Chem. Res. Toxicol. 1992,5, 614-619
674
InE ibition of 4-(Methylnitrosamino)-l-(3-pyridyl)-lbutanone Pulmonary Metabolism and Tumorigenicity in Mice by Analogues of the Investigational Chemotherapeutic Drug 4-lpomeanol Jyh-Ming Lin, Dhimant
H. Desai, Mark A. Morse, Shantu Amin,* and Stephen S. Hecht
Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received April 13, 1992
4-Ipomeanol (IPO) is an investigational chemotherapeutic drug with specific toxicity toward the lung. It is metabolically activated t o reactive intermediates by cytochrome P450 enzymes present in Clara cells. 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) is a highly carcinogenic tobacco-specific nitrosamine with organospecificity for the lung. Like IPO, which it resembles structurally, it is metabolically activated by cytochrome P450 enzymes of rat Clara cells. We synthesized nontoxic analogues of IPO and tested their activities as inhibitors of the metabolism and tumorigenicity of NNK. The IPO analogues synthesized were 4-hydroxy-lphenyl- 1-pentanone (HPP), 7-hydroxy-1-phenyl-1-octanone (HPO), 4-hydroxy-1 (2-thienyl)1-pentanone (HTP), and 4-hydroxy-l-(3-pyridyl)-l-pentanone (HPYP). When added to A/J mouse lung microsomal incubations, all compounds significantly inhibited the oxidative pathways of NNK metabolism-a-hydroxylation and pyridine N-oxidation-to varying extents. Inhibition of carbonyl reduction of NNK was generally less effective. Inhibition of a-hydroxylation by IPO, HPP, and H T P was more pronounced in incubations with lung microsomes than with liver microsomes. None of the IPO analogues showed significant toxicity when given to A/J mice at a dose of 25 pmol; IPO itself was lethal a t this dose. H P P and HPO, at doses of 25 pmol, significantly inhibited lung tumor multiplicity in mice treated with NNK; the other analogues and IPO itself were ineffective. The results of this study provide new leads for development of inhibitors of NNK metabolism and chemical probes for the active site of P450 enzymes in Clara cells.
-
Introduction 4-Ipomeanol (IPO,' Figure 1) is a naturally occurring pulmonary toxin isolated from sweet potatoes infected with the fungus Fusarium solani (1). Elucidation of the mechanism of its lung-specific toxicity has led to its development as a potential new drug for the treatment of lung cancer (I). IPO is metabolically activated to reactive intermediates by cytochrome P450 enzymes present in the Clara cells of the lung as well as by several human P450s (2-5). The binding of these metabolites to Clara cell macromolecules is associated with its toxicity. IPO shows remarkable specificity for binding and toxicity in the rat lung. 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK, Figure 1)is a potent pulmonary carcinogen in mice, rats, and hamsters (6, 7). Although NNK induces tumors of other organs as well, the lung is its major target. Because of ita relative abundance in tobacco smoke and strong carcinogenicity, NNK is believed to be involved in lung cancer induction in smokers (8). Like IPO, NNK is metabolized effectively by Clara cells of rat lung, and cytochrome P450 enzymes are known to be involved in its metabolic activation (9-12). We were struck by the Abbreviations: IPO,4-ipomeanol;NNK, 4-(methylnitrosamino)-l(3-pyridylbl-butanone;MS,mass spectra;TLC,thin-layerchromatography;THF,tetrahydrofuran;HPP,4-hydroxy-1-phenyl-1-pentanone; HPO,7-hydroxy-1-phenyl-1-octanone; HTP,4-hydroxy-l-(Z-thienyl)-lpentanone;.HPYP,4-hydroxy-l-(3-pyridyl)-l-pentanone; NCS, N-chlorosuccinimide.
&" H 3: '+
IPO
I
W
C 0
H
NNK
@$hc
,
HTP
HPYP
Figure 1. Structures of IPO,NNK, and analogues.
structural similarities of these two biologically active compounds that are metabolized by Clara cells. We are interested in developing agents which can inhibit NNK metabolic activation and potentially prevent NNK carcinogenesis (13).We hypothesized that IPO or its structural analogues might be competitive inhibitors of NNK metabolism in the lung, thus preventing its metabolic activation to the DNA adducts which are associated with its carcinogenicity. Since IPO is toxic, we synthesized several analogues of IPO (Figure 1) and examined their 0 1992 American Chemical Society
Ipomeanol Analogues a n d NNK
effects on the metabolism and tumorigenicity of NNK in the A/J mouse. The AIJ mouse was used for these studies because the mechanism of NNK tumorigenesis is well understood in this model, and tumors can be induced rapidly by a single dose of NNK (14,15). However, it should be noted that the preferential metabolic activation of NNK by Clara cells observed in rat lung is not evident in A/J mouse lung, where both Clara cells and type I1 cells efficiently activate NNK (16).
Experimental Section General Procedures. NMR spectra were determined in CDC13 on a Bruker AM 360 WB or Jeol FX9oQ spectrometer. Chemical shifts are expressed in ppm downfield from tetramethylsilane. MS were determined with a Hewlett-Packard Model 5988A instrument. High-resolution MS were determined on a VG-70SE instrument at the Institute of Environmental Medicine, New York University. Elemental analysis was carried out by Schwarzkopf Laboratories, New York, NY. TLC was performed on aluminum-supported precoated silica gel plates from EM Separations (Gibbstown,NJ). Most starting materials were obtained from Aldrich Chemical Co., Milwaukee, WI. IPO, 4-oxo-4-(3-pyridyl)butanal(15), and 2-phenyl-l,3-dithiane (1) were prepared according to literature procedures (17-19). Synthesis. (A) 2-(2-Thienyl)-l$-dithiane(2). 2-Thiophenecarboxaldehyde (6.0 g, 53.5 mmol) in benzene (100 mL) was mixed with 1,3-propanedithiol (6.14 g, 56.8 mmol) and p toluenesulfonic acid (20 mg). The solution was heated under reflux for 4 h, and HzO was removed by azeotropic distillation. The mixture was diluted with 200 mL of EtzO, washed with saturated NaHC03 (50 mL X 2), and dried (NazSO4). Removal of solvent and crystallization from MeOH yielded 2 (9.46 g, 87 % ): mp 77-78 "C [lit. (20)77-78 "C]; 'H NMR (CDC13) 6 1.84-2.28 (m, 2 H), 2.80-3.04 (m, 4 H), 5.38 (8, 1 H), 6.8-6.96 (m, 1 H, thiophene H4), 7.04-7.24 (m, 2 H, thiophene H3 and H5); MS m/z (re1 intensity) 202 (Mt, 74), 159 (lo), 128 (1001,127 (84), 84 (14), 45 (62). (B) 2 42-(2-Phenyl-l,3-dit hian-2-y1)et hyll- 1,3-dioxolane (3). A solution of 2-phenyl-l,3-dithiane (1, 1.55 g, 7.69 mmol) in anhydrous THF (50 mL) was treated under Nz at -78 "C with n-butyllithium (9.0 mL, 0.94 M, 8.46 mmol). After stirring for 1h at -78 "C, a solution of 2-(2-bromoethyl)-1,3-dioxolane (1.62 g, 8.96 mmol) in dry THF (20 mL) was added. This reaction mixture was stirred at -78 "C for 2 h and allowed to warm up to room temperature overnight. The mixture was poured into 100 mL of HzO, which was extracted with EtOAc (3 X 100 mL). The combined organic layers were dried (MgS04) and concentrated in vacuo to yield a crude product, which was chromatographed on silica gel with elution by hexane/EtOAc (20:l) to give 3 (1.9 g, 80%): mp 69-70 O C ; Rf0.18 [hexane/EtOAc (10:l)l; 'H NMR (CDC13) 6 1.40-1.74 (m, 2 H), 1.76-2.28 (m, 4 H), 2.56-2.76 (m, 4 H), 3.60-3.92 (m, 4 H), 4.72 (t, 1H, J = 4.0 Hz), 7.08-7.42 (m, 3 H), 7.68-7.92 (m, 2 H); MS m/z(re1intensity) 296 (M+,ll), 195 (24), 121 (42), 73 (100); high-resolution MS, calcd for M+ 269.0905, found 269.0910. In a similar manner, compound 4 was prepared as an oil in 76 % yield from 2-phenyl-1,3-dithianeand 2-(6-bromohexyl)-l,3dioxolane (see below): Rf 0.21 [hexane/EtOAc (19:l)l; lH NMR (CDC13) 6 1.0-1.62 (m, 8 H), 1.70-2.06 (m, 4 H), 2.42-2.70 (m, 4 H), 3.62-3.90 (m, 4 H), 4.70 (t, 1 H, J = 5.2 Hz), 7.04-7.40 (m, 3 H), 7.70-7.86 (m, 2 H);MS mlz (re1intensity) 338 (Mt, 18),231 (42), 193 (64), 73 (100). Anal. Calcd for ClaH2602Sz: C, 63.86; H, 7.74; S, 18.94. Found: C, 63.74; H, 7.76; S, 18.90. By using the above procedure, compound 5 was prepared in 61 % yield from 2-(2-thienyl)-1,3-dithiane(2) a n d 2-(2-bromoethyl)-1,3-dioxolane: mp 72-73 "C; lH NMR (CDC13) 6 1.64-2.31 (m, 6 H), 2.63-2.96 (m, 4 H), 3.79-3.93 (m, 4 H), 4.81 (t, 1H , J = 4.38Hz), 6.89-6.99 (m, 1H, thiophene H4), 7.17-7.30 (m, 2 H, thiophene H3 and H5); MS m/z (re1intensity) 302 (M+, lo), 228 (lo), 201 (18),127 (32), 109 (30), 86 (go), 73 (100); highresolution MS, calcd for Mt 302.0469, found 302.0472.
Chem. Res. Tonicol., Vol. 5, No. 5, 1992 675 (C) 2-(6-Bromohexy1)-1,3-dioxolane.Ethyl 6-bromohexanoate (15 g, 67.4 mmol) was dissolved in 200 mL of dry toluene and treated with diisobutylaluminum hydride (83.6 mL, 1.0 M) under an NZatmosphere at -78 "C. The mixture was stirred for 3 h a t -78 "C and then warmed to room temperature over 20 min. It was poured into 100mL of 2 N HzS04 and extracted with EhO (3 x 150 mL). The combined organic layers were washed with saturated NaHC03, dried (NazSOr),filtered, and concentrated to give 10.9 g of crude product, which was chromatographed on silica gel with hexane/EtOAc (151)as eluent to yield 6-bromohexanal (9.27 g, 76.8% ): lH NMR (CDCl3) 6 1.28-2.01 (m, 6 H), 2.40 (t, 2 H, J = 5.2 Hz), 3.28 (t, 2 H, J = 5.2 Hz), 9.64 [s, 1 H, CH(O)I. 6-Bromohexanal(10.6g, 59.1 mmol) was dissolved in benzene and mixed with 70 mL of ethylene glycol and p-toluenesulfonic acid (0.11 9). The mixture was heated under reflux for 6 h, and HzOwas removed by azeotropicdistillation. The reactionmixture was poured into 100mL of saturated NaHC03 and then extracted with hexane (3 X 100 mL). The combined organic layers were washed with saturated NaHC03, dried (NazSOd), filtered, and concentrated to give the crude product, which was chromatographed on silica gel with elution by hexane/EtOAc (19:l) to yield 2-(6-bromohexyl)-l,3-dioxalane (11.7 g, 88%): lH NMR (CDC13)6 1.25-2.0 (m, 8 H), 2.28 (t, 2 H, J = 5.2 Hz), 3.65-3.80 (m, 4 H), 4.80 (t, 1 H, J = 5.2 Hz). (D) 4-Phenyl-4-(1,3-dithian-2-yl)butyraldehyde (6). Aryl acetal 3 (1.73 g, 5.85 mmol) was dissolved in a mixture of 80 mL of 50% aqueous isopropylalcoholand glacial acetic acid (16mL). The mixture was heated under reflux for 24 h and poured into cold HzO(120mL). Extraction with CHC13 (3 X 100 mL), drying (MgSO& filtration, and concentration in vacuo yielded the crude product. This was chromatographed on silica gel with hexane/ EtOAc (201) as eluent to yield 6 as an oil (1.02 g, 70%): R/ 0.32 [hexane/EtOAc (10:l)l; lH NMR (CDCl3) 6 1.80-2.08 (m, 2 H), 2.3-2.52 (m, 4 H), 2.60-2.80 (m, 4 H), 7.08-7.46 (m, 3 H), 7.727.92 (m, 2 H), 9.54 [s, 1 H, CH(0)I; MS mlz (re1 intensity) 252 (M+, 22), 223 (4), 195 (44), 136 (loo), 121 (94), 77 (34). Likewise, aldehyde 7 was prepared from 4 in 81% yield as an oil: Rf0.35 [hexane/EtOAc (10:l)l; 'H NMR (CDC13)6 1.04-2.08 (m, 10 H), 2.25 (t,2 H, J = 9.0 Hz), 2.44-2.76 (m, 4 H), 7.08-7.44 (m, 3 H), 7.68-7.92 (m, 2 H), 9.54 [s, 1H, CH(0)I; MS m/z (re1 intensity) 294 (M+,221,220 (€9,195 (1001,136 (69), 121 (661,103 (42), 77 (31). By use of this method, aldehyde 8 was prepared in 92 % yield from5: Rf0.28 [hexane/EtOAc (10:l)l; 'HNMR (CDCl3) 6 1.762.12 (m, 2 H), 2.28-2.96 (m, 8 H), 6.84-7.0 (m, 1 H, thiophene H4), 7.12-7.32 (m, 2 H, thiophene H3 and H5), 9.58 [s, 1 H, CH(O)]; MS m/z (re1 intensity) 258 (Mt, 44), 201 (341,142 (54), 127 (loo), 107 (42). (E)4-Hydroxy-l-phenyl-l-(1,3-dithian-2-yl)pentane(9). The aryl aldehyde 6 (0.82,3.25mmol) was dissolvedin anhydrous THF (25 mL) and treated with methylmagnesium bromide (4.5 mL, 3.0 M, 13.5 mmol) under an NZatmosphere at -78 "C for 2 h. The reaction mixture was gradually allowed to warm up to room temperature overnight. The mixture was poured into 200 mL of HzO. Extraction with EtOAc (3 X 100 mL), drying over MgS04, filtration, and concentration in vacuo gave the crude product, which was chromatographed on silica gel with hexane/ EtOAc (5:l) as eluent to yield 9 (0.57 g, 65%): Rf 0.30 [hexane/ EtOAc (41)l; 'H NMR (CDC13) 6 1.08 (d, 3 H, CHs, J = 6.4 Hz), 1.18-1.52 (m, 3 H), 1.76-2.08 (m, 3 H), 2.48-2.76 (m, 4 H), 3.453.82 (m, 1 H), 7.08-7.44 (m, 3 H), 7.72-7.92 (m, 2 H); MS m/z (re1intensity) 268 (Mt, €9,195 (36), 161 (loo), 136 (88),121 (56), 103 (30), 77 (28). Similarly,10was prepared in 62 % yield as an oil from aldehyde 7: Rf0.23 [hexane/EtOAc (4:l)l; 'H NMR (CDCls) 6 1.06-1.52 (m, 11 H), 1.8-2.1 (m, 4 H), 2.40-2.76 (m, 4 H), 3.50-3.85 (m, 1 H), 7.0-7.44 (m, 3 H), 7.64-7.92 (m, 2 H); MS mlz (re1intensity) 310 (M+, 14), 195 (62), 136 (21), 121 (30), 91 (18), 45 (100). In a similar manner, compound 11 was prepared in 97 % yield as an oil from aldehyde 8: Rf 0.28 [hexane/EtOAc (41)l; lH NMR (CDC13) 6 1.15 (d, 3 H, J = 6.9 Hz), 1.36-1.68 (m, 4 H), 1.76-2.28 (m, 2 H), 2.62-3.12 (m, 4 H),3.45-3.82 (m, 1 H), 6.84-
676 Chem. Res. Toxicol., Vol. 5, No. 5, 1992 6.96 (m, 1 H, thiophene H4), 7.08-7.28 (m, 2 H, thiophene H3 and H5); MS m/z (re1intensity) 274 (M+,lo), 201 (28), 167 (80), 142 (loo), 109 (28), 45 (30). By the same method, HPYP was prepared in 57 % yield from aldehyde 15: R10.35 [hexane/EtOAc (1:l)l; 'H NMR (CDC13) 6 1.25 (d, 3 H, CH3, J = 6.4 Hz) 1.8-2.08 (m, 2 H, CHz), 3.14 (t,2 H,COCHz,J = 6.9 Hz), 3.85-3.98 [m, 1 H, CH(OH)],7.4 (m, 1 H, pyr H5), 8.24 (m, 1H, pyr H4), 8.75 (d, 1H, pyr H6), 9.18 (s, 1 H, pyr H2); MS m/z (re1 intensity) 179 (M+,3), 164 (22), 134 (19), 122 (50), 106 (100); high-resolution MS, calcd for M+ 180.1025, found 180.1029. (F)4-Hydroxy-1-phenyl-1-pentanone(12, HPP). The aryl alcohol 9 (0.85 g, 3.17 mmol) in acetonitrile (6 mL) was added quickly to a well-stirred solution of NCS (1.69 g, 12.7 mmol) and silver nitrate (2.42 g, 14.2 mmol) in aqueous acetonitrile (80%, 60 mL) at room temperature. The mixture was stirred for 15 min and then treated successively with saturated NazSO3, saturated Na2C03,and brine and extracted with 100 mL of CH2Cldhexane (1:l). The organic layer was dried over MgS04, filtered, and concentrated in vacuo to give a residue, which was chromatographed on silica gel with hexane/EtOAc (3:l) as eluent to yield 12 (3,20) (0.27 g, 48%): R, 0.31 [hexaneiEtOAc (3:l)l; 1H NMR (CDC13)6 1.27 (d, 3 H, CH3, J = 6.4 Hz), 1.8-2.1 (m, 2 H, CHz), 3.14 [t, 2 H, C(O)CHz, J = 7.5 Hz], 3.85-3.95 [m, 1 H, CH(OH)], 7.4-7.62 (m, 3 H, aromatic), 7.90-8.02 (m, 2 H, aromatic); MS m/z (re1 intensity) 178 (M+, 2), 161 (4), 133 (14), 120 (48), 105 (1001, 77 (491, 51 (14). Similarly, compound 13 (HPO) was prepared in 65% yield from aryl alcohol 1 0 R, 0.23 [hexane/EtOAc (3:l)l; lH NMR (CDC13)6 1.19 (d, 3 H, CH3,J = 6.12 Hz), 1.33-1.59 (m, 6 H, CHd, 1.7-1.83 (m, 2 H, CHzCH), 2.98 [t, 2 H, CHzC(O), J = 7.3 Hzl, 3.73-3.86 [m, 1H, CH(OH)], 7.4-7.6 (m, 3 H, aromatic), 7.9-8.0 (m, 2 H, aromatic); MS m/z (re1 intensity) 220 (M+, 2) 202 (6), 120 (66), 105 (loo), 77 (50),45 (40);high-resolution MS, calcd for (M+ - HzO) 203.1436, found 203.1436. Likewise, compound 14 (HTP) was prepared in 71% yield as an oil from aryl alcohol 11: R, 0.44 [hexane/EtOAc (2:l)l; 'H NMR (CDC13) b 1.25 (d, 3 H, CH3, J = 6.4 Hz), 1.8-2.05 (m, 2 H, CHz), 3.08 [t, 2 H, C(O)CHz, J = 7.1 Hz], 3.82-3.94 [m, 1 H, CH(OH)], 7.13 (dd, 1H, thiophene H4, J = 4.9,3.8 Hz), 7.64 (dd, 1 H, thiophene H5, J = 4.91,0.93 Hz), 7.75 (dd, 1 H, thiophene H3, J = 3.71, 0.92 Hz);MS m/z (re1 intensity) 284 (M+, 4), 139 (9), 126 (78), 111(loo), 97 (4),45 (22);high-resolution MS, calcd for M+ 185.0636; found 185.0640. Effects of IPO and Analogues on NNK Metabolism. A/J mouse liver and lung microsomes were prepared as described previously (22). Hepatic microsomes (1 mg of protein) or pulmonary microsomes (0.5 mg of protein) were incubated with the test compounds (100 or 500 pM) and [5JH]NNK (Chemsyn Science Laboratories, Lenexa, KS) (lOpM, 1.5pCi) in the presence of 100 mM sodium phosphate (pH 7.4), 5 mM sodium bisulfite, 25 mM glucose 6-phosphate, 5 mM NADP+, 5 mM EDTA, 15 mM MgC12, and 3.8 units/mL glucose-6-phosphate dehydrogenase in a total volume of 1.0 mL. All incubations were carried out in triplicate. Incubations were terminated after 15 min (hepatic microsomes) or 30 min (pulmonary microsomes) by the addition of 0.1 mL of 60% trichloroacetic acid. Following centrifugation at 2000g for 5 min, the supernatant of each sample was removed and filtered through 25-pm Gelman Acrodiscs (Ann Arbor, MI) and analyzed by HPLC (21). Statistical evaluation was performed with Student's t-test. Preliminary Toxicity Study. Groups of 4 female A/J mice (7 weeks old) were given single doses of 50,10,5,or 1pmol/mouse HPP, HTP, or HPYP by intragastric gavage in 0.1 mL of corn oil. Two weeks after dosing, surviving mice were sacrificed via cervical dislocation. The liver, lungs, and kidneys of each animal were examined for gross lesions, and body and liver weights were recorded. Bioassay for Inhibition of NNK Tumorigenesis. The protocol for tumor induction by NNK was identical to that described previously (13, 15). Female A/J mice, 6-8 weeks old, were maintained under standard conditions and fed AIN-76A diet containing 5% corn oil. They were divided into groups of
Lin et
a1.
Scheme I. Synthesis of HPP, HPO, and HTP
1
.z
nS
S
oy CH,
ArX(CH,),dH4H
Ncs* AONOJ, CHJCN
"-
3.5
0
6.8
CHI
Arx(CH2),iH-OH 12-14
9.11
Ar=Ph, M ;1.3,6,9,12 (HPP) Ar=Ph. n=S: 1,4,7.10.13 (HPO) At&Thlsnyl, n.2 2.5,8,11,14 (HTP)
20 as summarized in Table V. The test compounds were administered in 0.1 mL of corn oil by intragastric gavage. Doses are given in Table V. Two hours later, the mice were given an ip injection of either 0.1 mL of saline or 0.1 mL of saline containing 10 pmol of NNK. Sixteen weeks later, the mice were killed and lung adenomas were counted. Tumor multiplicity was evaluated statistically using Student's t-test.
Results The method illustrated in Scheme I was found to be useful for the preparation of three of the IPO analogues. The appropriate (haloalky1)dioxolanesand aryldithiaranes were condensed to give products 3-5. These were converted to the aldehydes 6-8 by selective deprotection with acetic acid in isopropyl alcohol. Reaction of 6-8 with methylmagnesium bromide followed by removal of the thiarane group with NCS provided the desired products in good yield. The pyridyl analogue, HPYP, was synthesized from 4-0~0-4-(3-pyridyl)butanal(l5) by selective reaction of the aldehyde carbonyl with methylmagnesium bromide.
15
HPYP
IPO and its analogues were teated for their ability to inhibit NNK metabolism in A/J mouse lung and liver microsomes. The major microsomal metabolites of NNK are illustrated in Figure 2. NNK N-oxide and NNAL are formed by pyridine N-oxidation and carbonyl reduction of NNK. They are both less tumorigenic than NNK in A/J mice (23, 24). OPB and HPB are produced by a-hydroxylation of the methylene and methyl carbons of NNK, respectively. Methylene hydroxylation also gives methanediazohydroxide, which leads to the formation of methylated DNA bases in vivo. This pathway is believed to be critical in A/J mouse lung tumorigenesis by NNK (14). Methyl hydroxlyation also yields 4-(3-pyridyl)-4oxobutanediazohydroxide, which pyridyloxobutylates mouse lung DNA and enhances the tumorigenic potential of the methylation pathway (14). Previous studies have demonstrated that pyridine N-oxidation and a-hydroxylation of NNK in mouse lung microsomes are mediated by cytochrome P450 enzymes (12). Initially, IPO or its analogues were added at concentrations of 10, 100, or 1000 pM to incubations of mouse liver or lung microsomes containing cofactors and 10 pM [5-3HlNNK. The results of these preliminary experiments showed that inhibition of metabolism was generally poor at 10 pM but was extensive at 1000 pM (data not shown). Thus, in subsequent experiments, IPO or ita analogues were added to the incubations at concentrations of 100 or 500 pM.
Ipomeanol Analogues and NNK N=O
0 W t
'
" I
Y
H
s
-
W
Chem. Res. Toxicol., Vol. 5, No. 5, 1992 677 IpO N 0*
NNK-N-oxide
C
H
N=O 3
e
f-
NNK
/
-0
N
Y
HI
3
NNAL
\
1
OPB
\ DNA pyridyloxobutylation
DNA-methylation
Figure 2. Major metabolic pathways of NNK in microsomes. Table I. Effects of IPO and Analogues on NNK Metabolism in A/J Mouse Lung Microsomesa~b inhibitor inhibitor added NNK metabolite control values concentration ( p M ) IPO HPP HPO HTP HPYP 8.30 f 0.32 OPBc 15.8 f 2.3 100 11.8 f 0.88 2.16 f 0.48 5.84 f 0.32 7.00 f 0.38 500 5.78 f 1.96 4.36 f 0.33 NDd 2.08 f 0.83 1.04 f 1.07 8.66 f 0.85 HPB 35.6 f 4.8 100 21.6 f 1.56 8.84 f 0.21 7.13 f 0.64 13.7 f 0.80 500
9.69 f 1.34
3.35 f 0.28
1.92 f 0.43
2.02 f 0.13
3.92 f 0.39
NNK N-oxide
42.6 f 4.2
100 500
24.3 f 2.60 9.61 f 1.27
6.16 f 4.12 2.74 f 0.27
14.22 f 0.76 3.48 f 0.38
7.30 f 1.15 2.04 f 0.54
16.7 f 1.09 5.56 f 1.02
NNAL
15.0 f 3.9
100 500
14.3 f 0.39 13.6 f 0.78
11.3 f 0.35 8.76 i 0.02
7.68 f 0.92 5.12 f 0.40
12.9 f 0.33 10.3 f 0.13
12.9 f 0.40 10.6 f 0.23
a Incubations were carried out for 30 min with 10 pM [5-3H]NNK,pulmonary microsomes (0.5 mg), cofactors, sodium bisulfite, and 0,100, or 500 pM inhibitor, as described in the Experimental Section. Values: pmol/(min.mg of protein) f SD, N = 3. OPB was determined as its sodium bisulfite adduct. ND, not detected.
Table 11. Effects of IPO and Analogues on NNK Metabolism in A/J Mouse Liver Microsomesn.b inhibitor inhibitor added NNK metabolitec control values concentration (pM) IPO HPP HPO HTP HPYP OPBd 76.5 f 4.21 100 68.2 f 1.80 51.5 f 5.21 14.6 f 0.82 47.2 f 2.33 43.3 f 10.70 HPB NNAL
500
44.7 i 2.94
24.2 f 1.82
2.46 f 2.23
18.2 f 0.66
15.8 f 1.21
41.0 f 2.47
100 500
38.8 f 2.48 24.9 f 1.81
28.1 f 3.09 14.5 f 0.94
8.88 f 0.39 2.88 f 0.39
24.2 f 0.14 10.4 f 0.44
21.9 f 2.86 9.62 f 0.72
134.6 f 3.33
100 500
135.7 f 2.31 135.4 f 1.97
99.0 f 2.42 75.2 f 1.42
124.7 f 6.35 99.1 f 5.03
122.8 f 1.11 108.5 f 1.50
64.0 f 3.91 37.7 f 2.01
a Incubations were carried out for 15 min with 10 p M [5-3H]NNK,hepatic microsomes (1 mg of protein), cofactors, sodium bisulfite, and 0,100, or 500 pM inhibitor, as described in the Experimental Section. Values: pmol/(min.mg of protein) f SD, N = 3. NNK N-oxide was
not detected in liver microsomal incubations. OPB was determined as its sodium bisulfite adduct.
The results of the assays carried out with mouse lung microsomes are summarized in Table I. All compounds inhibited pyridine N-oxidation and both a-hydroxylation pathways significantly (see Table 111). Inhibition of carbonyl reduction was generally less effective. In the incubations carried out with 100pM of IPO or ita analogues, differences among the inhibitors were apparent. The most effective inhibitors of the oxidative pathways were HPP, HPO, and HTP. HPO was the strongest inhibitor of ahydroxylation at the methylene carbon of NNK, whereas all three compounds strongly inhibited a-hydroxylation of the methyl carbon. HPP and HTP were the best inhibitors of pyridine N-oxidation. The results of the liver microsomal assays are summarized in Table 11. In agreement with previous results, NNK
N-oxide was not detected as a metabolite of 10 pM NNK. With the exception of 100pM IPO, all compounds inhibited the two a-hydroxylation pathways significantly (Table 111). Inhibition of NNAL formation was less pronounced in general, as in the lung microsomal experiments. HPO was the best inhibitor of all pathways. Comparisons of the results obtained in the lung and liver microsomal incubations indicated some selectivity for inhibition of the a-hydroxylation pathways (Table 111). IPO, HPP, and HTP all inhibited both a-hydroxylation pathways more effectivelyin lung than in liver microsomes. These results suggest that IPO, HPP, and HTP may be relatively specific inhibitors of cytochrome P450s which are present in AfJ mouse lung.
678 Chem. Res. Toxicol., Vol. 5,No.5, 1992
Lin et al.
Table 111. Percent Inhibition of NNK Metabolite Formation by IPO and Analogues in A/J Mouse Lung and Liver Microsomes NNKmetabolite
inhibitor concentration(pM)
IPO
HPP
100 500 100 500 100 500 100 500
25" 63b 3g6 73d 43c 77e 5 9
47c 72d 76d 91e 86d 94e 25 42n
OPB HPB NNK N-oxide NNAL
Significant inhibition compared to control, P < 1
X
lung HPO 86d 100'
75e 95' 67d 92' 49" 66b
compound corn oil HPP
HTP
HPYP
a
~
~~
~~
dose (pmol) 50 10 5 1 50 10 5 1 50 10 5 1
body weight (g) 21.1 f 0.5 20.4 f 1.2 20.3 f 1.3 20.6 f 1.3 21.3 f 0.5 20.6 f 1.3 19.0 f 1.2 19.6 f 1.4 19.7 f 0.8 20.7 f 1.9 18.9 f 1.5 20.8 f 0.8 20.9 f 2.2
~
HPYP
IPO
HPP
liver HPO
HTP
HPYP
63d 87d 80' 94e 83' 95e 14 32"
55c 93d 61d 89' 61d 87e 14 29
11" 42d 5 3gd
33c 68' 32b 65'
81' 97' 78' 93'
38d 76' 41e 75e
436 79' 47c 77'
0 0
26e 44O
52O 72'
7 26d
9C 19e
(Student's t-test). P < 1 x 10-3. P
Table IV. Effect of Some IPO Analogues on Body and Liver Weights of A/J Mice. ~
HTP
