Inhibition of Human Cytochrome P450 1A1-, 1A2-, and 1B1-Mediated

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Chem. Res. Toxicol. 2006, 19, 288-294

Inhibition of Human Cytochrome P450 1A1-, 1A2-, and 1B1-Mediated Activation of Procarcinogens to Genotoxic Metabolites by Polycyclic Aromatic Hydrocarbons Tsutomu Shimada* and F. Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine, 606 Light Hall, 2215 Garland AVenue, NashVille, Tennessee 37232-0146 ReceiVed October 20, 2005

Many chemicals in the environment can cause cancer, and polycyclic aromatic hydrocarbons (PAHs) are among the most ubiquitous. Cancer risk assessments require consideration of these in complex mixtures. PAHs require metabolic activation by cytochrome P450 (P450) enzymes, primarily 1A1, 1A2, and 1B1. We determined if individual PAHs and other procarcinogens affect the activities of human P450s 1A1, 1A2, and 1B1 by measuring 7-ethoxyresorufin O-deethylation (EROD) activity and metabolic activation of PAH dihydrodiols and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) to genotoxic metabolites in a Salmonella typhimurium NM2009 system. Of 23 PAHs examined, benz[a]anthracene (B[a]A), benzo[b]fluoranthene, and 5-methylchrysene were the most potent inhibitors of P450 1A2- and 1B1-catalyzed EROD activity, with IC50 values 1000 170 ( 26 350 ( 46 >1000 250 ( 52 250 ( 22 450 ( 72 330 ( 62 >1000 130 ( 21 >1000 56 ( 7 >1000 >1000 >1000 750 ( 120 >1000 >1000 135 ( 22 500 ( 77 >1000

>1000 >1000 >1000 7.7 ( 1.0 140 ( 15 24 ( 2.6 9.5 ( 1.4 35 ( 3.2 23 ( 2 >1000 51 ( 8 47 ( 12 >1000 41 ( 8 >1000 150 ( 20 >1000 >1,000 14 ( 2 150 ( 19 6.0 ( 0.7 >1000 >1000

>1000 >1000 >1000 9.1 ( 1.8 31 ( 6. 17 ( 3 4.9 ( 1.0 44 ( 8 27 ( 4 >1000 92 ( 23 8.8 ( 1.4 5.2 ( 0.6 15 ( 2 700 ( 140 70 ( 11 >1000 300 ( 54 15 ( 3 14 ( 2 8.0 ( 1.0 >1000 350 ( 47

15 ( 3 120 ( 26 200 ( 32 200 ( 29 170 ( 27 5.5 ( 0.7 >1,000 360 ( 55 >1000 >1000 >1000

9.0 ( 1.4 600 ( 72 200 ( 29 42 ( 7 90 ( 8 8.0 ( 1.3 140 ( 18 50 ( 11 900 ( 120 >1000 >1000

R-naphthoflavone (RNF) 1-ethynylpyrene 1-(1-propynyl)pyrene 2-ethynylphenanthrene 3-ethynylphenanthrene galangin 2,4,3′,5′-tetramethoxystilbene acacetin piceatannol resveratrol thiotepa

other compounds 60 ( 13 120 ( 35 >1000 >1000 >1000 65 ( 13 240 ( 44 100 ( 13 >1000 >1000 >1000

a A substrate concentration of 2.5 µM was used. Uninhibited EROD activities of P450 1A1, 1A2, and 1B1 were 46 ( 5, 9.5 ( 0.9, and 27 ( 3 nmol product formed/min/nmol P450, respectively. Chemicals (inhibitors) were added at concentrations ranging from 0.64 to 400 nM. Results are presented as IC50 ( SD.

were determined to be 135, >1000, and 350 nM for the inhibition of EROD by 5-Me chrysene, DB[a,h]A, and B[a]P, respectively, in the P450 1A1 system. When P450 1A2 was used, IC50 values of 6.0, >1000, and 140 nM, respectively, were estimated. IC50 values for inhibition of P450 1B1 were 8.0, 5.2, and 31 nM, respectively. Inhibition of EROD Activities by 23 PAHs and 11 Chemicals Reported to Inhibit P450 Family 1-Dependent Activities. Of the 23 PAH compounds examined, B[a]A, benzo[b]fluoroanthene, and 5-Me chrysene were the most potent in inhibiting P450 1A2 and 1B1; the IC50 values were 4000 >4000 >4000 3000 ( 420 >4000

84 ( 17 620 ( 81 290 ( 26 800 ( 160 330 ( 53 290 ( 67 900 ( 150 >4000 3500 ( 700 400 ( 65 420 ( 84 400 ( 38

250 ( 33 120 ( 26 70 ( 13 500 ( 83 170 ( 27 300 ( 55 560 ( 52 >4000 >4000 310 ( 68 150 ( 20 400 ( 55

100 ( 12 >4000 >4000

45 ( 10 >4000 >4000

>4000 >4000 >4000 >4000 >4000 >4000 >4000

>4000 >4000 >4000 >4000 >4000 55 ( 10 200 ( 26

>4000 >4000

>4000 >4000

PAH diols B[a]P-4,5-diol 3-OH B[a]P 9-OH B[a]P (+)-B[a]P-7,8-diol (-)-B[a]P-7,8-diol benzo[b]fluoroanthene-4,5-diol benzo[b]fluoroanthene-9,10-diol 7,12-DMBA-3,4-diol DB[a,l]P-11,12-diol chrysene-1,2-diol 3-MC-11,12-diol 5-Me chrysene-1,2-diol

amino and nitro PAHs 110 ( 38 >4000 >4000

6-aminochrysene 2-nitrochrysene 6-nitrochrysene

heterocyclic arylamines 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1) >4000 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) >4000 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) >4000 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) >4000 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) >4000 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) >4000 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) 1200 ( 132 mycotoxins >4000 >4000

aflatoxin B1 sterigmatocystin

a See Table 1 for uninhibited EROD activities of P450s 1A1, 1A2, and 1B1. Chemicals were added at concentrations ranging from 1.6 to 1000 nM. The substrate concentration used was 2.5 µM. Results are presented as IC50 ( SD.

Table 3. Inhibition of EROD Activity by Chemical Inhibitors in P450 1A1, 1A2, and 1B1 Systemsa

Table 4. IC50 Values for Inhibition of P450-Dependent Activation of Promutagensa

EROD activity inhibitor P450 1A1

P450 1A2

P450 1B1

none B[a]P RNF galangin none B[a]P 5-Me chrysene RNF galangin none B[a]P 5-Me chrysene RNF galangin

concn (µM) 0.8 0.16 0.16 0.4 0.016 0.032 0.032 0.08 0.016 0.016 0.016

Km (µM)

kcat (nmol/min/ nmol P450)

1.5 ( 0.3 0.63 ( 0.24 0.45 ( 0.13 0.32 ( 0.10 0.61 ( 0.11 0.87 ( 0.11 0.84 ( 0.16 0.37 ( 0.09 0.22 ( 0.05 0.54 ( 0.10 0.37 ( 0.05 0.26 ( 0.06 0.22 ( 0.04 0.23 ( 0.06

65 ( 2 21 ( 3 26 ( 2 18 ( 2 11 ( 1 4.1 ( 0.2 3.8 ( 0.2 4.9 ( 0.3 3.3 ( 0.2 29 ( 2 14 ( 1 11 ( 1 12 ( 1 16 ( 1

a EROD activities were determined at substrate concentrations between 0.0195 and 5.0 µM, and kinetic parameters were determined. Results are presented as ( SD estimated for kcat and Km (from GraphPad Prism).

procarcinogens used heres5-Me chrysene-1,2-diol, (()-B[a]P7,8-diol, DB[a,l]P-11,12-diol, and MeIQshave previously been shown to be very active in inducing umu gene expression after activation by P450s in this bacterial tester strain (14, 29). The parent PAHs (5-Me chrysene, B[a]P, and B[a]A) were slightly activated to genotoxic products in S. typhimurium NM2009 by P450s 1A1 and 1B1 (Figure 3A,G); activation of these three PAHs by P450 1A2 was very limited (Figure 3E) relative to the rates of activation measured when 5-Me chrysene1,2-diol, (()-B[a]P-7,8-diol, and DB[a,l]P-11,12-diol were used as substrates in the absence of inhibitors (Figure 3). The effects of the three PAHss5-Me chrysene, B[a]P, and B[a]Ason the activation of 5-Me chrysene-1,2-diol, (()-B[a]P-7,8-diol, and DB[a,l]P-11,12-diol by P450 1A1 and 1B1 and of MeIQ by P450 1A2 were determined (Figure 3 and Table 4). Inhibition

P450 1A1 IC50 (µM) inhibitor

5-Me chrysene1,2-diol

(()-B[a]P7,8-diol

DB[a,l]P11,12-diol

5-Me chrysene B[a]P B[a]A RNF galangin

8.1 ( 2.2 6.2 (1.8 3.3 ( 0.9 2.4 ( 0.8 0.93 ( 0.35

18 ( 4 18 ( 2 18 ( 3 4.9 ( 1.5 1.4 ( 0.3

7.9 ( 1.8 9.2 ( 1.7 7.3 ( 2.6 1.6 ( 0.5 0.46 ( 0.06

P450 1A2 IC50 (µM) MeIQ 8.9 ( 3.0 15 ( 4 0.94 ( 0.97 0.98 ( 0.30 0.39 ( 0.64

5-Me chrysene B[a]P B[a]A RNF galangin P450 1B1

IC50 (µM)

5-Me chrysene B[a]P B[a]A RNF galangin

5-Me chrysene1,2-diol

(()-B[a]P7,8-diol

DB[a,l]P11,12-diol

4.3 ( 0.4 2.2 ( 0.4 2.1 ( 0.6 2.2 ( 0.5 0.39 ( 0.10

9.4 ( 2.7 4.4 ( 0.7 6.4 ( 2.5 1.5 ( 0.2 0.39 ( 0.1

2.7 ( 0.7 2.2 ( 0.4 1.8 ( 0.3 1.0 ( 1.1 0.20 ( 0.50

a IC 50 values ((SD) were estimated and determined by nonlinear regression analysis using GraphPad Prism (GraphPad).

by RNF and galangin was compared with that of the three PAHs. The three PAHs inhibited P450 1A1-dependent activation of 5-Me chrysene-1,2-diol, (()-B[a]P-7,8-diol, and DB[a,l]P-11,12-diol with IC50 values of 3-8, ∼18, and 7-9 µM, respectively

292 Chem. Res. Toxicol., Vol. 19, No. 2, 2006

Shimada and Guengerich

Figure 4. Inhibition of metabolism by PAHs. Inhibition of metabolism A naphthyl moiety is shown as the PAH being activated to a genotoxin. AKR, aldo-ketoreductase (57). One PAH can inhibit the P450 activation of another at either the first or the latter step.

(Figure 3B-D and Table 4). RNF and galangin were more inhibitory (than the three PAHs) toward the activation of 5-Me chrysene-1,2-diol, (()-B[a]P-7,8-diol, and DB[a,l]P-11,12-diol catalyzed by P450 1A1 (Figure 3 and Table 4). Activation of MeIQ by P450 1A2 was most strongly inhibited by B[a]A with an IC50 value of 0.94 nM, which was comparable to RNF (0.98 nM) and galangin (0.39 nM) (Figure 3F and Table 4). The IC50 values with 5-Me chrysene and B[a]P were determined to be 9 and 15 µM, respectively, for the activation of MeIQ by P450 1A2. Three PAHs were stronger inhibitors of the activation of 5-Me chrysene-1,2-diol, (()-B[a]P-7,8-diol, and DB[a,l]P-11,12-diol by P450 1B1 than those by P450 1A1; the IC50 values with three PAHs in P450 1B1 were comparable to those of RNF but higher than that of galangin (Figure 3H-J and Table 4).

Discussion Urban atmosphere, diesel exhaust particles, tobacco smoke condensates, and broiled foods contain many types of carcinogens, and PAHs have been recognized to be the major ones (20, 22, 38). Most of the carcinogenicity studies have been done using single chemicals in experimental animals, and B[a]P, DB[a,h]A, DMBA, and 3-MC are reported to be potent carcinogens (4, 20). Complex mixtures of PAHs and other chemicals have been studied to determine if these mixtures cause increased or decreased capacities to form PAH-DNA adducts in vitro and tumor formation in vivo, as compared with single chemicals (27, 28, 39-45). The results reported to date are complex; in some cases, the carcinogenic activities and DNA-binding capacities are additive (20, 40-42, 46-48) but in other cases decreased when compared with individual carcinogens (20, 22, 25, 40, 46, 49-52). The complex mixtures contain different types of chemicals that cause alterations in the biological activities of PAHs (20, 22, 25). Because most of the PAHs require metabolic activation by P450s (and other enzymes) to exert their carcinogenic potentials, the nature of enzyme inhibitors and inducers in the mixtures has been studied extensively (15, 53-56). In general, the chemical inhibitors may be more important than the chemical inducers with respect to their low dose effects in modifying carcinogen metabolism and action (Figure 4) (37, 46, 58, 59). Numerous inhibitors of P450 1A1, 1A2, and 1B1 have been reported. These include flavonoids (RNF and galangin), 2,4,3′,5′tetramethoxystilbene, naturally occurring coumarins (bergamottin, imperatorin, and isopimpinellin), vinylic and acetylenic PAH compounds [e.g., 1-ethynylpyrene and 1-(1-propynyl)pyrene], and synthetic organoselenium compounds (34, 37, 46, 58, 6063). In vivo studies have shown that some of these inhibitors are able to suppress the tumor formation induced by PAHs and other carcinogens in experimental animals (61, 64-66). Several carcinogenic PAHs were found to be potent inhibitors of P450 1A2 and 1B1, with IC50 values