0 mmol) in diethyl ether (50 mL) under
nitrogen in 20 min at -40 °C. The temperature was allowed to
rise slowly to -15 to -10 °C. After 2 h, the reaction mixture
was dried over sodium sulfate and filtered at -20 °C. The
solvents were evaporated at 0 °C. The obtained yellow oil of
N-acetoxy-4MA was dissolved at 0 °C in THF (110 mL). Then
a suspension of 2′-deoxyguanosine monohydrate (1.14 g, 4.00
mmol) in water (40 mL) was added under vigorous stirring. The
mixture was warmed up in about 30 min to room temperature
and stirred for another 1.5 h. The solvents were then evapo-
rated in vacuo. The dark residue was taken up in water (400
mL) and extracted with diethyl ether (5 × 100 mL) and
1-butanol (5 × 100 mL). The 1-butanol phase was evaporated.
The orange residue was taken up in water/methanol 2:8 (10 mL)
and purified by semipreparative HPLC [250 × 12.5 mm, RP18
column, methanol gradient in water: 30-90% in 13 min, flow
3 mL/min]. The peak eluting at 10.5 min was collected. After
evaporation of the methanol and freeze-drying, pale yellow
needles of dG-4MA (70 mg) were obtained in 4.7% yield. Purity
by HPLC with UV detection at 254 nm was >95%. 1H NMR
(250 MHz, DMSO-d6): δ ) 1.99 (dd, J ) 12.8 Hz, J ) 5.7 Hz, 1
H), 2.24 (s, 3 H), 3.74 (m, 2 H), 3.92 (d, J ) 1.6 Hz, 1 H), 4.41
(m, 1 H), 5.33 (d, J ) 3.4 Hz, 1 H), 5.90 (t, J ) 4.5 Hz, 1 H),
6.32 (dd, J ) 5.7 Hz, 1 H), 6.34 (s, 2 H), 7.16 (d, J ) 8.4 Hz, 2
H), 7.48 (d, J ) 8.4 Hz, 2 H), 8.54 (s, 1 H), 10.51 (br s, 1 H). The
signal of 2′-Ha[dG] was covered by the DMSO-d6 signal. 13C
NMR (63 MHz, DMSO-d6): δ ) 20.7 (q, PhCH3), 38.5 (t, C-2′-
[dG]), 61.6 (t, C-5′[dG]), 71.6 (d, C-3′[dG]), 83.2 (d, C-1′[dG]),
87.5 (d, C-4′[dG]), 112.5 (s, C-5[dG]), 117.8 (d, C-2,6), 129.2 (d,
C-3,5), 129.6 (s, C-4), 138.7 (s, C-1), 143.9 (s, C-8[dG]), 149.8 (s,
C-4[dG]), 153.1 (s, C-2[dG]), 156.0 (s, C-6[dG]). UV (methanol):
λmax (log ꢀ) ) 202 nm (4.492), 282 (4.398). MS (DCI/isobutane,
70 eV): m/ z (%) ) 373 (7) [M+ + H], 257 (12) [M+ - D-ribose],
124 (13), 117 (100), 116 (17), 114 (11), 113 (41), 112 (16).
An im a l Exp er im en ts. Female Wistar rats (200-225 g)
were obtained from the Zentralinstitut fu¨r Versuchstierkunde
(Hannover, FRG). They had free access to feed (Altromin 1324)
and water. MDA‚2HCl and MDA-d4‚2DCl were given in H2O
and D2O by gavage to groups of two animals. Two control
animals received only H2O. After 24 h the animals were
anesthetized with diethyl ether, and blood (4-6 mL) was taken
by heart puncture. The livers were perfused with 0.9% saline
solution. Livers of male Wistar rats (190-235 g) given 5.6 µmol/
kg (0.3 mCi/kg) and 116.5 µmol/kg (3 mCi/kg) [14C]MDA by ip
were kindly provided by Dr. Eric Bailey (MRC Toxicology,
Leicester). The rats were sacrificed after 24 h.
Isola tion of Ra t Liver DNA. Liver tissue (1 g) in 5 mL
HM buffer (250 mM mannitol, 70 mM sucrose, 5 mM 4-(2-
hydroxyethyl)-1-piperazineethane-sulfonic acid, pH 7.4) was
homogenized in a teflon-potter-glass for 10 s at medium speed.
DNA was isolated as described by Gupta (20, 21) and dissolved
in water (Merck, HPLC water) for the GC-MS analysis and in
nucleotide buffer (8 mM calcium chloride, 20 mM sodium
succinate, pH 6.0) for the postlabeling analysis. An aliquot of
the solution (25 µL) was added to 1 mL of water and the DNA
determined by UV [DNA (mg/mL) ) extinction E260 nm/20].
About 1 mg of DNA was obtained from 1 g of liver by this
method.
DNA Bin d in g Assa y. Rat liver DNA was isolated from the
liver chromatin fraction and purified to constant specific
radioactivity by lysis, extraction with phenol/chloroform/isoamyl
alcohol, chromatography on hydroxylapatite, dialysis and pre-
cipitation with ethanol according to Sagelsdorff et al. (22, 23).
Using this method, about 10 mg of DNA could be isolated from
a rat liver. The specific activity of the DNA was determined by
scintillation counting of 1-5 mg of the isolated DNA.
[
14C]-
MDA-DNA adducts were assessed by HPLC analysis of di-
gested DNA using the following methods:
(i) Rat liver DNA (1 mg) in nucleotide buffer (1 mL) was
hydrolyzed with micrococcal endonuclease (5 U) and spleen
phosphodiesterase (0.1 U) for 16 h at 37 °C and was analyzed
with method A [Nucleosil C18 column (250 × 4.6 mm, 10 µm),
3% methanol in 50 mM AF buffer (pH 4.5) for 10 min, followed
by a linear gradient to 100% methanol in 35 min, flow 1.5 mL/
min]. The retention times for the unmodified deoxyribonucle-
otides: 2′-deoxycytidine 3′-phosphate (dCp), dGp, 2′-deoxythy-
midine 3′-phosphate (dTp), and 2′-deoxyadenosine 3′-phosphate
(dAp) were 3.5, 5.8, 6.9, and 12.4 min, respectively. Two minute
fractions were collected and assessed for radioactivity by scintil-
lation counting (60 min). Completeness of the hydrolysis was
checked by analyzing 10 µL of the hydrolysate with method A.
The amount of liberated nucleotides was calculated according
to the respective peak areas.
(ii) Alternatively, the DNA hydrolysates were analyzed after
the addition of the synthetic standards dGp-4MA, dGp-MDA,
and dGp-AcMDA with method B [Nucleosil C18 column (250 ×
4.6 mm, 5 µm), flow of 1.5 mL/min, 40 min gradient of 20-80%
methanol in 50 mM AF buffer, pH 4.5]. The synthesized adducts
dGp-4MA, dGp-MDA, and dGp-AcMDA eluted at 14.6, 18.9, and
20.4 min, respectively. Two minute fractions were collected and
assessed for radioactivity by scintillation counting (60 min).
N-(3′-Mon oph osph ate-2′-deoxygu an osin -8-yl)-4MA (dGp-
4MA). A cold solution of N-acetoxy-4MA (1.5 mmol) in THF (2
mL) was added to dGp (14.8 mg, 42.6 µmol) and triethylamine
(152 mg, 1.50 mmol) in water (2 mL) under nitrogen at 37 °C.
After 1 h, the reaction solution was poured into 50 mM AF
buffer, pH 5.1 (200 mL), and extracted with diethyl ether (5 ×
50 mL). Then the water phase was evaporated in vacuo. The
brown residue was taken up in 50 mM AF buffer (pH 5.1)/
methanol 3:1 (8.0 mL) and purified by HPLC [250 × 4 mm, RP18
column, methanol gradient in AF buffer (pH 5.1): 10-55% in
15 min, 55-85% in 1 min, flow 1 mL/min]. The peak eluting at
15.5 min was collected. After evaporation of the solvents in
vacuo, the residue was rechromatographed [250 × 4 mm, RP
select B column, THF/acetonitrile ) 9:1 gradient in water:
0-30% in 15 min, then 5 min 80%, flow 1 mL/min]. The peak
eluting at 13.5 min was collected. After evaporation of the
organic solvents and freeze-drying, slightly yellow crystals of
dGp-4-MA were obtained and dissolved in methanol/water 2:8
for storage. The purity of the product was checked by HPLC
and UV detection [250 × 4 mm, RP18 column, methanol
gradient in AF buffer (pH 5.1): 10-80% in 30 min, flow of 1
mL/min, tR ) 17.5 min]. The purity at wavelengths 245 and
280 nm was 97% and 99%, respectively. Structural elucidation
and quantitation of the yield: dGp-4MA solution (10 µL) in 30
mM sodium acetate buffer, pH 7.2 (500 µL), and 10 mM zinc
sulfate (20 µL) was incubated at 37 °C with a solution of NP1
(20 µL, ca. 20 U) in 30 mM sodium acetate buffer (pH 5.3). After
20 h the whole reaction mixture was analyzed by HPLC with
the same conditions described for the purity check (tR ) 20.2
(iii) The purines were liberated from the rat liver DNA (1
mg in 1 mL of nucleotide buffer) by the addition of 0.1 volume
of 1 M HCl and hydrolysis at 90 °C for 1h. The hydrolysate
was neutralized with NaOH and loaded on a Bondelut SAX
cartridge, which was previously treated with methanol (1 mL)
and water (2 × 1 mL). The column was then washed with 100
µL of water. The eluates containing the purines were combined.

DNA Adducts of 4,4′-Methylenedianiline
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1107
The apurinic DNA was eluted with 0.48 M sodium phosphate,
pH 3.2 (1 mL). The radioactivity of all collected fractions was
determined by scintillation counting. The purines were ana-
lyzed after the addition of the purine adducts G-4MA, G-MDA,
and G-AcMDA and the corresponding nucleotide adducts by
HPLC with method C [Nucleosil C18 column (250 × 4.6 mm, 5
µm), 35 min gradient 20-80% MeOH in 50 mM AF buffer (pH
4.5), flow of 1.5 mL/min]. G-4MA, G-MDA, and G-AcMDA
eluted at 16.4, 18.4, and 20.4 min, respectively. Fractions of 2
min were collected and assessed for radioactivity by scintillation
counting. The samples were counted for 60 min.
32P -P ostla belin g An a lysis. Rat liver DNA (10 µg in 12.5
µL) was hydrolyzed with micrococcal endonuclease (30 mU/µL)
and spleen phosphodiesterase (6 mU/µL). The deoxynucleotide
adducts were enriched by extraction with water saturated
double distilled 1-butanol in the presence of 3 mM tetrabutyl-
ammonium chloride (21, 24). After evaporation at reduced
pressure, the residue was dissolved in water (10 µL) and 32P-
labeled with 100 µCi [32P]ATP (60 pmol) and T4 polynucleotide
kinase (7.5 U) in a final volume of 15 µL, for 2 h at room
temperature. Excess [32P]ATP was digested with apyrase (20
mU). The adducts were separated by multidirectional TLC on
PEI cellulose with the following solvents: D1: 2.3 M NaH2PO4/
NaOH, pH 5.77 (overnight onto a 10 cm wick); D3: 1 M NaH2-
PO4/NaOH, 3.8 M urea, pH 3.5; and D4: 1 M NaH2PO4, 5 M
urea, pH 7.6. For the determination of the amount of nucle-
otides, an aliquot of the diluted DNA hydrolysate was 32P-
labeled (see above) and analyzed by TLC on PEI cellulose sheets
with 120 mM NaH2PO4 (pH 6.8) as eluent.
F igu r e 1. 1H NMR (400 MHz, DMSO-d6) of the synthesized
dG-AcMDA.
Sch em e 1. Syn th esis of d G a n d d Gp Ad d u cts of
MDA a n d AcMDA
Hyd r olysis of Liver DNA a n d An a lysis by GC-MS. DNA
(1 mg) isolated as described above was taken up in 1 mL of water
(Merck, HPLC water) and transferred to Teflon centrifuge tubes
(Nalgene). The solution was made basic with NaOH (final
concentration 0.1 M) and extracted twice with dichloromethane.
The internal standard (5 pmol), MDA-d4 for MDA dosed rats,
MDA for MDA-d4 dosed rats, and 4MA (24 µg) as carrier were
added, and the samples were hydrolyzed at 110 °C overnight.
The hydrolysate was extracted with dichloromethane (5 mL).
The organic phase was passed through a Pasteur pipet with
anhydrous sodium sulfate. HFBA (5 µL) was added. After 15
min, a methanolic solution of 4MA (10 µL ) 24 µg) was added.
After evaporation in a speed evaporator, the residue was taken
up in ethyl acetate (2 × 50 µL), transferred to a microinsert for
autosampler vials, and evaporated again to dryness. The
residue was dissolved in ethyl acetate (15 µL) and analyzed by
GC-MS with the conditions published recently (8).
Resu lts a n d Discu ssion
Syn th esis of Deoxygu a n osin e Ad d u cts. DNA ad-
ducts of MDA (Scheme 1) were synthesized following a
method Boche used for the preparation of DNA adducts
of other arylamines (25-30). The intermediate nitro-
AcMDA was obtained after oxidation of AcMDA with
mCPBA. The N-hydroxyarylamine of AcMDA was ob-
tained after reduction of nitro-AcMDA with hydrazine
hydrate on 5% Pd/C. Addition of pyruvonitrile to the
N-hydroxyarylamine yielded OAc-AcMDA, which was
added to a solution of dG or dGp. The dG adduct dG-
AcMDA was isolated with 1.1% yield. dG-MDA was
obtained by deacetylation of AcMDA with a pig liver
carboxylesterase following a method described by Martin
et al. (31) for the deacetylation of N′-acetyl-N-(2′-deoxy-
guanosin-8-yl)benzidine. The dG adduct of 4MA was
synthesized according to the procedure by Meier and
Boche (25). The corresponding dGp-4MA, dGp-AcMDA,
and dGp-MDA were synthesized following the method
used for the synthesis of the dG adducts. These products
were identified by comparing the retention times and the
UV spectra with the corresponding dG adducts after
dephosphorylation with NP1. The yields of dGp-AcMDA
and dGp-4MA were 3 times higher than for the corre-
sponding nucleoside adduct syntheses.
Our attempts to synthesize the deoxyadenosine (dA)
adduct with OAc-AcMDA, according to the method de-
scribed for the synthesis of dG-AcMDA, failed. The
synthesis may be successful, following the method pub-
lished recently for N-(3′-monophosphate-2′-deoxyadenosin-
8-yl)-4-aminobiphenyl (32, 33). Another approach is the
reaction of the OAc-AcMDA with calf thymus DNA
following the method for the synthesis of the dA adducts
of MOCA (13, 14). However, it should be noted that the
yields for dG-AcMDA (34) and N-(deoxyguanosin-8-yl)-
aniline (29) are 20 times lower using DNA instead of dG
as reactant. This might be also the case for dA adducts.
Sp ectr oscop ic P r op er ties. All synthesized com-
pounds were characterized by NMR and MS (Figures
1-3). The spectra of known derivatives of MDA have

1108 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Ta ble 1. Com p a r ison of 1H NMR Da ta (DMSO-d 6) of MDA Der iva tives a n d d G-MDA
ring with changing functional groups ring with N-acetyl group
Schu¨tze et al.
compdsa
(MDA derivatives)
2,6-H
3,5-H
NHx
Ph2CH2
2′,6′-H
3′,5′-H
NH-Ac
Ac
MDA
AcMDA
6.47
6.47
6.77
7.52
8.14
7.61
7.60
6.47
6.82
6.84
7.00
7.19
7.47
7.09
7.05
6.84
4.82
4.87
8.13
3.56
3.67
3.76
3.86
4.02
3.81
3.68
7.45
7.48
7.50
7.51
7.46
7.07
7.10
7.13
7.17
7.11
9.86
9.86
9.89
9.90
9.84
2.02
2.02
2.02
2.02
2.00
hydroxyarylaminea
hydroxamic acida
nitroarenea
dG-AcMDAb
dG-MDAb,c
8.56
8.54
4.84
a
Hydroxyarylamine ) N′-acetyl-N-hydroxy-MDA, hydroxamic acid ) N,N′-diacetyl-N-hydroxy-MDA, nitroarene ) N′-acetyl-4-amino-
4-nitrodiphenylmethane. The signals of the nucleoside protons are not listed in this table. c The signals of the aromatic ring with nitrogen
b
attached to C-8 of guanine are listed in the first row. The signals of the other aromatic protons are listed in the second row.
F igu r e 2. 13C NMR (63 MHz, DMSO-d6) of dG-MDA.
also been included in the tables, since NMR data were
not available from the literature. For the aromatic
protons, spectra of the AA′BB′ type are expected. How-
ever, in general the spectra were of the AB type and were
interpreted accordingly. The change of the proton shifts
due to different functional groups are shown in Table 1.
The acetylation of the amino group in MDA shifts the
proton in the meta and ortho position 0.23 and 0.98 ppm
downfield. Oxidation of MDA to the N-hydroxyarylamine
shifts the same protons 0.18 and 0.30 ppm downfield.
With the introduction of an acetyl and a hydroxy group
as in the hydroxamic acid, the protons shift 0.37 and 1.05
ppm downfield. A nitro group instead of an amino group
as in nitro-AcMDA shifts the meta and ortho protons 0.65
and 1.67 ppm downfield. Binding of the nitrogen at the
C-8 position of guanine shifts the protons 0.23 and 1.13
ppm downfield. These shifts are comparable to those
seen in the hydroxamic acid. Similar shifts have been
registered for the 4MA derivatives as shown in the
Experimental Procedures. The downfield shift of the
proton on the nitrogen of the aromatic amine by 4 ppm
in comparison to the parent aromatic amine was char-
acteristic for the dG adducts. The signals for the dG
protons correspond well with the C-8-dG adducts of
4-methoxyaniline (25, 30), 4-chloroaniline (25), aniline
(29), 4-aminoazobenzene (35), 4-aminobiphenyl (36), 3,2′-
dimethylaminobiphenyl (37), benzidine (31), 2-naphthyl-
amine (38), 2-aminofluorene (39), 2-aminophenanthrene
(40), 2,4-dimethylaniline (41, 42), and 2,6-dimethylaniline
(41). 1H NMR data have been used to elucidate the
conformation of dG adducts of arylamines (30, 42-44).
The comparison of the chemical shifts shows that the
conformation of the new adducts correspond to the
F igu r e 3. MS of dG-MDA using (A) negative and (B) positive
electrospray ionization on a Quattro-BQ mass spectrometer.
structures as elaborated by Meier and Boche for dG-4MA
(30, 43).
The 13C NMR spectra of the synthesized adducts were
interpreted according to the C-8 guanine adducts of 4MA
(42), 4-methoxyaniline (30), 4-chloroaniline (30), aniline
(29), and 4-aminobiphenyl (28). The carbons of the dG
unit are very similar for all compounds except for N2-
(2′-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo-
[4,5-b]pyridine (dG-PhiP) (44). The multiplicity of the
carbons was determined with DEPT experiments. For
the 13C NMR data (Table 2) of the aryl carbons of all MDA
derivatives, the largest differences are seen for the
carbons attached to the nitrogen ((5.90 ppm) and for the
carbon C4 in the para position ((8.2 ppm). The chemical
shifts of the carbons in the meta position change only
slightly ((0.6 ppm). The 13C chemical shifts of the carbon
in the ortho and para position shift downfield with the

DNA Adducts of 4,4′-Methylenedianiline
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1109
Ta ble 2. Com p a r ison of th e 13C NMR Da ta (DMSO-d 6) MDA Der iva tives a n d d G Ad d u cts of MDA a n d AcMDA
ring with changing functional groups
ring with the N-acetyl group
compds
(MDA derivatives)
C-1
C-2,6
C-3,5
C-4
CH2
C-1′
C-2′,6′
C-3′,5′
C-4′
CdO
CH3
MDA
AcMDA
146.8
147.0
150.5
138.3b
150.1
139.1
138.9
146.9
114.3
114.4
113.6
121.1
123.9
117.9
117.8
114.3
129.4
129.4
129.1
129.2
130.1
129.0
128.9
129.3
129.9
128.8
132.6
140.1
146.2
133.9
134.9
129.2
40.1
40.1
40.2
40.2
40.5
40.1
40.2
137.5
137.5
137.8
138.1
137.5
119.4
119.5
119.5
119.7
119.5
129.0
129.0
128.9
129.4
129.0
137.4
140.0
136.1
134.6
136.8
168.4
168.4
168.4
168.5
168.4
24.3
24.3
24.3
24.3
24.3
hydroxyarylaminea
hydroxamic acida
nitroarenea
dG-AcMDAc
dG-MDAc,d
a
Hydroxyarylamine ) N′-acetyl-N-hydroxy-MDA, hydroxamic acid ) N,N′-diacetyl-N-hydroxy-MDA, nitroarene ) N′-acetyl-4′-amino-
4-nitro-diphenylmethane. Broad signal. c The signals of the nucleoside carbons are not listed in this table. The signals of the aromatic
b
d
ring with nitrogen attached to C-8 of guanine are listed in the first row. The signals of the other aryl carbons are listed in the second
row.
F igu r e 4. GC-MS with negative chemical ionization of MDA-d4 released from liver DNA of control rats and of rats dosed with
MDA-d4. The dichloromethane extracts were derivatized with HFBA. The fragment ion 574 [570] corresponds to the molecular ion
minus HF of the di-HFBA derivative of MDA-d4 [MDA].
increasing electron withdrawing properties of the sub-
stituent (NO2 > NHAcOH > NAc > NHOH). The dG
substituent shifts the ortho carbon 3.5 ppm and the para
carbon of the arylamine 5.0 ppm downfield. These
downfield shifts are similar to those caused by an acetyl
group on nitrogen. Similar effects were seen for the DNA
adducts of 4MA (Experimental Procedures). The position
of the nitrogen binding on guanine was seen by the
disappearance of the H-8 of guanine at 7.93 ppm and a
8 ppm downfield shift of the C-8.
The mass spectra were acquired using different meth-
ods. The molecular ion of dG-4MA could be detected
using the DCI technique; however, for dG-MDA and dG-
AcMDA only the fragments resulting from the loss of the
deoxyribose unit could be recorded. Mass spectra were
also obtained by electrospray ionization. The molecular
ion was the major ion in the negative ion spectra. For
the positive ion spectra M+ - deoxyribose was the major
peak (Figure 3).
suggests an extension of the conjugation system of
guanine with MDA.
Hyd r olysis of d G Ad d u ct Sta n d a r d s. Recently the
parent aromatic amines have been released by base
hydrolysis from DNA of animals treated with 4-amino-
biphenyl (15) and with 2-amino-1-methyl-6-phenylimid-
azo[4,5-b]pyridine (PhIP) (16). This method was applied
to the quantification of synthetic dG adducts of MDA.
dG-MDA (0.6, 2.4, and 4.8 pmol) was hydrolyzed in Teflon
tubes overnight with 0.1 M NaOH in the presence of DNA
(1 mg) of control rats and 4MA (24 µg) as carrier. The
released MDA was extracted in CH2Cl2 and analyzed by
GC-MS. The release of MDA was linear over a range of
0-5 pmol; MDA (pmol) ) 0.02 + 0.68 × dG-MDA (pmol),
r2 ) 1.0. The yield for released MDA was 68 ( 5%. For
dG-AcMDA treated under the same conditions, MDA was
found with a yield of 79 ( 7%.
32P -P ostla belin g of d G Ad d u ct Sta n d a r d s. The
postlabeling method was optimized for the quantitation
of dGp-4MA, dGp-AcMDA, and dGp-MDA. The synthe-
sized 3′-monophosphate-dG adducts were incubated with
The UV spectra of the adducts shift by about 30 nm to
longer wavelengths compared to the spectrum of dG. This

1110 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Schu¨tze et al.
and phosphodiesterase (6 mU/µL), prior to the enrich-
ment and labeling procedure. This indicates that the
synthetic adducts are not stable under the conditions
used for DNA hydrolysis. Therefore, different hydrolysis
conditions were tested with calf thymus DNA spiked with
the synthetic adducts to increase the recoveries. DNA
(5-10 µg) was spiked with the synthetic adduct stan-
dards (20-600 fmol) and was hydrolyzed to the nucle-
otides by incubation with different amounts of micrococ-
cal nuclease (10-75 mU/µL) and spleen phosphodiesterase
(0.6-6 mU/µL) in a final volume of 12 µL of nucleotide
buffer for 3 h at 37 °C. The hydrolysate was extracted
with 1-butanol and 32P-postlabeled as described above.
The adduct recoveries were increased by a factor of 2,
using 15 mU/µL micrococcal nuclease and 1 mU/µL
phosphodiesterase. The DNA was not completely di-
gested with lower amounts of enzymes. The limit of
detection of the assay under the present conditions allows
the detection of about 1 fmol of adduct in 30 nmol of
normal nucleotides. This corresponds to an adduct level
of 1 adduct in 3 × 107 normal nucleotides (RAL ) 3.3 ×
10-8).
Liver DNA Ad d u cts in Vivo. Three approaches were
used for the analysis and quantification of liver-DNA
adducts: (i) HPLC analysis of DNA isolated from animals
dosed with radiolabeled MDA, (ii) analysis of the DNA
by 32P-postlabeling, and (iii) base hydrolysis of the DNA
and determination of the released MDA by GC-MS. In
rats given ip 14C-labeled MDA (5.6 µmol/kg, 0.3 mCi/kg;
116.5 µmol/kg, 3 mCi/kg), radioactivity was associated
with the purified liver DNA. Specific activities of 2.4 and
50.4 dpm/mg of DNA were found with the DNA isolated
from the low and high dosed animals. Repurification of
the DNA sample from the high dosed animal did not
result in a reduction of the specific DNA radioactivity.
This indicates that, during the DNA isolation process,
the DNA adducts formed with DNA were stable and all
noncovalently bound radiolabeled metabolites of MDA
were removed. For the animals dosed with 5.6 and 116.5
µmol [14C]MDA/kg body weight, the chemical binding
index (CBI) [(µmol of chemical bound/mol of DNA)/(mmol
of chemical applied/kg body weight)] was 1.05 and 2.3,
respectively. This classifies MDA as a weak genotoxic
carcinogen (46). DNA was hydrolyzed to the nucleotides
and analyzed by HPLC with method A. The correspond-
ing radioactivity profile (Figure 6A) showed that no
radioactivity coeluted with the normal nucleotides. The
major peak of the recovered radioactivity (78%) eluted
distant from the nucleotides, in the region known to
contain the more lipophilic DNA adducts. This indicates
covalent adduct formation of MDA. To investigate the
type of MDA adducts, DNA hydrolysate was analyzed by
HPLC with method B in the presence of the synthetic
adducts (Figure 6B). Additionally, the DNA spiked with
the synthetic adducts was chemically hydrolyzed to the
purines and analyzed by HPLC with method C (Figure
6C). The recoveries of the radioactivity for the two
analyses were 79% and 70%, respectively. The results
clearly showed that the radioactivity did not coelute with
one of the synthesized standards. The same DNA was
used for the analysis of DNA adducts by the optimized
postlabeling technique. No adducts corresponding to
dGp-4MA, dGp-MDA, or dGp-AcMDA could be detected.
The same result was obtained with DNA of female Wistar
rats dosed with 500 µmol/kg MDA‚2HCl by gavage. No
adducts corresponding to the synthetic standards could
be detected (Figure 5D), although the adduct level (see
F igu r e 5. 32P-Postlabeling analysis of (A) standard DNA
adducts: dG-MDA, dG-AcMDA, and dG-4MA; (B) control DNA;
(C) DNA of control rats spiked with adduct standards resulting
in a RAL of 1 × 10-6; and (D) DNA (10 nmol) from an MDA
treated rat (500 µmol/kg).
T4 polynucleotide kinase (PNK) (0.2-0.5 U/µL) and [32P]-
ATP (1-5 pmol/µL) for various time periods (1-3 h) to
check the labeling efficiency. The products were analyzed
by multidirectional TLC on PEI cellulose. After the
labeling reaction, a multidirectional chromatography on
PEI coated cellulose TLC sheets was chosen to resolve
the adducted nucleotides (Figure 5). The first develop-
ment (D1) with 2.3 M NaH2PO4/NaOH (pH 5.77) as
eluent was used to elute impurities and contaminating
normal nucleotides, while dGp-AcMDA and dGp-MDA
remained at the origin. The dGp-4MA slowly migrated
under this condition to 1-2 cm above the origin. The
development in the D3 and D4 solvent system resulted
in sufficient separation of the adducts (Figure 5A-D).
The best labeling efficiencies were obtained after an
incubation time of 2 h with 0.5 U/µL PNK and 4 µM [32P]-
ATP: ca. 70% for dGp-4MA and 60% for dGp-AcMDA and
20% for dGp-MDA. The methods of adduct enrich-
ments1-butanol extraction and NP1 digestionswere
tested with the dGp adduct standards. Using NP1
digestion, the recoveries for the adducts were much less
than 1%. This indicates that the respective dGp adducts
were dephosphorylated by NP1 as well as the normal
nucleotides. This confirms the results obtained with
other dGp adducts of aromatic amines (45). Using
1-butanol extraction, the recoveries for the adducts
improved with increasing concentrations of TBA. In the
presence of 3 mM TBA, dGp-4MA, dGp-AcMDA, and
dGp-MDA were recovered with 56%, 71%, and 23%,
respectively. Therefore, the overall procedure with 1-bu-
tanol extraction in the presence of 3 mM TBA and
labeling for 2 h with 0.5 U PNK/µL and 4 µM [32P]ATP
should result in recoveries of about 40%, 40%, and 4%
for dGp-4MA, dGp-AcMDA, and dGp-MDA, respectively.
This is in agreement with the experimentally found
recoveries of 32%, 30%, and 2%. These recoveries were
much lower (3%, 6%, and 0.8% for dGp-4MA, dGp-
AcMDA, and dGp-MDA, respectively) when the adducts
were incubated with micrococcal nuclease (30 mU/µL)

DNA Adducts of 4,4′-Methylenedianiline
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1111
F igu r e 6. Analysis of liver DNA isolated from an animal after treatment with radiolabeled MDA. The radioactive background
levels (11 cpm) obtained from experiments with control DNA were not subtracted. DNA was digested with spleen phosphodiesterase
and micrococcal nuclease and analyzed by HPLC (A) with method A. (B) The DNA hydrolysate was spiked with the adducts dGp-
4MA, dGp-MDA, and dGp-AcMDA and analyzed by HPLC with method B. (C) DNA spiked with the adducts dGp-4MA, dGp-MDA,
and dGp-AcMDA was chemically hydrolyzed and analyzed by HPLC with method C.
GC-MS analysis) is higher than the determination limit
found for the postlabeling method of the synthetic
standards spiked to control DNA (Figure 5C).
the DNA solution with dichloromethane prior to hydroly-
sis overnight eliminates noncovalently bound MDA.
The structure of the MDA-DNA adduct found in vivo
is still unknown. At the moment it is unclear if only C-8
adducts of guanine are cleavable with base. It is conceiv-
able that also the C-8 adducts with deoxyadenosine (dA)
release MDA after base treatment. The release of MDA
with base suggests that a MDA bond with the C-8 of a
purine base is present.
Livers of female Wistar rats treated with MDA‚2HCl
and/or MDA-d4‚2DCl were analyzed by GC-MS following
the third approach (Figure 4). DNA was isolated as
described for the postlabeling analyses. Livers from
animals dosed the same day with only the vehicle were
used as controls, in order to account for background levels
of MDA. Liver DNA of two female Wistar rats given 500
µmol/kg MDA‚2HCl was hydrolyzed in 0.1 M NaOH
overnight at 110 °C in the presence of the recovery
standard MDA-d4. GC-MS analysis of the HFBA deriva-
tized dichloromethane extracts detected 428 ( 40 fmol
of MDA/mg of DNA. In the control animals no MDA was
found. The experiment was repeated with livers from
animals dosed with 500 µmol/kg MDA-d4. In these rats
488 ( 19 fmol of MDA-d4/mg of DNA was found to be
bound. Taking into account a 68% yield of the method,
the CBI found in these cases was 0.82 and 1.0, respec-
tively. Chemical release of the parent compound from
protein or DNA adducts always raises the question about
the covalent character of the binding. DNA from rats
given MDA-d4 was thoroughly tested for the presence of
noncovalently bound MDA-d4. DNA was dissolved in 0.1
M NaOH, spiked with the internal standard 3,3′-meth-
ylenedianiline, and extracted with dichloromethane. No
MDA-d4 could be found in this first extract; but MDA-d4
was released after hydrolysis overnight (see above). In
addition, the workup procedure was tested with regard
to the detection of noncovalently bound MDA. DNA (1
mg) in 0.1 M NaOH was spiked with 2 pmol of MDA.
After incubation for 1 h at room temperature, the amines
were extracted twice with dichloromethane. The water
phase was then spiked with MDA-d4 (5 pmol) as internal
standard and 4MA (24 µg) as carrier, hydrolyzed over-
night at 110 °C, and extracted with dichloromethane.
MDA was quantified against MDA-d4. No MDA could
be detected in the final extract. Therefore, extraction of
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