Hemoglobin Binding of Bicyclic Aromatic Amines - American Chemical

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Chem. Res. Toxicol. 1998, 11, 471-483

471

Hemoglobin Binding of Bicyclic Aromatic Amines Gabriele Sabbioni* and Dietrich Schu¨tze† Walther-Straub-Institut fu¨ r Pharmakologie und Toxikologie, Ludwig-Maximilians-Universita¨ t Mu¨ nchen, Nussbaumstrasse 26, D-80336 Mu¨ nchen, Germany Received September 4, 1997

Aromatic diamino compounds, e.g., 4,4′-methylenebis(2-chloroaniline) (MOCA) and 4,4′methylenedianiline (MDA), are used as curing agents in the production of elastomers. Since MOCA and MDA are mutagenic and carcinogenic, substitutes are of great commercial interest. For benzidine it has been shown that ortho substitution with methyl groups yields the nonmutagenic 3,3′,5,5′-tetramethylbenzidine. Therefore, MDA analogues with large substituents in the ortho position have been synthesized. The substituents are supposed to inhibit the formation of the N-hydroxyarylamines which are the putative genotoxic intermediates. We investigated the biological availability of the N-hydroxylamines of ortho-substituted diamines and of known carcinogenic diamines in female Wistar rats, by determining hemoglobin (Hb) adducts. Hb from rats dosed with 0.5 mmol/kg diamine and from controls was isolated and hydrolyzed in base. The released diamine and monoacetyldiamine were quantified by HPLC with electrochemical detection and/or GC/MS. MDA, 4,4′-oxydianiline (ODA), 4,4′ethylenedianiline, and 4,4′-thiodianiline (TDA) bound to hemoglobin as diamine and as monoacetyl-diamine. 4,4′-Methylenebis(2,6-dimethylaniline), 4,4′-methylenebis(2,6-diethylaniline), MOCA, and 4,4′-sulfonyldianiline (dapsone) bound only as diamine to Hb. 4,4′Methylenebis(2,6-dichloroaniline) did not bind to Hb. Thus, the presence of two substituents in the ortho position and the presence of electron-withdrawing groups in the para position to the amino group drastically reduced the formation of Hb adducts. The amount of hemoglobin adducts was compared to their carcinogenic potency. The extent of hemoglobin binding of the bicyclic diamines (dapsone, 3,3′-dichlorobenzidine, MDA, MOCA, TDA, ODA, and benzidine) increases with their carcinogenic potency.

Introduction Aromatic diamino compounds, e.g., 4,4′-methylenebis(2-chloroaniline) (MOCA)1 and 4,4′-methylenedianiline (MDA) (Chart 1), are used as curing agents in the production of polyurethane elastomers. Most compounds with the MDA structure are mutagenic and carcinogenic (1-3). MDA (4), 4,4′-methylenebis(2-methylaniline), 4,4′methylenebis(2-ethylaniline), 4,4′-methylenebis(2-fluoroaniline), MOCA (5), 4,4′-ethylenedianiline (EDA) (6), 4,4′-oxydianiline (ODA), and 4,4′-thiodianiline (TDA) (4) are mutagenic in Salmonella typhimurium TA98 (7). Dapsone, MDA, MOCA, ODA, and TDA are animal * Corresponding author. Tel/Fax: (089) 51452250. E-mail: [email protected]. † Present address: Boehringer Ingelheim Pharma KG, Birkendorferstrasse 65, D-88397 Biberach an der Riss, Germany. 1 Abbreviations: 4,4′-methylenebis(2-chloroaniline) (MOCA), 4,4′methylenedianiline (MDA), hemoglobin (Hb), 4,4′-oxydianiline (ODA), 4,4′-thiodianiline (TDA), 4,4′-sulfonyldianiline (dapsone), 4,4′-ethylenedianiline (EDA), 4,4′-methylenebis(2,6-dimethylaniline) (Me,MeMDA), 4,4′-methylenebis(2,6-diethylaniline) (Et,Et-MDA), 4,4′-methylenebis(2,6-dichloroaniline) (Cl,Cl-MDA), 4-ethylaniline (4EtA), 4,4′methylenediphenyl diisocyanate (MDI), 2,4,6-trimethylaniline (246TMA), 4,4′-methylenedianiline dihydrochloride (MDA‚2HCl), 4,4′-oxydianiline dihydrochloride (ODA‚2HCl), 4,4′-methylenebis(2-chloro-6-methylaniline) (Cl,Me-MDA), N′-acetyl-4,4′-methylenedianiline (AcMDA), N′acetyl-4,4′-ethylenedianiline (AcEDA), N′-acetyl-4,4′-oxydianiline (AcODA), N′-acetyl-4,4′-methylenebis(2,6-dimethylaniline) (Me,Me-AcMDA), N′-acetyl-4,4′-methylenebis(2,6-diethylaniline) (Et,Et-AcMDA), N′acetyl-4,4′-methylenebis(2,6-dichloroaniline) (Cl,Cl-AcMDA), N′-acetyl4,4′-methylenebis(2-chloro-6-methylaniline) (Cl,Me-AcMDA), N′-acetyl4,4′-thiodianiline (AcTDA), N′-acetyl-4,4′-sulfonyldianiline (Acdapsone), half-wave oxidation potential (E1/2), electrochemical detector (ECD), single-ion monitoring (SIM), heptafluorobutyric acid anhydride (HFBA), benzidine (Bz), 3,3′-dichlorobenzidine (DcBz).

carcinogens (8, 9). In rats, MOCA binds to DNA, albumin, and Hb (10, 11). Cheever et al. (12, 13) demonstrated that blood protein and DNA binding of MOCA increases proportionally with the dose. MDA binds to Hb of rats (14) and to liver DNA [chemical binding index ) 2.3 (15)]. Kugler-Steigmeier (16) found DNA adducts of 4,4′-methylenebis(2-ethyl-6-methylaniline). The adduct could result from the oxidation of the amino group or also from the C-oxidation at the exocyclic carbon. Substitutes for MOCA and MDA, which are less mutagenic and carcinogenic, are of great commercial interest. For benzidine it has been shown that ortho substitution with methyl groups yields the nonmutagenic 3,3′,5,5′-tetramethylbenzidine (1-3). Therefore, MDA analogues with large substituents in the ortho position have been synthesized. The substituents are supposed to inhibit the formation of the N-hydroxylamines and the reaction thereof with DNA. We propose the quantitation of hydrolyzable Hb adducts as a fast method to estimate the biologically available N-hydroxylamines. For the present work, we investigated the formation of hydrolyzable Hb adducts with MDA, ODA, TDA, dapsone, 4,4′-methylenebis(2,6-dimethylaniline) (Me,Me-MDA), 4,4′-methylenebis(2,6-diethylaniline) (Et,Et-MDA), and 4,4′-methylenebis(2,6dichloroaniline) (Cl,Cl-MDA) in rats.

Materials and Methods Animal Experiments. Female Wistar rats (200-225 g) were obtained from the Zentralinstitut fu¨r Versuchstierkunde

S0893-228x(97)00164-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/30/1998

472 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 Chart 1. Structures of the Compounds

(Hannover, FRG). They had free access to feed (Altromin 1324) and water. The test compounds (0.1 mL/100 g of body weight) were administered as solutions (0.5 M) by gavage to groups of two animals. The compounds were given in the following solvents: MDA and EDA in 1,2-propanediol; TDA and dapsone in tricapryllin; MDA‚2HCl and ODA‚2HCl in water; Me,MeMDA‚2HCl and Et,Et-MDA‚2HCl in water/1,2-propanediol, 1:3; Cl,Cl-MDA in DMSO. 4,4′-Methylenediphenyl diisocyanate (MDI) was given ip and/or by gavage in tricapryllin. After 24 h the animals were anesthetized with ether, and blood (4-6 mL) was taken by heart puncture with a heparinized syringe. Chemicals. Silica gel 60 (0.063-0.2 mm) and silica gel 60 (0.040-0.063 mm) from Merck (Darmstadt, Germany) were used for chromatography and flash chromatography, respectively. The TLC plates DC ALUGRAM SIL G/UV254 were obtained from Macherey-Nagel (Du¨ren, Germany). Dapsone, diethyl ether, dioxane, MDI, and acetic acid anhydride were obtained from Merck (Darmstadt, Germany); ammonium formate, benzidine, dapsone, 4-ethylaniline (4EtA), MDA, ODA, 2,4,6-trimethylaniline (246TMA), and triethylamine were from Fluka (Buchs, Switzerland); ethyl acetate p.a. and tetrahydrofuran were from Riedel-de Haen (Selze, Germany); and TDA were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 4-Ethylaniline was distilled over a 15-cm Vigreux column (bp 98-101 °C/18 Torr). To obtain colorless needles, MDA was recrystallized three times from water. EDA was recrystallized three times from ethanol/ water. Benzidine (Bz) was recrystallized from ethanol. MDI was distilled over a Vigreux column (15 cm). The colorless liquid with a boiling point at 163-165 °C/0.3 Torr [lit. (17) bp 150 °C/ 0.3 Torr; lit. (18) bp 170 °C/0.2 Torr] was collected. At room temperature the liquid solidifies: mp 39-42 °C [lit. (18) mp 38 °C; lit. (19) mp 46 °C]. The purity (>97%) of the recrystallized and distilled compounds was determined by GC/MS, except for MDI. The mass spectra correspond to the spectrum present in the reference library NBS spectra library by Hewlett-Packard (HP59988K). MDA-d4 was synthesized according to Schu¨tze et al. (20). Me,Me-MDA and Et,Et-MDA were a gift from Lonza (Visp, Switzerland).

Sabbioni and Schu¨ tze Caution: The aromatic amines used in the present work are potentially carcinogenic. They should be handled with protective clothing in a well-ventilated fume hood. Instrumentation. 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 Select B (250 × 4 mm, 5 µm) from Merck (Darmstadt, Germany), Nucleosil C18 (250 × 12.5 mm, 10 µm) from Macherey-Nagel (Du¨ren, Germany), and Supersphere RP18 (125 × 2 mm, 3 µm) from Bischoff (Leonberg, Germany). NMR spectra were recorded on a Bruker AC 250 spectrometer. The 13C signals were distinguished by a DEPT (distortionless enhancement by polarization transfer) experiment. Internal standard was tetramethylsilane 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). 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). 4,4′-Methylenebis(2,6-dichloroaniline) (Cl,Cl-MDA). A 35% formaldehyde solution (1.85 g, 61.7 mmol) was added to a suspension of 2,6-dichloroaniline (20 g, 132 mmol) in water (20 mL) and 6 M HCl (21 mL). The reaction mixture was stirred for 18 h at 100 °C and then cooled to 4 °C. The precipitate was filtered and washed with cold water. To release the free base, the precipitate was suspended in water (300 mL) and an excess of sodium carbonate was added in portions. The precipitated free base was filtered, washed with water, and recrystallized from dimethylformamide (950 mL). After drying over silica gel, 12.5 g [61%, lit. (21) 70%] of colorless needles of Cl,Cl-MDA was obtained, mp 267-269 °C [Lit. (21) mp 260-262 °C]. 1H NMR (250 MHz, DMSO-d6): δ 3.62 (s, 2 H, Ph2CH2), 5.43 (s, 4 H, NH2), 7.13 (s, 4 H, arom H). IR (KBr): ν 3480, 3360 cm-1 (NH), 2960, 2880 (CH), 1645, 1520 (CdC), 1095 (CCl). UV (methanol): λmax (log ) 246 nm (4.344), 302 (3.825). MS (70 eV): m/z (%) 338 (20) [M + 4]+, 337 (11), 336 (41) [M + 2]+, 335 (15), 334 (31) [M+], 303 (26), 301 (87), 299 (100), 297 (21), 265 (30), 263 (45), 229 (40), 228 (25), 178 (38), 174 (60), 149 (20), 138 (33), 114 (23). 4,4′-Methylenebis(2-chloro-6-methylaniline) (Cl,MeMDA). A 35% formaldehyde solution (2.12 g, 70.6 mmol) was added to a suspension of 2-chloro-6-methylaniline (20.0 g, 141 mmol) in water (22 mL) and 6 M HCl (24 mL). The reaction mixture was stirred for 18 h at 100 °C and then cooled to 4 °C. The orange precipitate was filtered and washed with cold water. To release the free base, the precipitate was suspended in water (100 mL) and an excess of sodium carbonate was added in portions. The precipitated free base was filtered, washed with water, and recrystallized from ethanol (1600 mL). After drying over silica gel, 14.3 g (69%) of colorless needles of Cl,Me-MDA was obtained, mp 210-212 °C. 1H NMR (250 MHz, DMSO-d6): δ 2.09 (s, 6 H, CH3), 3.55 (s, 2 H, Ph2CH2), 4.84 (s, 4 H, NH2), 6.77 (d, J ) 1.7 Hz, 2 H, H-5), 6.90 (d, J ) 1.7 Hz, 2 H, H-3). 13C NMR (63 MHz, DMSO-d ): δ 18.1 (q, CH ), 38.6 (t, Ph 6 3 2 CH2), 117.0 (s, C-2), 123.2 (s, C-6), 126.2 (d, C-3), 128.9 (d, C-5), 130.0 (s, C-4), 140.2 (s, C-1). IR (KBr): ν 3450, 3360 cm-1 (NH), 3030, 3000 (dCH), 2940, 2870 (CH), 1640, 1500 (CdC), 1085 (CCl). UV (methanol): λmax (log) 245 nm (4.330), 295 (3.719). MS (70 eV): m/z (%) 296 (25) [M + 2]+, 295 (18), 294 (45) [M+], 279 (23), 261 (33), 260 (20), 259 (100), 257 (32), 243 (29), 224 (17), 223 (39), 209 (24), 208 (21), 178 (18), 156 (25), 154 (81), 153 (18), 152 (18), 129 (34), 118 (33), 117 (25), 112 (17), 104 (52), 91 (45), 89 (20), 78 (19), 77 (49), 65 (22). 4,4′-Methylenedianiline Dihydrochloride (MDA‚2HCl). Concentrated HCl (10 mL, 120 mmol) was added to 4,4′methylenedianiline (5.00 g, 25.2 mmol) in methanol (30 mL) under ice cooling. After 4.5 h, the fine needles were filtered and recrystallized from 90% ethanol. After drying over silica gel, colorless needles of MDA‚2HCl (1.04 g, 15%) were obtained.

Hemoglobin Binding of Bicyclic Aromatic Amines The needles melt and decompose at 260-270 °C [lit. (22) mp 288 °C dec]. 1H NMR (250 MHz, D2O): δ ) 3.80 (s, 2 H, Ph2CH2), 7.24 (d, J ) 8.4 Hz, 4 H, H-2,6), 7.32 (d, J ) 8.4 Hz, 4 H, H-3,5). 4,4′-Oxydianiline Dihydrochloride (ODA‚2HCl). Concentrated HCl (50 mL) was added slowly to 4,4′-oxydianiline (10.0 g, 50.0 mmol) in ethanol (80 mL) and 2 M HCl (50 mL). The formed precipitate was filtered and washed with a little cold ethanol. After recrystallization in 1-propanol/water (9:1) and drying in a desiccator under silica gel, slightly brown needles (8 g, 59%) of ODA‚2HCl were obtained, which melt under decomposition at 255-260 °C. 1H NMR (250 MHz, D2O): δ 7.10 (d, J ) 9 Hz, 4 H, H-2,6), 7.35 (d, J ) 9 Hz, 4 H, H-3,5). N′-Acetyl-4,4′-methylenedianiline (AcMDA). Triethylamine (2.02 g, 20.0 mmol) and acetic anhydride (3.06 g, 30.0 mmol) were added to MDA (3.97 g, 20.0 mmol) in dioxane (50 mL). The reaction was followed by TLC [silica gel, ethyl acetate, Rf (MDA) ) 0.61, Rf (AcMDA) ) 0.42]. A precipitate was formed. After 1 h, 1 M NaOH (50 mL) and water (350 mL) were added. The precipitate was filtered and dried over silica gel in a desiccator. An aliquot of the crude product was purified by HPLC [Nucleosil C18 (250 × 12.5 mm, 10 µm), 0.02 M phosphate buffer, pH 4.5/methanol (43:57), flow 2.5 mL/min]. The combined fractions containing AcMDA (tR ) 12.5 min) were evaporated in vacuo until crystals started to precipitate and then stored at 4 °C for 24 h. The fine colorless needles were filtered and dried over silica gel in a desiccator. AcMDA (430 mg) was obtained with a melting point at 138-139 °C [lit. (23) mp 121122 °C; lit. (24) mp 135.5-136 °C]. 1H NMR (80 MHz, CDCl3/ TMS): δ 2.15 (s, 3 H, CO-CH3), 3.80 (s, 2 H, Ph2CH2), 3.5 (br s, 3 H, NH), 6.5-7.4 (m, 8 H, arom H). 1H NMR (250 MHz, DMSO-d6): δ 2.02 (s, 3 H, CO-CH3), 3.67 (s, 2 H, Ph2CH2), 4.87 (s, 2 H, NH2), 6.47 (d, J ) 8.3 Hz, 2 H, H-2,6), 6.84 (d, J ) 8.3 Hz, 2 H, H-3,5), 7.07 (d, J ) 8.4 Hz, 2 H, H-3′,5′), 7.45 (d, J ) 8.4 Hz, 2 H, H-2′,6′), 9.86 (s, 1 H, NH-CO-CH3). 13C NMR (63 MHz, DMSO-d6): δ 24.3 (CO-CH3), 40.1 (Ph2CH2), 114.4 (C-2,6), 119.4 (C-2′,6′), 128.8 (C-4), 129.0 (C-3′,5′), 129.4 (C-3,5), 137.4 (C-4′ or C-1′), 137.5 (C-1′ or C-4′), 147.0 (C-1), 168.4 (COCH3). MS (70 eV): m/z (%) 241 (17), 240 (100) [M+], 239 (11), 198 (52), 197 (99), 182 (38), 181 (16), 180 (25), 106 (59), 104 (14), 93 (12), 77 (13). N′-Acetyl-4,4′-ethylenedianiline (AcEDA). Triethylamine (1.01 g, 10.0 mmol) and acetic anhydride (1.53 g, 15.0 mmol) were added to ethylenedianiline (EDA) (2.12 g, 10.0 mmol) in dioxane (80 mL). The reaction was followed by TLC [silica gel, ethyl acetate, Rf (EDA)) 0.61, Rf (AcEDA) ) 0.48]. After 10 min a precipitate was formed. The reaction was stopped after 6 h of stirring at room temperature. The reaction mixture was basified with NaOH (50 mL, 1 M) and extracted with dichloromethane (4 × 25 mL). The combined organic phases were dried over magnesium sulfate and evaporated. A small portion of the brown oil was purified by HPLC [Nucleosil C18 (250 × 12.5 mm, 10 µm), 0.02 M phosphate buffer, pH 4.5, with 5% methanol/methanol (45:55), flow 2.5 mL/min]. The fraction with AcEDA (tR ) 10.2 min) was evaporated, basified with 1 M NaOH, and extracted with diethyl ether (5 × 10 mL). The extract was dried over MgSO4 and evaporated to dryness. The residue was recrystallized from ethanol/water (30:70) and dried over silica gel. Slightly yellow needles (80 mg) of AcEDA were yielded: mp 151-152 °C [lit. (23) mp 146-147 °C]. HPLC purity (245 nm) >99%. 1H NMR (80 MHz, CDCl3/TMS): δ 2.13 (s, 3 H, CO-CH3), 2.78 (s, 4 H, Ph-(CH2)2-Ph), 3.3 (br s, 3 H, NH), 6.5-7.4 (m, 8 H, arom H). MS (70 eV): m/z (%) 254 (5) [M+], 106 (100). N′-Acetyl-4,4′-oxydianiline (AcODA). Triethylamine (2.02 g, 20.0 mmol) and acetic acid anhydride (3.06 g, 30.0 mmol) were added to ODA (4.00 g, 20.0 mmol) in dioxane (100 mL). The progress of the reaction was followed by TLC [silica gel, ethyl acetate/hexane (6:4), Rf (ODA) ) 0.28, Rf (AcODA) ) 0.10]. After 30 min, a colorless precipitate formed; after 1 h, the reaction mixture was basified with 1 M NaOH (100 mL) and diluted with

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 473 water (400 mL). The formed precipitate was filtered off, rinsed with water, and dried over silica gel in a desiccator. A portion of the reaction mixture was purified by HPLC [Nucleosil C18 (250 × 12.5 mm, 10 µm), 0.02 M phosphate buffer, pH 4.5/methanol (52:48), flow 2.5 mL/min]. The AcODAcontaining fractions (tR ) 14.1 min) were evaporated to dryness. The colorless residue was taken up in 1 M NaOH (50 mL) and extracted with diethyl ether (5 × 20 mL). The extract was evaporated. Then the residue was taken up in a little acetone and filtered. Evaporation of acetone under a stream of nitrogen was followed by drying over silica gel. Slightly yellow needles of AcODA (49 mg) were obtained: mp 128-131 °C [lit. (25) mp 131-133 °C]. 1H NMR (250 MHz, DMSO-d6): δ 2.06 (s, 3 H, CO-CH3), 4.99 (s, 2 H, NH2), 6.63-7.54 (m, 8 H, arom H), 9.90 (s, 1 H, Ac-NH). 13C NMR (63 MHz, DMSO-d6): δ 23.7 (COCH3), 114.7 (C-2,6), 116.8 (C-3′,5′), 120.3 (C-2′,6′ or C-3,5), 120.4 (C-3,5 or C-2′,6′), 133.6 (C-1′), 145.4 (C-1 or C-4), 146.1 (C-4 or C-1), 154.1 (C-4′), 167.7 (CO-CH3). IR (KBr): ν 3450, 3300, 3270 cm-1 (NH), 3080, 3060 (dCH), 1670 (CdO), 1230 (dCO). UV (methanol): λ max (log ) 203 nm (4.613), 253 (4.339). MS (70 eV): m/z (%) 243 (17), 242 (100) [M+], 201 (16), 200 (100), 171 (24), 108 (45), 80 (16), 65 (26). N′-Acetyl-4,4′-methylenebis(2,6-dimethylaniline) (Me,Me-AcMDA). Triethylamine (1.01 g, 10.0 mmol) and acetic acid anhydride (1.53 g, 15.0 mmol) were added to 4,4′-methylenebis(2,6-dimethylaniline) (2.54 g, 10.0 mmol) in dioxane (15 mL). The progress of the reaction was followed by TLC [silica gel, ethyl acetate/hexane (8:2), Rf (Me,Me-MDA) ) 0.75, Rf (Me,MeAcMDA) ) 0.25]. A colorless precipitate formed after 1 h. The reaction mixture was basified with 1 M NaOH (10 mL) and diluted with water (100 mL). After separation of the precipitate, washing with water, and drying in a desiccator over silica gel, colorless crystals were obtained. A portion (1.5 g) of this product mixture was purified by column chromatography on silica gel [150 g of silica gel, ethyl acetate/hexane (8:2)]. Evaporation of the solvents in vacuo, recrystallization of the residue from ethyl acetate/hexane (8:2), and drying over silica gel gave 410 mg (26%) of slightly yellow, fine needles of Me,Me-AcMDA, mp 208211 °C. 1H NMR (250 MHz, DMSO-d6): δ 2.02 (s, 3 H, COCH3), 2.04 (s, 6 H, 2,6-CH3 or 2′,6′-CH3), 2.07 (s, 6 H, 2′,6′-CH3 or 2,6-CH3), 3.61 (s, 2 H, Ph2CH2), 4.35 (s, 2 H, NH2), 6.66 (s, 2 H, H-3,5), 6.84 (s, 2 H, H-3′,5′), 9.12 (s, 1 H, Ac-NH). 13C NMR (63 MHz, DMSO-d6): δ 17.8 (2,6-CH3), 18.1 (2′,6′-CH3), 22.5 (CO-CH3), 40.3 (Ph2CH2), 120.6 (C-2,6), 127.6 (C-3′,5′ or C-3,5), 128.1 (C-3,5 or C-3′,5′), 128.3 (C-4), 132.9 (C-1′), 134.7 (C-2′,6′), 140.4 (C-4′), 142.1 (C-1), 167.8 (CO-CH3). IR (KBr): ν 3490, 3410, 3280 cm-1 (NH), 3040 (dCH), 2980, 2940, 2870 (-CH), 1655 (CdO), 1545, 1500 (CdC). UV (methanol): λmax (log ) 206 nm (4.763), 236 (4.151), 289 (3.313). MS (70 eV): m/z (%) 297 (21), 296 (100) [M+], 281 (42), 254 (18), 253 (38), 240 (26), 239 (87), 238 (41), 223 (26), 222 (28), 208 (15), 134 (65), 133 (18), 91 (16). N′-Acetyl-4,4′-methylenebis(2,6-diethylaniline) (Et,EtAcMDA). Triethylamine (1.01 g, 10.0 mmol) and acetic acid anhydride (1.53 g, 15.0 mmol) were added to 4,4′-methylenebis(2,6-diethylaniline) (3.10 g, 10.0 mmol) in dioxane (20 mL). The progress of the reaction was followed by TLC [silica gel, ethyl acetate/hexane (6:4), Rf(Et,Et-MDA) ) 0.86, Rf (Et,Et-AcMDA) ) 0.38]. The reaction mixture was basified after 1 h with 1 M NaOH and diluted with water (100 mL). The formed precipitate was separated, washed with water, and dried over silica gel in a desiccator. The residue was dissolved in ethyl acetate/hexane (6:4) and filtered. After evaporation of the solvents, 1.84 g of colorless crystals was obtained. A portion of the product mixture (1 g) was purified by column chromatography (150 g of silica gel, ethyl acetate/hexane, 6:4). After evaporation of the solvents, the residue was recrystallized from ethyl acetate/hexane (6:4). Et,Et-AcMDA (210 mg, 11%) was obtained as slightly yellow needles, mp 168-170 °C. 1H NMR (250 MHz, DMSO-d6): δ 1.06 (t, J ) 7.5 Hz, 6 H, 2′,6′-CH2-CH3 or 2,6-CH2-CH3), 1.11 (t, J ) 7.5 Hz, 6 H, 2,6-CH2-CH3 or 2′,6′-CH2-CH3), 2.02 (s, 3 H, CO-CH3), 2.44 (q, J ) 7.5 Hz, 4 H, 2,6-CH2-CH3 or 2′,6′-CH2CH3), 2.45 (q, J ) 7.5 Hz, 4 H, 2′,6′-CH2-CH3 or 2,6-CH2-

474 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 CH3), 3.70 (s, 2 H, Ph2CH2), 4.36 (s, 2 H, NH2), 6.70 (s, 2 H, H-3,5), 6.88 (s, 1 H, H-3′,5′), 9.10 (s, 1 H, Ac-NH). 13C NMR (63 MHz, DMSO-d6): δ 13.3 (2,6-CH2-CH3), 14.5 (2′,6′-CH2CH3), 22.4 (CO-CH3), 23.7 (2,6-CH2-CH3), 24.3 (2′,6′-CH2CH3), 40.4 (Ph2CH2), 125.9 (C-3′,5′ or C-3,5), 126.0 (C-3,5 or C-3′,5′), 126.0 (C-2,6), 128.4 (C-4), 131.6 (C-1′), 140.7, 140.8, and 140.9 (C-4′, C-1, C-2′,6′), 168.5 (CO-CH3). IR (KBr): ν 3460, 3390, 3290 cm-1 (NH), 3030 (dCH), 2990, 2960, 2800 (-CH), 1665 (CdC), 1540, 1490 (CdC). UV (methanol): λmax (log ) 207 nm (4.772), 237 (4.077), 290 (3.409). MS (70 eV): m/z (%) 353 (12), 352 (61) [M+], 323 (24), 309 (41), 293 (27), 264 (21), 162 (100), 160 (45), 148 (15), 147 (25), 146 (44), 132 (18), 130 (16). N′-Acetyl-4,4′-methylenebis(2,6-dichloroaniline) (Cl,ClAcMDA). Triethylamine (2.86 g, 28.3 mmol) and acetic acid anhydride (3.85 g, 37.7 mmol) were added to a suspension of 4,4′-methylenebis(2,6-dichloroaniline) (6.34 g, 18.9 mmol) in dioxane (300 mL). The mixture was refluxed for 48 h (the solution became clear with heat). The progress of the reaction was followed by TLC [silica gel, ethyl acetate/hexane (4:6), Rf (Cl,Cl-MDA) ) 0.86, Rf (Cl,Cl-AcMDA) ) 0.34]. The mixture was then cooled to room temperature, basified with 1 M NaOH (50 mL), and diluted with water (150 mL). The formed colorless precipitate was filtered off, rinsed with water, and dried in a desiccator over silica gel. A portion of the crystals (1.50 g) was purified by column chromatography (150 g of silica gel, ethyl acetate/dichloromethane, 1:15). The collected fraction was evaporated and recrystallized from chloroform. Fine, colorless, long needles (49.0 mg, 4.1%) of Cl,Cl-AcMDA were obtained, mp 267-269 °C (sublimation). 1H NMR (250 MHz, DMSO-d6): δ 2.04 (s, 3 H, CO-CH3), 3.78 (s, 2 H, Ph2CH2), 4.99 (s, 2 H, NH2), 7.23 (s, 2 H, H-3,5), 7.43 (s, 2 H, H-3′,5′), 9.75 (s, 1 H, Ac-NH). 13C NMR (63 MHz, DMSO-d6): δ 22.3 (CO-CH3), 37.9 (Ph2CH2), 118.0 (C-2,6), 128.09 (C-3′,5′ or C-3,5), 128.13 (C-3,5 or C-3′,5′), 128.9 (C-4), 131.1 (C-1′), 133.3 (C-2′,6′), 139.5 (C-1), 142.8 (C-4′), 167.9 (CO-CH3). IR (KBr): ν 3440, 3320, 3230 cm-1 (NH), 2940, 2860 (-CH), 1665 (CdO), 1520, 1490 (CdC), 1080 (CCl). UV (methanol): λmax (log ) 244 nm (4.120), 304 (3.429). MS (70 eV): m/z (%) 380 (6), 378 (15), 376 (11) [M+], 343 (18), 341 (18), 338 (18), 336 (44), 335 (15), 334 (31), 303 (24), 302 (16), 301 (77), 300 (23), 299 (100), 298 (15), 297 (22), 265 (29), 264 (24), 263 (42), 229 (34), 228 (32), 207 (22), 201 (27), 193 (18), 192 (27), 176 (58), 175 (16), 174 (87), 173 (15), 164 (30), 150 (28), 140 (21), 139 (26), 138 (51), 124 (18), 113 (15), 111 (20), 104 (18), 102 (34), 100 (23), 76 (14), 75 (24), 73 (16), 52 (17). N′-Acetyl-4,4′-methylenebis(2-chloro-6-methylaniline) (Cl,Me-AcMDA). Cl,Me-MDA (5.90 g, 20.0 mmol) was dissolved in dioxane (150 mL) by slightly heating. Triethylamine (2.02 g, 20.0 mmol) and acetic acid anhydride (3.06 g, 30.0 mmol) were added. The reaction was monitored by TLC [silica gel, ethyl acetate/n-hexane (6:4), Rf (Cl,Me-MDA) ) 0.87, Rf (Cl,MeAcMDA) ) 0.39]. After 24 h at room temperature a colorless precipitate was present. After addition of 1 M NaOH (50 mL) and water (150 mL) further precipitate formed. The precipitate was filtered, washed with water, and dried in a desiccator over silica gel. For the isolation of pure Cl,Me-AcMDA, 1.5 g was purified by column chromatography (150 g of silica gel, ethyl acetate/n-hexane, 3:2). After evaporation of the solvents, the residue was recrystallized from ethyl acetate/n-hexane (4:1). Fine, long, colorless needles of Cl,Me-AcMDA (260 mg, 17%) were obtained, mp 238-239 °C. 1H NMR (250 MHz, DMSOd6): δ 2.03 (s, 3 H, CO-CH3), 2.10 (s, 3 H, 6-CH3 or 6′-CH3), 2.11 (s, 3 H, 6′-CH3 or 6-CH3), 3.69 (s, 2 H, Ph2CH2), 4.88 (s, 2 H, NH2), 6.83 (s, 1 H, 5-H), 6.98 (s, 1 H, 3-H), 7.04 (s, 1 H, 5′H), 7.14 (s, 1 H, 3′-H), 9.43 (s, 1 H, Ac-NH). 13C NMR (63 MHz, DMSO-d6): δ 18.1 (6-CH3), 18.3 (6′-CH3), 22.4 (CO-CH3), 38.9 (Ph2CH2), 117.0 (C-2), 123.4 (C-6), 126.4 (C-5′), 126.5 (C-5), 128.8 (C-3′ or C-3), 128.9 (C-3 or C-3′), 129.1 (C-4), 131.4 (C-2′), 131.7 (C-1′), 137.8 (C-6′), 140.5 (C-1), 141.6 (C-4′), 168.0 (CO-CH3). IR (KBr): ν 3460, 3360, 3280 cm-1 (NH), 3040 (dCH), 2960, 2880 (-CH), 1670 (CdO), 1540, 1510 (CdC). UV (methanol): λmax (log ) 244 nm (4.238), 295 (3.492). MS (70 eV): m/z (%)

Sabbioni and Schu¨ tze 340 (4), 338 (24), 336 (38) [M+], 303 (28), 302 (21), 301 (90), 294 (18), 293 (18), 281 (16), 279 (21), 261 (33), 260 (18), 259 (100), 257 (30), 243 (28), 208 (18), 207 (25), 180 (16), 156 (21), 154 (59), 118 (19), 91 (16). N′-Acetyl-4,4′-thiodianiline (AcTDA). Triethylamine (236 mg, 2.34 mmol) and acetic acid anhydride (421 mg, 3.51 mmol) were added to 4,4′-thiodianiline (507 mg, 2.34 mmol) in diethyl ether (10 mL). A white precipitate formed. The progress of the reaction was followed by TLC [silica gel, ethyl acetate, Rf (TDA) ) 0.76, Rf (AcTDA) ) 0.48]. After 2 h the solution was basified with 1 M sodium hydroxide (15 mL) and extracted with dichloromethane (3 × 20 mL). The combined organic extracts were dried over MgSO4 and evaporated in vacuo. The brown solid (570 mg) was redissolved in ethyl acetate and purified by flash chromatography with ethyl acetate on silica gel. Brownish needles of AcTDA were obtained (127 mg, 20%). 1H NMR (250 MHz, DMSO-d6): δ 2.00 (s, 3 H, COCH3), 5.45 (s, 2 H, NH2), 6.58 (d, J ) 8.4 Hz, 2 H, H-2,6), 7.02 (d, J ) 8.6 Hz, 2 H, H-3′,5′), 7.13 (d, J ) 8.4 Hz, 2H, H-3,5), 7.47 (d, J ) 8.6 Hz, 2 H, H-2′,6′), 9.92 (s, 1 H, NH-Ac).13C NMR (63 MHz, DMSO-d6): δ 24.1 (COCH3), 114.9 (C-2,6), 116.5 (C-4), 119.9 (C-2′,6′), 128.0 (C-3′,5′), 133.0 (C-4′), 135.6 (C-3,5), 137.4 (C-1′), 149.8 (C-1), 168.4 (COCH3). N′-Acetyl-4,4′-sulfonyldianiline (Acdapsone). Triethylamine (209 mg, 2.07 mmol) and acetic acid anhydride (421 mg, 3.51 mmol) were added to 4,4’-sulfonyldianiline (507 mg, 2.34 mmol) in THF (10 mL). The reaction mixture was refluxed. The progress of the reaction was followed by TLC [silica gel, ethyl acetate, Rf(dapsone) ) 0.74, Rf(Acdapsone) ) 0.38]. After 52 h, the solution was basified with 1 M NaOH (15 mL) and extracted with dichloromethane (3 × 12 mL). The combined organic extracts were dried over MgSO4 and evaporated in vacuo. The brown solid (570 mg) was redissolved in ethyl acetate and purified by flash chromatography with ethyl acetate on silica gel. Orange crystals of Acdapsone were obtained (148 mg, 25%). 1H NMR (250 MHz, DMSO-d ): δ 2.06 (s, 3 H, CO-CH ), 6.13 6 3 (s, 2 H, NH2), 6.60 (d, J ) 8.8 Hz, 2 H, H-2,6), 7.51 (d, J ) 8.7 Hz, 2 H, H-2′,6′), 7.70-7.79 (m, 4 H, H-3,5, H-3′,5′), 10.31 (s, 1 H, -NH-Ac). 13C NMR (63 MHz, DMSO-d6): δ 24.3 (CO-CH3), 113.1 (C-2,6), 119.0 (C-2′,6′), 126.3 (C-4), 127.9 (C-3′,5′), 129.4 (C-3,5), 137.0 (C-4′), 143.1 (C-1′), 153.6 (C-1), 169.2 (CO-CH3). Electrochemical Determination of the Half-Wave Oxidation Potential (E1/2). E1/2 of the aromatic amines was determined on an electrochemical detector (ECD), model 5100A, by ESA (Bedford, MA). The diamines and the monoacetyl compounds were injected onto a reversed-phase column Select B (Merck; 250 × 4 mm, 5 µm) with a flow of 1 mL/min. ODA, MDA, and EDA eluted at 7, 10, and 22 min with 47% methanol in sodium phosphate buffer (50 mM, pH 7.4). AcODA, AcMDA, and AcEDA eluted at 8, 10, and 17.5 min with 50% methanol in sodium phosphate buffer (50 mM, pH 7.4). The diamines TDA, AcTDA, dapsone, and Acdapsone were analyzed with the conditions described for the analysis of the respective hemoglobin binding but with sodium phosphate buffer at pH 7.4 instead of at pH 4.5. The electrode potential was decreased stepwise (0.05 V) from 1 to 0.4 V. The peak integrals obtained (average of two injections) were plotted against the electrode potential. The half-wave oxidation potential was obtained from the resulting hydrodynamic voltammograms. Determination of Hb Adducts of MDA and AcMDA. For base hydrolysis of animals dosed with 0.5 mmol of MDA/kg of body weight, Hb (50 mg) was dissolved in 3 mL of 0.1 M NaOH/ 0.01% SDS in the presence of the surrogate internal standards EDA (1088 pmol) and AcEDA (184 pmol). For neutral hydrolysis, Hb (50 mg) was dissolved in 3 mL of 0.1 M sodium phosphate buffer, pH 7.4, and 0.01% SDS in the presence of the recovery standards EDA (435 pmol) and AcEDA (73.7 pmol). For animals dosed with 0.07 mmol/kg of body weight, the amount of the internal standards was reduced 5-fold. The samples were stirred with magnetic bars for 1 h at room temperature. The hydrolysates were then extracted with diethyl ether (2 × 3 mL). Possible interphases were eliminated by a freeze-thaw cycle.

Hemoglobin Binding of Bicyclic Aromatic Amines

Figure 1. Quantification by HPLC with electrochemical detection of MDA and AcMDA released with base from hemoglobin of animals dosed with MDA. EDA and AcEDA were added as surrogate internal standards, and 4-ethylaniline (4EtA) was added as volumetric internal standard. The combined extracts were evaporated almost to dryness in a speed evaporator. The residue was taken up in 200 µL of 0.02 M sodium phosphate buffer, pH 8.0/methanol (70:30), and 4-ethylaniline (716 pmol for base hydrolysis; 71.6 pmol for neutral hydrolysis) was added as volumetric internal standard. Each sample was analyzed three times by HPLC on a RP select B column (250 × 4 mm, 5 µm) with 50 mM sodium phosphate buffer, pH 4.5/methanol (50:50) and a flow rate of 1 mL/ min (Figure 1). The compounds eluted at tR(MDA) ) 7.5 min, tR(AcMDA) ) 10 min, tR(4-ethylaniline) ) 11.5 min, tR(EDA) ) 14.5 min, and tR(AcEDA) ) 17 min. The electrode was set at 0.8 V with a gain of 80. For animals dosed with 0.07 mmol/kg of body weight, the amount of the surrogate internal standards was reduced 5-fold. The compounds were quantified against calibration curves obtained with standard solutions. The obtained values were corrected with the recovery factors of MDA and AcMDA determined in separate experiments. The recovery from the base hydrolysis was performed with 76.8-1228 pmol of MDA, 12.6-202 pmol of AcMDA, and the surrogate internal standards EDA (1088 pmol) and AcEDA (184 pmol). The average recovery for MDA was 56 ( 10% and for AcMDA 68 ( 4%. The recovery for EDA was 49 ( 7% and for AcEDA 72 ( 3%. The response factor MDA/EDA was 1.1 ( 0.07 and for AcMDA/AcEDA 1.0 ( 0.08. The recovery from the neutral hydrolysis was determined with 15.4 pmol of MDA, 10.1 pmol of AcMDA, 109 pmol of EDA, and 18.4 pmol of AcEDA. The recoveries for MDA and AcMDA were 35 ( 9% and 56 ( 7%. The recoveries of EDA and AcEDA were 26 ( 8% and 43 ( 5%. The response factors for MDA/ EDA and AcMDA/AcEDA were 1.34 ( 0.08 and 1.30 ( 0.16. For further structural identification, 1 g of Hb from a 0.5 mmol/kg dosed rat was hydrolyzed in base and analyzed by LC/

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 475 MS and LC/UV. The HPLC conditions given above were used, but with a photodiode array UV detector instead of an electrochemical detector. The retention times and the UV spectra were identical with the standard compounds. For the LC/MS analysis the sample was injected on a Supersphere RP18 column (125 × 2 mm, 3 µm; Bischoff) with a 30-min 30-60% methanol gradient in 50 mM ammonium formate buffer, pH 7.3. MDA and AcMDA eluted at 12 and 14.5 min with a flow rate of 0.4 mL/min. The temperature and helium pressure of the interface were 70 °C and 42-44 psi, respectively. The electron energy was set at 70 eV and the ion source at 250 °C. The retention times and the mass spectra were identical to those of the standards. Determination of Hb Adducts of MDI. Two animals were given MDI (0.5 mmol/kg of bw) per gavage and two animals given MDI (0.5 mmol/kg of bw) per ip. The procedure for the determination of MDA adducts was followed, except the amounts of the surrogate internal standards were modified: EDA (435 pmol) and AcEDA (184 pmol) for the base hydrolysis and EDA (76.8 pmol) and AcEDA (12.6 pmol) for the neutral hydrolysis. Determination of Hb Adducts of EDA and AcEDA. The procedure used for the determination of the Hb adducts of MDA was followed. For the base hydrolysis, MDA (767 pmol) and AcMDA (126 pmol) were used as surrogate internal standards and 4-ethylaniline (716 pmol) was used as volumetric internal standard. The quantification of the compounds was performed with calibration curves obtained with standard solutions. The obtained values were corrected with the recovery factors determined in a separate experiment. For the base hydrolysis, this experiment was performed with 535-1088 pmol of EDA, 73.7184 pmol of AcEDA, and the surrogate internal standards MDA (767 pmol) and AcMDA (126 pmol). The recovery factors were 0.91 ( 0.08 for EDA/MDA and 1.0 ( 0.1 for AcEDA/AcMDA. The structures of the compounds found in vivo were confirmed by HPLC with UV detection or with MS detection as described above. The retention times of EDA and AcEDA on the Supersphere RP18 column used for the LC/MS analysis (Figure 2) were 16.5 and 18.5 min for EDA and AcEDA, respectively. The retention times, the UV spectra, and the MS spectra of the compounds found in vivo were identical to the ones found in vitro. Determination of Hb Adducts of ODA and AcODA. The procedure for the determination of Hb adducts of EDA was used. Bz was taken as volumetric internal standard instead of 4-ethylaniline. For the base hydrolysis, 788 pmol of MDA and 520 pmol of AcMDA were added as internal standards. The neutral hydrolyses were performed in the presence of 158 pmol of MDA and 104 pmol of AcMDA. Bz was added as volumetric internal standard, 688 pmol for the base hydrolysis and 172 pmol for the neutral hydrolysis. Each sample was analyzed three times by HPLC on a RP select B column (250 × 4 mm, 5 µm) with 50 mM sodium phosphate buffer, pH 4.5/methanol (57: 43) and a flow rate of 1 mL/min: tR(ODA) ) 8 min, tR(Bz) ) 10.5 min, tR(MDA) ) 12 min, tR(AcODA) ) 14 min, tR(AcMDA) ) 18 min. The electrode was set at 0.7 V with a gain of 80. The recovery factors for ODA and AcODA from 40 mg of Hb in base or at pH 7 with MDA and AcMDA as surrogate internal standards were determined against a calibration curve with standard solutions. The following amounts were used for the base hydrolysis [neutral hydrolysis] 155-4580 pmol [115-458 pmol] of ODA, 103-4130 pmol [103-413 pmol] of AcODA, 788 pmol [158 pmol] of MDA, 520 pmol [104 pmol] of AcMDA. All samples were analyzed as described above and analyzed by HPLC-ECD. The recovery factors for MDA/ODA were 2.93 ( 0.26 [1.03 ( 0.07] and for AcMDA/AcODA 1.4 ( 0.1 [0.88 ( 0.1]. The structures of the compounds found in vivo were confirmed by HPLC with UV detection or with MS detection as described above (Figure 3). The retention times of ODA and AcODA on the Supersphere RP18 column used for the LC/MS analysis were 11 and 15 min with a 25-80% (35 min) methanol gradient in 50 mM ammonium formate buffer. Determination of Hb Adducts of Me,Me-MDA and Me,Me-AcMDA. For the base hydrolysis, Hb (50 mg) was dissolved

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Figure 2. Identification by LC/MS of EDA and AcEDA released from hemoglobin of animals dosed EDA. The chromatogram and the MS spectra of the in vivo samples were compared to a mix with standards. In addition the MS of EDA found in the MS library is presented. All chromatograms are normalized for a better comparison. in 3 mL of 0.025 M NaOH/0.01% SDS. Et,Et-MDA (17.6 pmol) and Et,Et-AcMDA (19.0 pmol) were added as surrogate internal standards. For the neutral hydrolysis, Hb (50 mg) was dissolved in 3 mL of 0.1 M sodium phosphate buffer, pH 7.4/0.01% SDS. Et,Et-MDA (8.78 pmol) and Et,Et-AcMDA (9.48 pmol) were added as surrogate internal standards. All samples were stirred for 1 h at room temperature and then extracted with diethyl ether (2 × 3 mL) by vortexing. For a good separation of the phases, the samples were frozen in liquid nitrogen and centrifuged while thawing. The combined organic phases were evaporated to dryness in a speed evaporator. The residue was transferred with ethyl acetate to microinserts in vials for the autosampler of the GC/MS. The ethyl acetate was evaporated in a speed evaporator. The residue was taken up in ethyl acetate (25 µL). The analyses were performed via on-column injection on an Ultra-2 column (12 m × 0.2 mm, 0.33-µm film thickness; Hewlett-Packard) equipped with a phenylmethyl retention gap (0.32 mm; Analyt, Mu¨llheim). The oven temperature was set initially at 80 °C for 30 s, then with 50 °C/min to 200 °C, 1 min at 200 °C, and finally 20 °C/min to 300 °C. The interface temperature was at 280 °C. The flow was kept

constant with a helium pressure of 0.6 bar at 50 °C. The compounds were detected with the following mass spectrometer setting: ion source temperature 250 °C, electron energy at 70 eV, single-ion monitoring (SIM) with dwell time 100 ms. The following mass fragments were registered: m/z 254 (Me,MeMDA, tR ) 6.9 min), 296 (Me,Me-AcMDA, tR ) 8.1 min), 310 (Et,Et-MDA, tR ) 7.7 min), 352 (Et,Et-AcMDA, tR ) 8.7 min) (Figure 4). For the quantification of the compounds, calibration curves were obtained by spiking hemoglobin with Me,Me-MDA and Me,Me-AcMDA and the surrogate internal standards. The calibration curve consisted of four levels. Each point corresponds to the average of two experiments. The calibration curve for the base [neutral] hydrolysis was performed with Me,MeMDA (0-12.4 [0-4.1] pmol), Me,Me-AcMDA (0-17.2 [0-4.3] pmol), Et,Et-MDA (17.6 [8.78] pmol), and Et,Et-AcMDA (19.0 [9.48] pmol). The following calibration curves were obtained for the base hydrolysis: Me,Me-MDA, base hydrolysis, y ) 0.785x - 0.041, r ) 1.0; neutral extraction, y ) 0.965x + 0.003, r ) 0.99. Four single ions were monitored for each compound to confirm the structural identification [SIM, dwell time ) 50

Hemoglobin Binding of Bicyclic Aromatic Amines

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 477

Figure 3. Identification of ODA and AcODA released from animals dosed ODA. For the identification of the substances, the samples were pooled and analyzed by HPLC equipped with a photodiode detector on a RP Select B column (250 × 4 mm, 5 µm) with 50 mM sodium phosphate, pH 4.5/methanol (57:43). ms, m/z 254, 239, 223, 134 (Me,Me-MDA); 296, 281, 253, 239 (Me,Me-AcMDA); 310, 295, 281, 162 (Et,Et-MDA); 352, 309, 293, 162 (Et,Et-AcMDA)]. For quantification the single ions m/z 254, 296, 310, 352 were used. Determination of Hb Adducts of Et,Et-MDA and Et,EtAcMDA. Me,Me-MDA (10.4 pmol) and Me,Me-AcMDA (10.8 pmol) were added as surrogate internal standards. The same procedure used for the determination of Me,Me-MDA and Me,Me-AcMDA was applied. Determination of Hb Adducts of Cl,Cl-MDA and Cl,ClAcMDA. For the base hydrolysis, Hb (40 mg) was dissolved in 3 mL of 0.1 M NaOH/0.01% SDS. For the neutral hydrolysis, Hb (40 mg) was dissolved in 3 mL of 0.1 M sodium phosphate buffer, pH 7.4/0.01% SDS. All samples were stirred for 1 h in the presence of Cl,Me-MDA (33.8 pmol) and Cl,Me-AcMDA (29.6 pmol). The samples were extracted with diethyl ether. The combined organic phases were evaporated with the speed evaporator. The residue was taken up in ethyl acetate (25 µL) and analyzed by GC/MS. An aliquot (2 µL) was applied by splitless injection on a HP-1 column (12 m × 0.2 mm, 0.33-µm film thickness; Hewlett-Packard) with a phenyl retention gap (1 m × 0.25 mm; Macherey Nagel). The injector temperature was set at 320 °C, and the split (1:10) was opened after 45 s. The oven temperature was initially at 45 °C for 1 min, then with 30 °C/min to 300 °C; interface temperature 300 °C; helium

with 1.0 bar pressure at 45 °C. The compounds were detected with a mass spectrometer (70 eV, ion source temperature 200 °C) with SIM (dwell time ) 100 ms, m/z 259, 299, 301). The following compounds were detected: Cl,Me-MDA, tR ) 8.2 min (m/z 259); Cl,Me-AcMDA, tR ) 8.9 min (m/z 259, 301); Cl,ClMDA, tR ) 8.4 min (m/z 299, 301); Cl,Cl-AcMDA, tR ) 9.3 min (m/z 299, 301). The samples were quantified against a calibration line obtained from the extracts of Hb spiked with different amounts of Cl,Cl-MDA (0-4 pmol) and Cl,Cl-AcMDA (0-4 pmol) and the surrogate internal standards Cl,Me-MDA (33.8 pmol) and Cl,Me-AcMDA (29.6 pmol). The absolute recoveries were not determined for these compounds. Determination of Hb Adducts of TDA and AcTDA. Two rats received TDA (0.5 mmol/kg) by gavage. Hb (50 mg) was dissolved in 3 mL of 0.1 M NaOH/0.01% SDS. MDA (1.51 nmol) was added as recovery standard. After 1 h of stirring at room temperature, the hydrolysate was extracted with diethyl ether (2 × 4 mL). Phosphate buffer (150 µL, pH 4.5, 50 mM) was added to the organic phase. After evaporation with a speed evaporator for 10 min at 45 °C, methanol (100 µL) was added, mixed with the water residue in the reaction tube, and transferred to a small vial. The tube was rinsed with an additional 150 µL of methanol. The volumetric internal standard 246TMA (1.48 nmol) was added. The samples were analyzed by HPLC

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Figure 4. GC/MS analyses of hemoglobin hydrolysate extracts from a control rat (A) and from a rat dosed with Me,Me-MDA (B). Et,Et-MDA and Et,Et-AcMDA were added as surrogate internal standard. The following mass fragments were registered: m/z 254 (Me,Me-MDA), 296 (Me,Me-AcMDA), 310 (Et,Et-MDA), 352 (Et,Et-AcMDA). with electrochemical detection (0.8 V). An aliquot (50 µL) was injected on a reversed-phase column (RP Select B) (250 × 4 mm, 1 mL/min flow, 50 mM sodium phosphate buffer, pH 4.5, and 53% methanol): tR(MDA) ) 5.1 min, tR(TDA) ) 6.4 min, tR(AcTDA) ) 9.1 min, tR(246TMA) ) 10.9 min. TDA and AcTDA were quantified against a calibration curve obtained from an experiment (n ) 3) of hemoglobin spiked with AcTDA (0, 2.07 nmol), TDA (0, 1.37 nmol), and MDA (1.51 nmol). The recoveries of TDA (1.37 nmol), AcTDA (2.07 nmol), and MDA (1.51 nmol) were 58 ( 3%, 82 ( 3%, and 55 ( 2%. To determine the presence of noncovalently bound TDA and AcTDA, the same experiments were performed in phosphate buffer (pH 7.4) instead of NaOH. Determination of Hb Adducts of Dapsone. Two rats received dapsone (0.5 mmol/kg) by gavage. Hb (50 mg) was dissolved in 3 mL of 0.1 M NaOH/0.01% SDS. MDA (500 pmol) was added as recovery standard. After 1 h of stirring at room temperature, the hydrolysate was extracted with diethyl ether (2 × 4 mL). Phosphate buffer (150 µL, pH 4.5, 50 mM) was added to the organic phase. After evaporation with a speed evaporator for 10 min at 45 °C, methanol (100 µL) was added, mixed with the water residue in the reaction tube, and transferred to a small vial. The tube was rinsed with additional methanol (150 µL). Bz (1.08 nmol) was added as volumetric internal standard. The samples were analyzed by HPLC with electrochemical detection (0.9 V). An aliquot (50 µL) was injected on a reversed-phase column (RP Select B) (250 × 4 mm, 1 mL/min flow, 50 mM sodium phosphate buffer, pH 4.5, and 40% methanol): tR(dapsone) ) 5.9 min, tR(Acdapsone) ) 9.8 min,

tR(Bz) ) 11.05, tR(MDA) ) 12.3 min. Dapsone and Acdapsone were quantified against a calibration curve obtained from experiments (n ) 3) of hemoglobin spiked with dapsone (0, 0.604 nmol), Acdapsone (0, 0.344 nmol), and MDA (0.504 nmol). The recoveries of dapsone (0.604 nmol), Acdapsone (0.344 nmol), and MDA (0.504 nmol) were 81 ( 12%, 53 ( 7%, and 56 ( 8%. The limit of detection with this method is 5 ng of dapsone and 40 ng of Acdapsone per sample. To determine the presence of noncovalently bound dapsone, the same experiments were performed in phosphate buffer (pH 7.4) instead of NaOH. Structural confirmation was obtained by GC/MS. Hb (40 mg) of rats was hydrolyzed in 0.1 M NaOH (0.01%) in the presence of the surrogate internal standard MDA-d4 and then extracted in dichloromethane (5 mL) following a procedure published recently for the analysis of MDA (20, 26). The organic extract was dried over a Pasteur pipet with sodium sulfate and then reacted with heptafluorobutyric acid anhydride (10 µL). After evaporation at the speed evaporator, the residue was taken up in ethyl acetate and analyzed by splitless injection with GC/ MS on a RTx-5MS (0.25 mm × 15 m, 0.5-µm film thickness; Restek) column with methyl-silyl-deactivated retention gap (1.5 m × 0.25 mm). The compounds were analyzed using the following conditions: injector and interface temperatures were 320 °C; initial oven temperature was set for 1 min at 50 °C, then increased at 40 °C/min to 320 °C; and held for 2 min at this temperature. The compounds were identified and quantified using a mass spectrometer as detector in the negative chemical ionization mode, with methane as reactant gas (ca. 1.4 Torr, ion source 250 °C, electron energy 240 eV) and SIM

Hemoglobin Binding of Bicyclic Aromatic Amines (dwell time ) 100 ms, low-mass resolution): tR(MDA-d4) ) 6.39 (m/z 574), tR(dapsone) ) 7.63 (m/z 620). The monoacetylated dapsone could not be analyzed by this method. Standard dapsone di-HFBA shows the following fragments: m/z (%) 640 (1) [M-], 639 (4) [M - 1]-, 622 (8), 621 (24) [M - 19]-, 620 (100) [M - 20]+, 301 (10), 269 (9).

Results Synthesis of Arylamines and N′-Acetylarylamines. Commercially unavailable ortho-substituted MDAs were synthesized by reacting the correspondingly substituted aniline with formaldehyde. Cl,Cl-MDA was synthesized following a procedure by Collins et al. (21). The N′acetylarylamines were obtained by reacting the dianilines with acetic acid anhydride in the presence of triethylamine. The N′-acetylarylamines had to be separated from the dianiline or the N,N′-diacetylarylamine by HPLC (AcMDA, AcEDA, AcODA) or by flash chromatography on silica gel (MeMe-MDA, Et,Et-MDA, AcTDA, Acdapsone). Spectroscopic Data. The synthesized arylamines and the N′-acetylarylamines were characterized by NMR, MS, IR, and UV. The data are listed in Materials and Methods. Except for AcMDA the spectroscopic values of the other N′-acetylarylamines were not available in the literature. The signals of the 1H and 13C NMR spectra were assigned with the help of the increment rules (27, 28) and by comparison of the spectra of the diamines with the spectra of the monoacetyldiamines. For signals which are close together, (0.2 ppm for 1H NMR signals and (1.5 ppm for 13C NMR signals, the assignment of the signals is tentative. In these cases further NMR experiments would be necessary, which are out of the scope of the present work. The 13C NMR signals for the ortho carbon and the para carbon of the N-acetylated Me,Me-MDA were shifted 12 and 14 ppm downfield compared to the unacetylated Me,Me-MDA. According to the increment rules, these signals should have been shifted only 3 and 4 ppm downfield. The same effect was observed for the other ortho-disubstituted compounds: Et,Et-AcMDA, Cl,Me-AcMDA, Cl,Cl-AcMDA. This means that the acetyl group of the ortho-disubstituted compounds is forced out of the plane of the aromatic ring. Determination of Hb Adducts. Hb adducts of dianilines were investigated in female Wistar rats. The animals were sacrificed after 24 h. After this time period, no further increase of Hb binding is expected, as shown by Suzuki et al. (29) for five arylamines, which are more lipophilic than the amines of the present study. Aromatic amines were released from Hb by mild base hydrolysis in the presence of surrogate internal standards. The aromatic amines were enriched by solvent extraction. Solid-phase extraction of MDA from plasma or blood yields lower recoveries. This was confirmed recently by Shintani (30). The recoveries of the compounds have been determined under basic and neutral conditions. At pH > 11 the sulfinamide adducts of arylamines with cysteine of Hb are cleaved readily. Under physiological conditions (pH 7.4), the sulfinamide adducts are stable. Therefore, extraction with organic solvents at physiological conditions will extract noncovalently bound amines which were present in blood. If the amounts of arylamines extracted at neutral and basic pH are similar, the found amines are not covalently bound to Hb. The amines in the extracts were quantified by HPLC with electrochemical detection or by GC/MS. To obtain

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 479

the maximum sensitivity with the electrochemical detector, the oxidation potential was optimized for the analytes. This was performed by plotting the area of the compounds obtained at different voltage. The half-wave oxidation potential was obtained from such a hydrodynamic voltammogram: ODA, 0.47 V; AcODA, 0.51 V; TDA, 0.48 V; AcTDA, 0.54 V; MDA, 0.60 V; AcMDA, 0.66 V; EDA, 0.63 V; AcEDA, 0.62 V; dapsone, 0.80 V. Determination of Hb Adducts of MDA. Wistar rats were dosed with 0.5 and 0.07 mmol of MDA/kg. The Hb hydrolysates with the surrogate internal standards, EDA and AcEDA, were extracted with diethyl ether and analyzed by HPLC with ECD detection (Figure 1). The peaks were standardized with the volumetric internal standard 4-ethylaniline; and assigned according to their retention time. With standards, the detection limit is at 50 fmol of MDA and 100 fmol of AcMDA. For structural confirmation, a larger amount of Hb of animals dosed with 0.5 mmol/kg MDA was hydrolyzed and analyzed by HPLC with UV or MS detection. The UV and MS spectra of standard MDA (AcMDA) and of MDA (AcMDA) released from Hb were identical. Extraction at physiological pH yielded only 5% of MDA and 10% of AcMDA found after base extraction. The hemoglobin binding index (HBI) listed in Table 1 was not corrected with these small values. The identity and the levels of the found compounds were confirmed by GC/MS following a method published recently (20, 26). Hb Binding of MDA after Exposure to MDI. Two rats received MDI by ip, and two rats were dosed with MDI by gavage. Hb was analyzed as described for MDAdosed rats. The samples were analyzed by HPLC with electrochemical detection in the presence of the internal surrogate standards EDA and AcEDA. In rats dosed MDI by gavage, MDA and AcMDA were found after neutral and base extraction of Hb. The amount after neutral extraction was one-fourth of the levels found after base extraction. For the calculation of the HBI the amount found after neutral extraction was subtracted from the amount after base extraction. After dosing by ip, the HBI for MDA was 0.025 and for AcMDA 0.038. After the oral dose, the HBI for MDA was 0.011 and for AcMDA 0.029. The identity and the levels of the found compounds were confirmed by GC/MS following a method published recently (20, 26). Therefore, most probably MDI hydrolyzes to MDA in the animals. MDA is then partially N′-acetylated and N-oxidized and then covalently bound to Hb. Hb Binding of EDA. The same conditions used for the quantification of MDA were used for the quantification of Hb adducts with EDA. MDA was taken as recovery standard for EDA and AcMDA for AcEDA. The peaks were assigned according to their retention times. Larger amounts of Hb were hydrolyzed for structural identification. The extract was analyzed by HPLC with photodiode array detector and by LC/MS (Figure 2). The UV spectra and the MS correspond to the ones of standard compounds. The extraction of the Hb solutions at physiological pH yielded 1.5% EDA and 8-9% AcEDA compared to the base hydrolysis. These small amounts were not taken into account for the calculation of the HBI. The HBI values for EDA and AcEDA amount to 14.7 and 3.4. Hb Binding of ODA. MDA and AcMDA were used as surrogate internal standards, and Bz was used as volumetric internal standard. After extraction with

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Table 1. Hb Binding Index (HBI)a,b of MDA Analogous Compounds and Their Corresponding Monocyclic Derivatives compound

dose (mmol/kg)

method of analysis

4,4′-methylenedianiline (MDA) 4,4′-methylenedianiline (MDA) 2HCl 4,4′-methylenedianiline (MDA) 2HCl 4,4′-oxydianiline (ODA) 2HCl 4,4′-ethylenedianiline (EDA) 4,4′-methylenebis(2-chloroaniline) (MOCA), lit.c 4,4′-methylenebis(2,6-dichloroaniline) (Cl,Cl-MDA) 4,4′-methylenebis(2,6-dimethylaniline) (Me,Me-MDA) 2HCl 4,4′-methylenebis(2,6-diethylaniline) (Et,Et-MDA) 2HCl 4,4′-thiodianiline (TDA) 4,4′-sulfonyldianiline (dapsone) benzidine (Bz), lit.h 3,3′-dichlorobenzidine (DcBz) 2HCl, lit.h 3,3′,5,5′-tetramethylbenzidine, lit.h aniline, lit.i 4-methylaniline, lit.i 4-ethylaniline, lit.j 2,6-dimethylaniline, lit.j 2,4,6-trimethylaniline, lit.j 4-methylmercaptoaniline, lit.j 2-chloro-4-methylaniline, lit.j 2-chloroaniline, lit.j 2,6-dichloraniline, lit.j

0.50 0.50 0.07 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.47 0.60 0.50 0.50 0.50 0.50 0.50 0.50 0.50

HPLC-ECD HPLC-ECD HPLC-ECD HPLC-ECD HPLC-ECD GC/MS GC/MS GC/MS GC/MS HPLC-ECD HPLC-ECD HPLC-ECD HPLC-ECD HPLC-ECD GC-FID GC-FID GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS GC/MS

HBI of cleavage products diamine N-acetylamine 2.4 ( 0.4 3.1 ( 0.2 2.5 ( 0.5 10.4 ( 2.9 14.7 ( 3.2 5.1 ( 0.6 e 0.012 ( 0.003 0.009 ( 0.002 8.2 ( 1.3 0.13 ( 0.02 2.4 ( 0.1 2.0 ( 0.7 nd

0.6 ( 0.1 0.6 ( 0.1 1.7 ( 0.4 6.2 ( 1.6 3.4 ( 0.6 ndd nd f

nd 7.4 ( 0.6 18.9 ( 3.5 1.5 ( 0.6 nd

HBI total 3.0 ( 0.5 3.7 ( 0.3 4.2 ( 0.9 16.6 ( 4.5 18.1 ( 3.8 5.1 ( 0.6 e 0.012 ( 0.003 0.009 ( 0.002 15.6 ( 1.9 0.13 ( 0.02 24.3 ( 3.7g 3.5 ( 1.5 22.0 ( 3.0 4.3 ( 1.0 5.8 ( 1.6 1.1 ( 0.3 0.2 ( 0.04 3.8 ( 0.5 1.0 ( 0.4 0.5 ( 0.1 e

a HBI, Hb binding index [compound bound (mmol/g of Hb)/dose (mmol/kg of body weight)]. The HBI and the standard deviation were determined from duplicate Hb hydrolyses of each animal. b Two female Wistar rats were dosed by gavage and sacrificed after 24 h. c Reference 10. d The compound could not be detected (detection limit < 1 pmol/sample). e Extraction of Hb at pH 7.4 yielded the same amount as at pH > 11. f Detection of small amounts was impossible due to contamination eluting at the same time. g Sum of benzidine (Bz) + N′-acetyl-Bz + 4-aminobiphenyl (HBI ) 3.0 ( 0.1) which is a metabolite of Bz.) h Reference 42. i Reference 48. j References 31, 44, 45.

diethyl ether, the compounds were analyzed by HPLC with ECD. The detection limits for ODA and AcODA were 50 and 100 fmol. The recoveries for ODA and AcODA, which are 20% and 53%, are lower than for MDA and AcMDA. For ODA, the recovery increases for larger concentrations. The highest concentration taken for the recovery experiment corresponds to the value found in the animal experiment. Therefore, the ODA and AcODA content in the extracts was calculated from the recovery ratio MDA/ODA ) 2.3 and AcMDA/AcODA ) 1.3. Hb of four Wistar rats dosed 0.5 mmol/kg ODA‚2HCl was analyzed by HPLC with ECD after base and neutral hydrolysis. For the quantification by HPLC, the compounds were identified according to their retention time. All the residues of the extracts were also analyzed by HPLC equipped with a photodiode array detector for further identification. The UV of the in vivo samples correspond to the UV of the standards (Figure 3). In addition, 200 mg of Hb was hydrolyzed and analyzed by LC/MS. Extraction at neutral pH gave 4-6% ODA and AcODA of the amount found after base hydrolysis. These small amounts were not taken into account for the calculation of the HBI. After base hydrolysis an HBI of 10.4 for ODA and 6.2 for AcODA was determined. Hb Binding of Me,Me-MDA. Et,Et-MDA and Et,Et-AcMDA were used as surrogate internal standards. The hydrolysates were extracted with diethyl ether and analyzed by GC/MS without derivatization (Figure 4). Me,Me-MDA and Me,Me-AcMDA were quantified against a calibration line obtained with the surrogate internal standards and different amounts of Me,Me-MDA (0-12 pmol) and Me,Me-AcMDA (0-17 pmol). The absolute recoveries were not determined for these compounds. The detection limit for Me,Me-AcMDA was 1 pmol per sample. Hb of animals dosed 0.5 mmol/kg Me,Me-MDA‚2HCl was analyzed after base and neutral hydrolysis. Extraction of neutral Hb hydrolysates yielded no Me,Me-MDA and

Me,Me-AcMDA. Extracts of basic Hb hydrolysates contained 3.6-5.9 pmol of Me,Me-MDA/50 mg of Hb. No Me,Me-AcMDA could be found. An HBI of 0.012 was determined for Me,Me-MDA. Hb Binding of Et,Et-MDA. Hb adducts of Et,EtMDA were determined according to the methods used for Me,Me-MDA. Me,Me-MDA and Me,Me-AcMDA were used as surrogate internal standards. Et,Et-MDA and Et,Et-AcMDA were quantified against a calibration line obtained with the surrogate internal standards and different amounts of Et,Et-MDA (0-14 pmol) and Et,Et-AcMDA (0-4 pmol). The determination limits for Et,Et-MDA and Et,Et-AcMDA were 0.13. Hb binding in the magnitude found for Me,Me-MDA and Et,Et-MDA cannot be excluded. The HBI values found for the MDA analogues were compared to those for the corresponding monocyclic arylamines. For these comparisons, the arylamines with an alkyl group in the para position, 4-methylaniline (4MA) and 4-ethylaniline (4EtA), are of interest because they cannot be hydroxylated in the para position. The HBI of 4EtA is larger than that for 4MA (31). This is comparable to the effect seen for MDA compared to EDA. Two methyl groups in the ortho position reduce hemoglobin binding drastically as seen for 2,6-dimethylaniline, 2,4,6-trimethylaniline, and Me,Me-MDA. For 3,3′,5,5′tetramethylbenzidine no Hb adducts were found. oChloro substitution leads to lower HBIs as seen for 2-chloroaniline and DcBz, except for MOCA. Two chlorines in the ortho position abolish hemoglobin binding: 2,6-dichloroaniline and Cl,Cl-MDA. Structure-Activity Relationships. The experimentally determined half-wave oxidation potentials (E1/2) of the diamines correspond well with values determined by Beilis (43). The amount of N′-acetyldiamine bound to Hb increases with the oxidizability of the diamines. In general the ease of N-acetylation increases with the oxidizability of arylamines without substituents in the ortho position. This was demonstrated by Andres et al.

482 Chem. Res. Toxicol., Vol. 11, No. 5, 1998

Sabbioni and Schu¨ tze

Acknowledgment. We are most grateful for the technical assistance of Michael Werner and Siegfried Schneider.

References

Figure 5. Correlation (r ) 0.80) between carcinogenicity and hemoglobin binding of bicyclic arylamines (sum of bound Nacetyldiamine and diamine).

(32) for the N-acetylation of para-substituted monocyclic arylamines in liver cytosol of rabbits. The apparent Km (32) values increase with the basicity of the acceptor amine and thus with the oxidizability of the amine (31, 44, 45). A comparison of the metabolites of ODA and MDA excreted in the 72-h urine in male SpragueDawley rats confirms this trend (46). After oral dose of the diamines, the weight ratio of the diamine:N′-acetyldiamine:N,N′-diacetyldiamine was 1.0:5.4:66.1 and 1.0: 2.6:1.0 for ODA and MDA, respectively. Most diamines studied for the present work are mutagenic. The mutagenicity values are listed in Table 2 and expressed as revertants per µmol of compound. The experiments were performed with S. typhimurium strains TA98 and TA100 after activation with rat liver homogenate (S9-mix). Bz, DcBz, EDA, MDA, MOCA, ODA, and TDA are mutagenic (4, 6); Me,Me-MDA, Et,Et-MDA, dapsone, and 3,3′,5,5′-tetramethylbenzidine are not mutagenic (5, 49). No data were found for Cl,Cl-MDA. MOCA was only mutagenic in TA100 (4, 6). Wu (47) found that MOCA is mutagenic as well in TA98, but with 10 times lower mutagenicity. The mutagenic potency order of the diamines is the same in the TA98 and TA100 strains, except for Bz. The Spearman rank order correlation of the mutagenicity data obtained from the two strains gives a correlation coefficient r ) 0.943 (P value ) 0.0167). The mutagenic potency is inversely related to the amount of hemoglobin binding. For the TA100 strain, the Spearman rank order correlation is significant: r ) -0.75, P value ) 0.0384. In contrast, dapsone, Me,Me-MDA, and Et,Et-MDA bind to a small extent to HBI but are not mutagenic. The carcinogenicity of most of the compounds presented in this study was tested in rats and mice. A good measure for carcinogenic potency is the TD50 values, which correspond to the daily dose which leads to tumors in 50% of the animals (9). The HBIs of Bz and DcBz were obtained from the literature (38). This small data set shows a good linear correlation (r ) 0.80) between hemoglobin binding and carcinogenicity of the tested compounds (Figure 5). The Spearman rank order correlation is highly significant: r ) 0.964, P value < 0.001. However, it should be noticed that the carcinogenicity and hemoglobin binding were determined in different rat strains and not in the same experiment.

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