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[CANCER RESEARCH57, 4757-4764, November 1, 19971 Inhibition of Human P450-catalyzed Oxidations of and Procarcinogens by Synthetic Organoselenium Compounds'

Tsutomu Shimada,2 Karam El-Bayoumy, Pramod Upadhyaya, Thomas R. Sutter, F. Peter Guengerich, and Hiroshi Yamazaki Osaka Prefectural institute of Public Health, Osaka 537, Japan IT. S.. H. Y.J: Department of Environmental Health Sciences, Division of Chemical Carcinogenesis. American Health Foundation, Valhalla, New York 10595 (K. E., P. Ui; Jo/ma Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205 fT. R. 5.1; and Center in Molecular . Vanderbilt University School of , Nashville, Tennessee 37232 (F. P. G.J

ABSTRACT nium compounds are also active in inhibiting chemically induced tumor formation in laboratory , but these selenoamino acids The effects of synthetic chemopreventive organoselenium compounds had comparable chemopreventive activity to inorganic selenium com 1,2-, 1,3-, and 1,4-phenylenebis(methylene)seleneeyanate (o-, m-, and p xSC, respectively),benzyl selenocyanate(BSC),and dibenzyl diselenide pounds, and in some instances, they were more toxic than inorganic (DDS) and inorganic sodium selenite on the oxidation of xenobiotics and selenium compounds (5). Several synthetic organoselenium com procarcinogens by human cytochrome P450 (P450 or CYP) were pounds have been developed recently, and El-Bayoumy et a!. (9) have determined in vitro. Spectral studies showed that BSC and three XSC shown that the synthetic organoselenium compounds XSC3 inhibit compounds (but not sodium selenite or DDS) induced type II difference 7,12-dimethybenz[a]anthracene-induced tumors and DNA adduct for spectrum when added to the suspension of microsomes isolated from mation in female Sprague Dawley mammary gland; further studies Ø-naphthoflavone-treated , with m-XSC being the most potent in Inducing spectral interactions with P450 enzymes; m-XSC also produced by Ip et a!. (10) documented this observation. However, mechanisms a type II spectral change with human liver microsomes. o-, m-, andp-XSC underlying the effects of these selenium compounds on the inhibition Inhibited 7-ethoxyresorufin O-deethylatlon catalyzed by human liver ml of chemically induced tumor formation still remain unclear. crosomes when added at concentrations below 1 g.tMlevels, but BSC and P450 comprises a superfamily of enzymes that catalyze oxidation DDS were less effective. AH of these compounds inhibited the oxidation of of a great number of chemicals such as , toxic model substrates for human P450s to varying extents. We studied the chemicals, and carcinogens and endobiotic chemicals such as , effects of these compounds on the of procarcinogens by recom fatty acids, vitamins, and prostaglandins (1 1). A number of studies binant human CYP1A1, 1A2, and 1B1 enzymes using Salmonella typhi murium NM2009 tester strain for the detection of DNA damage. The three have suggested that most of the chemical carcinogens in the environ XSCs were found to be very potent inhibitors of metabolic activation of ment are not active themselves in interacting with intracellular DNA 3-amino-1,4-dlmethyl-5H-pyrldo[4,3-bjindole, 2-amino-3,5-dimethylimi but induce tumors only after metabolic activation by a variety of dazo[4,5-flqulnoline, and 2-aminoanthracene, catalyzed by CYP1A1, 1A2, -metabolizing enzymes including P450 (12, 13). During the past and 1B1, respectively. The potency of Inhibition of m-XSC on CYP1B1- decade, significant roles of several human P450 enzymes in the dependent activation of 2-aminoanthracene was compatible to those of activation of procarcinogens and promutagens have been determined a-naphthoflavone. These inhibitory actions may, in part, account for the in many laboratories; CYP1A1, lA2, 1BI, 2A6, 2El, and 3A4 have mechanisms responsible for cancer prevention by organoselenium corn pounds in laboratory animals. been reported to be the major enzymes involved in the activation of most of the procarcinogens and promutagens that are metabolized by P450 enzymes in humans (14, 15). Among these P450 enzymes INTRODUCTION determined, CYP1A1 and lBl have been shown to be expressed in Epidemiological studies have shown that positive correlations exist extrahepatic tissues in experimental animals and humans (1 6, 17). in blood selenium levels and cancer susceptibilities in humans, al Particular interests are the observation that both P450s have been though understanding of the roles of selenium in the prevention of shown to be expressed in mammary glands and to be able to cancer susceptibilities remains unclear (1—4). Inorganic selenium activate mammary carcinogens such as 7,12-dimethylbenz[a]an compounds such as sodium selenite, when administered to experimen thracene, fluoranthene-2,3-diol, benzo[c]phenanthrene-3,4-diol, tel animals at levels above dietary requirements, have been shown to dibenzo[a,l}pyrene-l 1,12-diol, and benzo[g]chrysene-l 1,12-diol. prevent tumor formation caused by carcinogenic polycyclic hydrocar The resulting diol have been shown to be potent mam bons in various organs including mammary glands, liver, skin, colon, mary carcinogens in the rat (18, 19). stomach, oral cavity, bladder, and pancreas (5—8).Because of the In this study, we examined the effects of several organoselenium severe toxic effects of these inorganic selenium compounds to exper compounds as well as inorganic sodium selenite on the activities of imental animals and also possibly to humans, most studies have been xenobiotic oxidation and procarcinogen activation by human liver done using naturally occurring organoselenium compounds (e.g., sel microsomes and by recombinant human CYP1A1, 1A2, and lBl enomethionine and selenocysteine), examining them for abilities to enzymes. Selenium compounds used in this study include sodium prevent tumor formation caused by environmental carcinogens. The selenite, BSC, DDS, and o-, m-, and p-XSC, and procarcinogenic results collectively indicate that the naturally occurring organosele substrates for the P450 enzymes used were 2-aminoanthracene, 2- aminofluorene, Trp-P-l, and MeIQ. Received 4/10/97; accepted 9/8/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Health and Welfare of Japan, the Develop 3 The abbreviations used are: o-, m-, and p-XSC, 1,2-, 1,3-, and 1,4-phenylenebis mental and Creative Studies from Osaka Prefectural Government, and by USPHS Grants (methylene)selenocyanate, respectively; P450 or CYP, cytochrome P450; BSC, benzyl CA46589, CA70792, CA44353, ES00267, and ESO6O7I. selenocyanate; DDS, dibenzyl diselenide; aNF, a-naphthoflavone (7,8-benzoflavone); 2 To whom requests for reprints should be addressed, at Osaka Prefectural Institute of @NF,@-naphthoflavone(5,6-benzoflavone);MeIQ,2-amino-3,5-dimethylimidazo(4,5- Public Health; 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka 537, Japan. Fax: 81-6- f]quinoline; Trp-P-l, 3-amino-l,4-dimethyl-5H-pyridoi4,3-blindole; DLPC, L-a-dilau 972-2393; E-mail: [email protected]. royl-sn-glycero-3-phosphocholine. 4757

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MATERIALS AND METHODS P450-dependent activation of procarcinogens to reactive metabolites that cause induction of umu expression in tester strain S. typhimurium Chemicals. 7-Ethoxyresorufin,7-ethoxycoumarin,coumarin, chlorzoxa NM2009 was determined in reconstituted CYP1A1- or 1A2-containing sys zone, and were purchased from Sigma Chemical Co. (St. Louis, tems or microsomes expressing CYP1B1 as described previously (16). MO), and S-warfarinand (±)-bufuralolwerefrom UltraFineChemicalsCo. Standard incubation mixtures consisted of 5 pmol P450 with 5—10 @Mpro (Manchester, United ). Organoselenium compounds were prepared carcinogen in a final volume of 1.0 ml of 100 mai potassium phosphate buffer and purified as described previously (20). Other substrates, their oxidation (pH 7.4) containing an NADPH-generating system and 0.75 ml of bacterial products, and reagents used in this study were obtained from sources as suspension as described previously (16). Incubation conditions for yeast mi described previously or of the highest qualities commercially available (21— crosomal and reconstitution systems were described above. The induction of 23). umu gene expressionby activatedcarcinogensis presentedas units of @3- Preparation. The human CYP1BI cDNA clone was introduced galactosidase activity/min/nmol P450 (21, 37). into Saccharomyces cerevisiae, and microsomes containing CYP1B 1 Spectral studies of interaction of selenium compounds and liver microsomal were prepared as described (17, 24). Recombinant human CYP1A1 and lA2 P450 enzymes were determined by recording difference spectrum using a were purified to homogeneity from membranes of in which Shimadzu UV-300 spectrophotometer. P450 was estimated spectrally by the modified P450 cDNAs had been introduced (25, 26). NADPH-P450 methods of Omura and Sato (38). Protein concentrations were estimated by the and cytochrome b3 were purified from liver microsomes of method of Lowry et a!. (39). treated rabbits by the method of Yasukochi and Masters (27) as modified by Kinetic parameters for the 7-ethoxycoumarin O-deethylation by rat and Taniguchi et aL (28). human liver microsomal P450 enzymes were estimated using a non-linear Human liver samples were obtained from organ donors or patients under regression analysis program (K'cat; BioMetallics, Princeton, NJ). going liver resection as described previously (22). Male Sprague Dawley rats (weighing about 200 g) obtained from Nihon Clea Co. (Osaka) were treated i.p. with j3-naphthoflavone (50 mg/kg, daily for 3 days) or phenobarbital (80 RESULTS mg/kg, daily for 3 days). Rats were starved overnight before killing on the fourth day. Liver microsomes were prepared as described and suspended in 10 Spectral Interaction of Selenium Compounds with Liver Mi mMTris-Cl buffer (pH 7.4) containing 1.0 mMEDTA and 20% glycerol (vlv; crosomes of Rats Treated with IINF and Phenobarbital and of Ref. 29). Humans. Spectral interaction of several organoselenium com Enzyme Assays. Standard incubation mixtures consisted of human P450 pounds with liver microsomes of f3NF-treated rats showed that enzymes (2—10pmol P450) with several substrates in a final volume of three XSC compounds and BSC induced type II spectral changes 0.25—1.0mlof 100 mM potassium phosphate buffer (pH 7.4) containing an with P450 enzymes in liver microsomes (Fig. 1A). Inorganic so NADPH-generating system consisting of 0.5 nmi NADP@, S mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase/mI (22). In dium selenite, at 0. 1 m@i,did not cause spectral changes with rat the case of yeast microsomes, NADPH-P450 reductase (2—10pmol) and liver microsomes. DDS was found to interact with liver micro sodium cholate (final concentration, 0.02%) were added to improve catalytic somes causing unique, but uncharacterized, spectral changes, prob activities (see “Results―;16). Oxidation of substrates by CYPIA1 and lA2 ably due to the turbidity of microsomes after interacting with DDS were determined in reconstituted systems containing P450 (2—10pmol)and (Fig. lB). NADPH-P450 reductase (2—10pmol)with DLPC as described (16, 30). Liver f3NF treatment of rats was found to increase spectral changes with microsomal incubations included microsomes (0.5 mg protein/ml) in 100 mr@i m-XSC, but phenobarbital did not (Fig. 2A). The same spectral change potassium phosphate buffer (pH 7.4) containing the NADPH-generating sys was seen with human liver microsomes (Fig. 2B). tem and various concentrations of drug substrates (21, 22, 31). Effects of Selenium Compounds on Xenobiotic Oxidations Cat The O-deethylation of ethoxyresorufin (10 @xM),7-hydroxylationof couma alyzed by Human Liver Microsomes. Effects of several selenium rim(50 ELM),andO-deethylation of 7-ethoxycoumarin (50 xM)were assayed fluorometrically according to the methods as described (29, 32). The methods compounds on the xenobiotic-oxidizing activities were determined in used for 1‘-,4-,and 6- of bufuralol ( concentration, 0.2 human liver microsomes (Fig. 3). The substrates used included 7- mM), 7- of S-warfarin (100 @xM),6-hydroxylation of chlorzoxa ethoxyresorufin (marker substrate for CYP1A2), coumarin zone (0.5 mM),and 6(3-hydroxylationof testosterone (0.2 mM)were described (CYP2A6), S-warfarin (CYP2C9), bufuralol (CYP2D6 for 1‘-hy previously (23, 33—36). droxylation and CYPIA2 for 6-hydroxylation), 7-ethoxycoumarin

.. ::::.B \@‘

Fig. 1. Spectral interactions of several organoselenium compounds with liver microsomes of I3NF-treatedrats. Liver microsomal suspension [I nmol P450/mI in 100 msi potas sium phosphate buffer (pH 7.4) containing 20% glycerol] was placed in two cuvettes, and the spectrum was recorded 0 between 340 and 510 nm at 25'C. The difference spectrum was obtained by adding 0. 1 msi (final) of o-XSC (0), m xSC (I), or p-XSC (Lx)in A and sodium selenite (0), BSC (I), or DDS (Lx)in B into the sample cuveue (10 p1 of DMSO was added to the reference cuvette).

@@@ -o.o1@ — .I . 380 400 440 480 360 400 440 480 Wavelength (nm) 4758

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A B 0.010 0.010

0.005 0.005

Fig. 2. Spectral interactions of m-XSC with liver micro somes of untreated rats (•)andrats treated with @NF(0)or “2 phenobarbital (i@)in A and of a human sample HL-16 (I) in @o.0oo 0.@\J\@M. B. Experimental conditions were described in the legend to Fig.1.

-0.005 -0.005

360 400 440 480 360 400 440 480 Wavelength (mn)

(CYP2E1 and CYP1A2), (CYP2E1), and testosterone deethylation by human liver microsomes, whereas the inhibition (CYP3A4; Ref. 22). The effects of aNF on these monooxygenation effects were lesser than those by XSCs and aNF. Sodium selenite reactions were also determined and compared with human liver mi did not inhibit the 7-ethoxyresorufin 0-deethylation at concentra crosomes. tions up to 1 p.M. The three XSCs were found to be the potent inhibitors of Selenium compounds used in this study did not cause strong inhi 7-ethoxyresorufin O-deethylation in liver microsomes of human bition of coumarmn 7-hydroxylation and bufuralol I ‘-hydroxylationat sample HL-l6; the potencies to inhibit the catalytic activities by concentrations up to 1 p.M. although these compounds inhibited con these selenium compounds were almost the same as those by aNF siderably S-warfarin 7-hydroxylation, 7-ethoxycoumarin 0-deethyla (Fig. 3A). BSC and DDS also inhibited 7-ethoxyresorufin 0- tion, and chlorzoxazone 6-hydroxylation. Testosterone 6@3-hydroxy

@1I @s50 0

@ 0 ‘ I 0 0.5 1.0

0 0 0.5 1.0 Chemical concentration (pM)

Fig. 3. Effects of several organoselenium compounds on the liver microsomal activities of 7-ethoxyresorufin O-deethylation (A; human sample HL- 16, with activity of 26 pmol product formed/mn/mg protein in the absence of organoselenium compounds), coumarin 7-hydroxylation (B; HL-l I with activity of 79 pmollmin/mg protein), S-warfarmn 7-hydroxylation(C; HL-16 with activity of 12 pmol/min/mg protein), bufuralol I'- and 6-hydroxylation (D and E, respectively; HL-18 with activities of58 and 36 pmollmin/mg protein, respectively), 7-ethoxycoumarmn O-deethylation (F; HL-1 1 with activity of 240 pmol/min/mg protein), chlorzoxazone 6-hydroxylation (G; HL-l I with activity of 4.5 nmol/min/mg protein), and testosterone 6@-hydroxylation(H; HL-l8 with activity of 1.3nmol/min/mg protein). The chemicals tested were BSC (0), DDS (•),o-XSC(h), m-XSC (A), p-XSC ([1. sodium selenite (U), and aNF (small U). 4759

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Fig. 4. Effects of several organoselenium compounds on the kinetics of 7-ethoxycoumarmn O-deethylation catalyzed by liver microsomes (10 pmol P450/ml of incubation mixture) of rats treated with 3NF (A) and of I a human sample HL-64 (B). The organoselenium compounds (final concentration, 0.5 p@M)testedwere BSC (0), DDS (•),o-XSC (is), m-XSC (A), p-XSC (1:), and aNF (small U).Control experiments (U) in the absence of these chemicals were also determined.

-60 -30 0 30 60 -60 -30 0 30 60 us (7-ethoxycoumarin,mM)

lation activity was inhibited very significantly by sodium selenite, Effects of Selenium Compounds on Activation of Procarcino m-XSC, and p-XSC and moderately by DDS, o-XSC, and aNF. gens Catalyzed by Recombinant Human CYP1A1, 1A2, and 1B1 Effects of Selenium Compounds on Kinetics of 7-Ethoxycou in S. typhimurium NM2009. Using optimal conditions for xenobiotic mann O-Deethylation by Liver Microsomes of @3NF-treatedRats oxidations by CYP1B1 in yeast microsomes and by CYP1A1 and 1A2 and by Human Liver Microsomes. BSC, DDS, and o-, m-, and in reconstituted systems, we examined and compared the effects of p-XSCs, when added to the incubation mixtures containing liver several selenium compounds, aNF, and other typical P450 inhibitors microsomes of J3NF-treated rats (Fig. 4A) and human sample HL-64 on procarcinogen activation by these P450 enzymes in S. typhimurium (Fig. 4B), inhibited 7-ethoxycoumarin 0-deethylation in a noncom NM2009 as a tester bacterial strain for DNA damage. petitive or uncompetitive manner; the inhibition mechanism was We first determined the cytotoxic effects of these selenium com somewhat different from that of aNF in rat and human liver micro pounds and (YNF to the and whether these chemicals cause somes. genotoxic effects in the tester strain (Fig. 5). The three XSC com Optimal Conditions for Catalytic Activities of Human CYP1B1 pounds were cytotoxic in the bacteria, with p-XSC being the strong in Yeast Microsomes. Because the above results suggested that these est. Sodium selenite and BSC were also cytotoxic to the cell, but aNF selenium compounds inhibit xenobiotic-oxidizing activities catalyzed and DDS did not cause cytotoxic effects up to a concentration of 100 by P450 enzymes of CYP1 subfamilies selectively, we determined the p.M. These seven compounds determined were not genotoxic toward S. effects on activation of procarcinogens catalyzed by recombinant typhimurium NM2009 (data not shown). CYP1A1, lA2, and lBl in S. typhimurium NM2009 as a tester strain The potencies of inhibition of activation of 2-aminoanthracene for the detection of DNA damage. CYP1A1 and 1A2 were isolated from membranes of E. coli in which respective human P450 cDNAs have been introduced, and CYP1B 1 was a recombinant protein in microsomes of yeast in which human CYP1B1 cDNA has been introduced. To develop optimal conditions for the procarcinogen activation by CYP1B1 in yeast microsomes, the effects of rabbit NADPH-P450 reductase, cytochrome b5, DLPC, and cholate on 7-ethoxyresorufin 0-deethylation activities were determined. CYP1B1-dependent 7- ethoxycoumarin 0-deethylation activities were highest when both NADPH-P450 reductase and cholate were present in the incubation C mixture. DLPC and cytochrome b5 did not cause an increase in the C activities catalyzed by CYP1B1 in yeast microsomes (data not shown). Such increases in xenobiotic oxidations by CYP1B1 in yeast mi crosomes were found to be dependent on the concentrations of cholate in the reaction mixture when rabbit NADPH-P450 reductase was also .01 .1 1 10 100 present (data not shown). The optimal concentrations of cholate were Chemical concentration (pM@ found to be —0.01—0.02% in the reaction mixture when 7- ethoxyresorufin 0-deethylation, 7-ethoxycoumarin 0-deethylation, Fig. 5. Cytotoxic effects of several organoselenium compounds and aNF in S. typhi murium NM2009 tester strain. Different concentrations of these chemicals were added to and activation of 2-aminofluorene were determined with recombinant the incubation mixture containing bacterial strain (absorbance at 600 nm of —0.3),andthe CYP1B 1. Bufuralol hydroxylation catalyzed by CYP1B 1 in yeast bacterial suspension was incubated for 2 h at 37'C. The cytotoxicity was determined by measuring absorbance at 600 nm, using 100% as the reading in the absence of added @ microsomes was also enhanced when both NADPH-P450 reductase chemicals. The organoselenium compounds tested were BSC (0), DDS (•),o-XSC and cholate were present in the reaction mixture (data not shown). m-XSC (A), p-XSC (0), and aNF (small •). 4760

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None (control)

a-Naphthoflavone (5OpM)@ Metyrapone (5O1uM) Piperonylbutoxide (5Op Sulfaphenazole (5O@M) (504 Diethyidithiocarbamate (50 4-Methylpyrazole Ketoconazole Fig. 6. Effects of several chemicals on the activation of 2-aminoan thracene catalyzed by recombinant CYPIB1. Sodium selenite Benzyl selenocyanate (2pM) Dlbenzyl diselenide (2pM) o-XSC (2pM) m-XSC (2pM) p-XSC (2pM) @ (Chemicals added) 50 100 Activation of 2-aminoanthracene by CYP1B1 (% of control)

catalyzed by CYP1B1 were determined using selenium compounds when compared with CYP1B1 and lAl activities, were less potent in and aNF and other typical P450 inhibitors (Fig. 6). The classical P450 inhibiting activation of MeIQ catalyzed by CYP1A2. inhibitors used were aNF (an inhibitor for CYP1 subfamily), me Comparison of the Inhibition of Effects of m-XSC and aNF on tyrapone (CYP2B), piperonyl butoxide (CYP2B and others), sulfa the 7-Ethoxyresorufin O-Deetbylation by CYP1B1, 1A1, and 1A2 phenazole (CYP2C9), quinidine (CYP2D), diethyldithiocarbamate and by Human Liver Microsomes. The effects of m-XSC and aNF and 4-methylpyrazole (CYP2E1 and CYP2A), and ketoconazole on 7-ethoxyresorufin 0-deethylation by CYP1BI , lAl, and lA2 and (CYP3A). These chemicals (at a final concentration of 50 p.M),except by human liver microsomes were compared (Fig. 8). aNF was found for aNF and sulfaphenazole, did not cause severe inhibition of the to be a potent inhibitor of 7-ethoxyresorufin 0-deethylation catalyzed activities of 2-aminoanthracene activation by CYP1B1 in S. lyphi by CYP1B1, lAl, and lA2 and by human liver microsomes. m-XSC, murium NM2009. In contrast, aNF and sulfaphenazole inhibited on the other hand, was relatively weak in inhibition of activities by CYP1B1 activities more than 80% at a concentration of 50 p.M. CYP1A2 and by human liver microsomes, although it significantly Because several selenium compounds were cytotoxic to the bacterial inhibited the activities catalyzed by CYP1B1 and lAl. cells, we examined these compounds at 2 p.M. All three XSCs were very effective in inhibiting activation of 2-aminoanthracene by DISCUSSION CYP1B1, although sodium selenite and DDS did not cause inhibition of CYP1B1 activities at 2 p.M. BSC inhibited ‘—50%ofthe CYP1B1 Present studies showed that three XSC compounds were very activities. potent inhibitors of drug and xenobiotic oxidation activities cata The abilities of the three XSCs to inhibit activation of 2-aminoan lyzed by various forms of human P450 enzymes. These chemicals thracene by CYP1B1 were compared with those of Trp-P-l by were found to strongly inhibit 7-ethoxyresorufin 0-deethylation CYP1A1 and of MeIQ by CYP1A2 (Fig. 7). The three XSCs were all and testosterone 6f3-hydroxylation and moderately inhibit S-war potent inhibitors for CYP1B1 and 1A1 in the activations of 2-amino farm 7-hydroxylation, bufuralol 6-hydroxylation, 7-ethoxycouma anthracene and Trp-P-l, respectively. However, these three XSCs, rim 0-deethylation, and chlorzoxazone 6-hydroxylation in human

Q H

Fig. 7. Effects of o-XSC (•),m-XSC (i@),and p-XSC (A) on the activation of 2-aminoanthracene U catalyzed by CYP1B1 (A), the activation of Tip P-I by CYPIAI (B). and the activation of MeIQ by 0@ CYP1A2 (C). 0 .—

Concentration of chemicals (pM@ 4761

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@ CI 0

1

Fig. 8. Effects of m.XSC (0) and aNF (•)on7-ethoxyresoru fin O-deethylation catalyzed by recombinant CYP1BI in yeast 50 microsomes (A), CYP1A1 (B), and CYP1A2 (C) in reconstituted systems and human liver microsomes (sample HL-16; D). J11

0 0 m-XSC or ocNF (pM)

liver microsomes. However, coumarin 7-hydroxylation and bu metabolites that induce the SOS response in Salmonella tester strains furalol I ‘-hydroxylation (in human liver microsomes) were not (16). Of particular interest is the observation that dibenzo[a,l}pyrene severely affected by these three XSC compounds. One of the most 11,12-diol, 5-methylchrysene-trans-l,2-diol and benzo[a]pyrene important outcomes of this study was that these synthetic or trans-7,8-diol, which are suggested to be groups of the most potent ganoselenium compounds inhibited 7-ethoxyresorufin 0-deethyla inducers of mammary tumors and lung cancers in experimental ani tion very markedly in human liver microsomes; the concentrations mals (18, 19, 49), have been determined to be activated extensively by required to cause 50% inhibition of activities were below 0.25 p.M. human CYP1B1. The inhibitory potencies of these compounds were found to be CYP1A1 is reported to be expressed in extrahepatic organs includ similar to aNF, which is well accepted to be a potent inhibitor of ing prostate, mammary gland, intestine, thymus, colon, adrenal, ovary, P450 of CYP1A and lB families (14, 21, 40). uterus, lung, and testis, whereas CYP1A2 is expressed primarily in With the use of recombinant human P450 enzymes, we found that microsomal fractions of the liver (22, 50, 51). Both enzymes play the three XSCs used in this study had relatively higher abilities to major roles in the of a variety of procarcinogens and inhibit xenobiotic and procarcinogen oxidations with CYP1A1 and promutagens, and recent studies have demonstrated that CYP1A1 and lBl than CYP1A2 and other human P450 enzymes. Particularly, the 1A2 are able to oxidize drugs such as theophylline, , bu XSCs very significantly inhibited the metabolic activation of Trp-P-l furalol, chlorzoxazone, , zoxazolamine, acetaminophen, an by CYPIA1 and of 2-aminoanthracene by CYP1B1, at very low tipyrine, lidocaine, phenacetin, propranolol, and warfarin (22, 50, 52, concentrations. CYP1A2 activities were also highly susceptible to 53). Very recently, we have shown that CYP1B1 catalyzes the oxi these XSC compounds, e.g., 7-ethoxyresorufin 0-deethylation and dation of benzo[a]pyrene, 7-ethoxyresorufin, theophylline, caffeine, activation of MeIQ by CYP1A2 and 7-ethoxyresorufin 0-deethylation bufuralol, and 7-ethoxycoumarin (30). These chemicals have been by human liver microsomes. The observation that the three XSC reported to be known substrates for human CYP1A1 and 1A2, except compounds and BSC caused greater spectral changes with liver mi that bufuralol is catalyzed mainly by CYP2D6, whereas 4- and 6- crosomes of rats treated with @3NFthan those of untreated rats and hydroxylation of bufuralol are catalyzed by CYP1AI and 1A2 en phenobarbital-treated rats indicated that these selenium compounds zymes (22, 23, 50—52,54). may have higher affinities for f3NF-inducible P450 enzymes in rat The three XSCs used in this study showed high toxicity in bacterial liver microsomes. m-XSC, which was most effective in inducing tester strain S. zyphimurium NM2009. Because these compounds have spectral changes with rat liver microsomes, also caused type II spec been shown to be less toxic in experimental animals (9), the possi tral change with liver microsomes of a human sample (HL-l6), which bility exists that these selenium compounds may be metabolized by had high levels of CYP1A2 in liver microsomes (Fig. 2B). mammals to form less toxic metabolites. After absorption, sodium Recently, CYPJBJ cDNA was isolated and characterized in humans selenite is reduced by reduced through GSSeSG to the (17, 41), mice (42, 43), and rats (44, 45). CYP1B1 proteins have been volatile and highly toxic H2Se, which can be incorporated into pro isolated from a embryo fibroblast cell line and from rat adrenal teins and/or further methylated to dimethylselenide; the latter can be microsomes (46—48).Both the rat and mouse counterparts have been detected in the exhaled air (55). In contrast, however, in our prelim shown to catalyze the oxidation of carcinogenic polycyclic aromatic mary investigation, we have demonstrated that less that 1% of sele hydrocarbons such as 7,12-dimethylbenz[a]anthracene (46—48).The nium can be detected in the exhaled air, which may, in part, account human CYPIBI cDNA sequence has been reported to be about 80% for the lower toxicity of p-XSC as compared to sodium selenite (56). similar to the mouse and rat orthologues, and the expression of the In laboratory animals, we demonstrated that the organoselenium CYPJBJ mRNA has been observed in many organs including kidney, compounds can inhibit tumorigenesis induced by a variety of chem prostate, mammary gland, pituitary, thymus, spleen, adrenal, colon, ical carcinogens (9, 10, 20). In the rat mammary tumor model system ovary, uterus, , heart, lung, intestine, and testis (17, 45). CYP1B1 using 7,l2-dimethybenz[a]anthracene, we found that p-XSC has a may be very important in understanding chemical carcinogenesis in better chemopreventive index (more effective and less toxic) than humans, because this P450 enzyme can catalyze the activation of those of BSC and sodium selenite (9, 10). Dibenzyl diselenite has diverse procarcinogenic and promutagenic chemicals to genotoxic never been tested in this system. Previous studies also indicate 4762

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. HUMAN P450 ACTIVITIES AND ORGANOSELENIUMCOMPOUNDS that o- and m-XSC appear to be equally effective but both are better synthetic organoselenium compound, l,4-phenylenebis(methylene)selenocyanate. inhibitors of 7,12-dimethylbenz[a]anthracene-DNA adducts forma Cancer Res., 52: 2402—2407,1992. 10. Ip, C., El-Bayoumy, K., Upadhyaya, P., Ganther, H., Vadhanavikit, S., and tion in the rat mammary gland (9, 20, 56). Unfortunately, o- and Thompson, H. Comparative effect of inorganic and organic selenocyanate derivatives m-XSC have not yet been tested in efficacy studies. Collectively, in mammary cancer chemoprevention. Carcinogenesis (Lond.), 15: 187—192,1994. these results suggest that organoselenium compounds inhibit 7,12- 11. Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, 0., Coon, M. J., Estabrook, R. W., Gunsalus, I. C., dimethylbenz[a]anthracene-induced mammary tumors in the rat either and Nebert, D. W. P450 superfamily: update on new sequences, gene mapping, by inhibiting phase I activation or by inducing phase II accession numbers and nomenclature. Pharmacogenetics. 6: 1—42,1996. 12. Pelkonen, 0., and Nebert, D. W. Metabolism of polycyclic aromatic hydrocarbons: enzymes that are involved in the metabolism of this mammary car etiologic role in carcinogenesis. Pharmacol. Rev., 34: 189—222,1982. cmogen. The present study clearly demonstrates that the efficacy of 13. Dipple, A., and Bigger. C. A. H. Mechanism of action of food-associated polycyclic these organoselenium compounds is due, in part, to their inhibitory aromatic hydrocarbon carcinogens. Mutat. Res., 259: 263—276,1991. 14. Guengench, F. P., and Shimada, T. Oxidation of toxic and carcinogenic chemicals by effects on P450s that are involved in the metabolic activation of human cytochrome P-450 enzymes. Chem. Res. Toxicol., 4: 391—407, 1991. 7,12-dimethylbenz[a]anthracene. However, on the basis of the in vitro 15. Gonzalez, F. J., and Gelboin, H. V. Role ofhuman P450 in the metabolic results obtained in this study, it would be premature to rank the in vivo activation ofchemical carcinogens and toxins. Drug Metab. Rev., 26: 165—183,1994. 16. Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P., chemopreventive potency of the organoselenium compounds exam and Sutter, T. R. Activation of chemically diverse procarcinogens by human cyto med in this study. Moreover, we also demonstrated the efficacy of chrome P450 IB1. Cancer Res., 56: 2979—2984, 1996. 17. Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y-Y. P., Jabs, W., Li, X., Yin, H., Cody, BSC and p-XSC against the development of colon cancer induced by C. W., and Greenlee, W. F. Complete cDNA sequence of a human dioxin-inducible azoxymethane and lung tumors induced by the tobacco-specific mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromo nitrosamine, 4-(methylnitrosamine)-l-(3-pyridyl)-1-butanone. Al some 2. J. Biol. Chem., 269: 13092—13099, 1994. 18. Amin, S., Krzeminski, J., Rivenson, A., Kurtzke, C., Hecht, S. S., and El-Bayoumy, though studies in the colon and lung tumor model were only limited K. Mammary carcinogenicity in female CD rats of fjord region diol epoxides of to these two agents, the chemopreventive efficacy of both BSC and benzo[cjphenanthrene, benzo[g]chrysene and dibenzo[a.1]pyrene. Carcinogenesis p-XSC can be explained, in part, by the results of this study, which (Lond.), 16: 1971—1974,1995. 19. Hecht, S. S., El-Bayoumy. K., Rivenson, A., and Amin, S. Potent mammary card indicate that these agents are capable of inhibiting P450 enzymes nogenicity in female CD rats of a fjord region diol- of benzo(c]phenanthrene required for activating azoxymethane and 4-(methylnitrosamine)-l- compared to a bay region diol-epoxide of benzo[a]pyrene. Cancer Res.. 54: 2 1—24, (3-pyridyl)-1-butanone (57). 1995. 20. El-Bayoumy, K., Upadhyaya, P., Chae, Y-H., Sohn, 0-S., Rao, C. V., Faila, E., and In conclusion, the present results showed that several synthetic Reddy, B. S. Chemoprevention of cancer by organoselenium compounds. J. Cell selenium compounds such as the three XSCs, BSC, and DDS Biochem. Suppl., 22: 92—100,1995. 21. Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P. Human liver inhibited activities of xenobiotic and procarcinogen oxidations microsomal cytochrome P450 enzymes involved in the bioactivation of procarcino catalyzed by human P450 enzymes in vitro. Of these selenium gens detected by umu gene response in Salmonella typhimurium TA1535/pSKIOO2. compounds examined, the three XSCs were the most potent in Cancer Res., 49: 3218—3228, 1989. 22. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. Interindi inhibiting the xenobiotic and procarcinogen oxidation activities by vidual variations in human liver cytochrome P450 enzymes involved in the oxidation P450 enzymes in human liver microsomes and by recombinant of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 human P450 enzymes. Spectral studies showed that BSC and the Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther., 270: 414—423,1994. 23. Yamazaki, H., Guo, Z., Persmark, M., Mimura, M., Inoue, K., Guengerich, F. P., and three XSC compounds, but not sodium selenite and DDS, induced Shimada, T. Bufuralol hydroxylation by cytochrome P450 206 and 1A2 enzymes in type II difference spectrum when added to liver microsomes iso human liver microsomes. Mol. Pharmacol., 46: 568—577, 1994. 24. Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. C., Walker, N. J., and Sutter, T. R. lated from f3-naphthoflavone-treated rats, with m-XSC being most 17@3-Estradiolhydroxylation catalyzed by human cytochrome P450 1B I. Proc. Natl. potent in inducing spectral interactions with P450 enzymes. m Acad. Sci. USA, 93: 9776—9781, 1996. XSC also caused type II spectral change with human liver micro 25. Guo, Z., Gillam, E., Ohmori, S., Tukey, R. H., and Guengerich, F. P. Expression of modified human cytochrome P450 lAl in Escherichia coli: effects of 5' substitution, somes. These results suggest that some of the synthetic organose stabilization, purification, spectral characterization, and catalytic properties. Arch. lenium compounds inhibit the activation of environmental Biochem. 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Tsutomu Shimada, Karam El-Bayoumy, Pramod Upadhyaya, et al.

Cancer Res 1997;57:4757-4764.

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