0090-9556/02/3003-262–269$3.00 AND DISPOSITION Vol. 30, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 458/966047 DMD 30:262–269, 2002 Printed in U.S.A.

INDUCTION OF CYP1A1 AND CYP1B1 IN T-47D HUMAN BREAST CANCER CELLS BY BENZO[A]PYRENE IS DIMINISHED BY ARSENITE

DAVID C. SPINK, BARBARA H. KATZ, MIRZA M. HUSSAIN, BARBARA C. SPINK, SUSAN J. WU, NING LIU, RONALD PAUSE, AND LAURENCE S. KAMINSKY

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York

(Received May 15, 2001; accepted November 30, 2001)

This article is available online at http://dmd.aspetjournals.org

ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) and metals are often BAP; higher levels caused reduced rates of metabolism due to

environmental cocontaminants, yet there have been relatively few inhibition of CYP1A1 and CYP1B1. NaAsO2 caused pronounced studies of combined effects of PAHs and metals on cytochrome decreases in the induction of CYP1A1 and CYP1B1 by 3 ␮M BAP ␮ P450 (P450)-catalyzed metabolism. We examined the effects of because cotreatment with 10 M NaAsO2 inhibited the rates of the

NaAsO2 in combination with benzo[a]pyrene (BAP) on CYP1A1 and 2- and 4-hydroxylation pathways by 86 and 92%, respectively. CYP1B1 in T-47D human breast cancer cells by using estrogen Western immunoblots showed diminished levels of BAP-induced

metabolism as a probe of their activities. Exposure to BAP caused CYP1A1 by coexposure to NaAsO2. The levels of the CYP1A1 and elevated rates of the 2- and 4-hydroxylation pathways of estrogen CYP1B1 mRNAs induced by BAP were not significantly affected by

metabolism, indicating induction of both CYP1A1, an coexposure to NaAsO2; however, heme 1 mRNA levels

2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-in- were markedly induced by coexposure to BAP and NaAsO2. These duced metabolism peaked 9 to 16 h after exposure and returned to results indicate a post-transcriptional inhibitory effect of arsenite near-basal levels by 48 h. Concentration-response studies showed on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly maximal induction of the 2- and 4-hydroxylation pathways at 3 ␮M resulting from reduced heme availability.

Among the environmental contaminants of most concern due to bolic activation catalyzed by cytochromes P450 (P450) and epoxide their toxicity, including carcinogenicity, are heavy metals, such as (Wood et al., 1976; Kapitulnik et al., 1978). Covalent arsenic, lead, and mercury, and polycyclic aromatic hydrocarbons adducts formed by the reaction of PAH diol epoxide metabolites with (PAHs1) typified by benzo[a]pyrene (BAP). PAHs and heavy metals guanine in mutational hotspots of critical such as that of the p53 are often cocontaminants in the environment, yet there have been tumor suppressor (Denissenko et al., 1996), if not efficiently repaired, relatively few studies of the combined toxic and particularly the may initiate tumorigenesis. carcinogenic effects elicited by PAHs and heavy metals. The carci- Several members of the P450 superfamily in conjunction with nogenicity of BAP and other PAHs is a consequence of their meta- epoxide hydrolase have been shown to catalyze the metabolism of BAP to carcinogenic intermediates. In extrahepatic tissues, CYP1A1 This research was supported by the U.S. Environmental Protection Agency’s and CYP1B1 are thought to be the most important in Science to Achieve Results program through Grant R827180010 and by U.S. catalyzing the formation of mutagenic intermediates from BAP and a Public Health Service Grants CA81243 and ES04913. Although the research described in this article has been funded in part by the U.S. Environmental number of other PAHs, including several that are potent mammary Protection Agency’s STAR program, it has not been subjected to the agency’s gland carcinogens in rodents. CYP1B1 appears to be more active than required peer and policy review and therefore does not necessarily reflect the CYP1A1 in the conversion of a number of PAHs to genotoxic inter- views of the agency, and no official endorsement should be inferred. N.L., who is mediates (Shimada et al., 1996). In the presence of epoxide hydrolase, affiliated with the Division of Environmental Health, Department of Preventive both CYP1A1 and CYP1B1 catalyze the conversion of BAP to its Medicine, Jiangxi Medical College, Nanchang, China, is grateful to the World 7,8-dihydrodiol, and both enzymes can in turn metabolically activate Health Organization for support. this BAP metabolite to a mutagenic form (Shimada et al., 1996, 1999; 1 Abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BAP, ben- Kim et al., 1998). 7,12-Dimethylbenz[a]anthracene-induced carcino- zo[a]pyrene; P450, ; AhR, aromatic hydrocarbon receptor; E2, ␤ genesis, an extensively studied model of PAH-induced carcinogene- 17 -estradiol; OHE2, hydroxyestradiol; MeOE2, methoxyestradiol; DMSO, di- methyl sulfoxide; RT-PCR, reverse transcription-polymerase chain reaction; sis, recently was found to be dependent on CYP1B1: the CYP1B1 GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain knockout mouse is refractory to 7,12-dimethylbenz[a]anthracene-in- reaction; HO-1, heme oxygenase 1; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; duced carcinogenesis (Buters et al., 1999). CPR, NADPH-cytochrome P450 reductase; MTT, 3-[4,5-dimethylthiazol-2-yl]- PAHs are not only substrates of human CYP1A1 and CYP1B1 but 2,5-diphenyltetrazolium bromide. also are inducers of these enzymes through binding to and activation of the aromatic hydrocarbon receptor (AhR). The regulation of ex- Address correspondence to: Dr. David C. Spink, Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509. E-mail: [email protected] pression of both CYP1A1 and CYP1B1 is complex because transcription not only involves the AhR but also a number of tran- 262 EFFECTS OF BAP AND ARSENITE ON CYP1A1 AND CYP1B1 IN T-47D CELLS 263

scription factors, and is potentially influenced by the actions of Biochemicals, Indianapolis, IN) with the primers of Premkumar et al. (1995). transcriptional coactivators and corepressors. For reasons that are not Quantitation was achieved by monitoring the fluorescence of intercalated currently known, in some human Ah-responsive cells, CYP1A1 is SYBR Green I at each cycle. All mRNA levels were analyzed in triplicate, inducible but not CYP1B1, in other cells CYP1B1 is inducible but not each representing three separate reverse transcriptions of RNA isolated from individual wells of control or treated T-47D cells. CYP1A1, and in yet others both enzymes are inducible (Christou et Assay of Cellular Estrogen Metabolism. For the analysis of E2 metabo- al., 1994; Spink et al., 1994, 1998b; Do¨hr et al., 1995; Kress and lism, confluent cultures of T-47D cells in six-well plates were exposed to the Greenlee, 1997). There are several potential points, including tran- indicated concentrations of BAP, NaAsO2, 2,3,7,8-tetrachlorodibenzo-p-di- scription, translation, and incorporation of the heme prosthetic group, oxin (TCDD), or the solvent vehicle 0.1% v/v DMSO in 2 ml of medium per at which other chemicals, including environmental cocontaminants, well. After the specified time period for induction, media were ␮ could potentially modulate expression and catalytic activity of replaced with media containing 1 or 5 ME2. At the indicated times, these CYP1A1, CYP1B1, and other P450s. In several recent studies, an media were recovered and treated with b-glucuronidase/sulfatase for hydroly- effect of arsenite on expression of cytochromes P450 in hepatocyte sis of the metabolite conjugates as previously described (Spink et al., 1994). cultures was reported (Jacobs et al., 1999; Vakharia et al., 2001), These enzyme-treated samples were subjected to solid-phase extraction and although the mechanism responsible for this effect has not been preparation of the metabolite-trimethylsilyl derivatives. The metabolite deriv- determined. atives were analyzed by gas chromatography/mass spectrometry with stable- isotope dilution and selected-ion monitoring as described (Spink et al., 1994, Human CYP1A1 and CYP1B1 are also 17␤-estradiol (E ) hydroxy- 2 1998b). Rates of metabolite formation were normalized to cellular protein lases. CYP1A1 is primarily an E2 2-hydroxylase (Spink et al., 1992), content as determined by the bicinchoninic acid assay method (Smith et al., whereas CYP1B1 is primarily an E2 4-hydroxylase with a lesser 1985) by using a commercial reagent (Pierce Chemical, Rockford, IL). activity at C-2 (Spink et al., 1994; Hayes et al., 1996). The resultant Determination of Microsomal E2 Hydroxylase Activity. To evaluate the

catechol estrogens, 2- and 4-hydroxyestradiol (2- and 4-OHE2), are inhibitory effects of BAP and NaAsO2 on human CYP1A1 and CYP1B1, the converted to 2- and 4-methoxyestradiol (2- and 4-MeOE2)bythe E2 hydroxylase activities of the two enzymes were determined essentially as action of constitutively expressed catechol O-methyltransferase. This described (Spink et al., 1992). Human recombinant CYP1A1 and CYP1B1, both coexpressed with human NADPH/cytochrome P450 in metabolism of E2 can be used as a probe of the AhR-regulated expression of CYP1A1 and CYP1B1 in human breast epithelial and Sf9 cells (Supersomes), were obtained from GENTEST (Woburn, MA). Mi- crosomal incubations (250 ␮l) contained 5 mM MgCl ,10␮MofE as breast tumor cells (Spink et al., 1998b). In this study, we used the 2 2 substrate, 10 pmol of cDNA-expressed CYP1A1 or CYP1B1, and 2 mM T-47D line of estrogen receptor-␣-positive breast tumor cells, which ascorbic acid, and were buffered at pH 7.4 with 100 mM sodium phosphate. rapidly metabolize BAP (Merrick et al., 1985) and express both The reaction mixtures were preincubated at 37°C for 5 min in the presence of CYP1A1 (Vickers et al., 1989) and CYP1B1 (Spink et al., 1998b) in added BAP or NaAsO2, and enzymatic reactions were initiated with NADPH response to exposure to Ah-receptor activation, to investigate the at a final concentration of 1.4 mM. After 10 min, the reactions were terminated effects of BAP alone or in combination with NaAsO2 on the expres- by the addition of two volumes of ice-cold 30 mM ascorbic acid, addition of sion of CYP1A1 and CYP1B1. internal standards, and immediate extraction with ethyl acetate. The extracts

were dried over anhydrous sodium sulfate, evaporated to dryness under N2, and Materials and Methods trimethylsilyl derivatives were prepared. The derivatized samples were ana- Cell Culture and Treatments. T-47D cells were obtained from the Amer- lyzed by gas chromatography/mass spectrometry with selected-ion monitoring. ican Type Culture Collection (Rockville, MD) and were maintained in a 37°C Western Immunoblot Analysis of NADPH-P450 Reductase (CPR) and

incubator with a humidified atmosphere containing 5% CO2. For experimental CYP1A1. Cultures in six-well plates were exposed to BAP and NaAsO2 as protocols, the medium was DC5 (Gierthy et al., 1996) consisting of Dulbecco’s indicated, after which the cell monolayers were washed with phosphate- modified Eagle’s medium, without phenol red, and with 5% bovine calf serum buffered saline and solubilized by addition of gel sample buffer (250 ␮l/well), (Cosmic calf serum; Hyclone Laboratories, Logan, UT), 10 mM nonessential which did not contain reducing agent or dye. Equal amounts of protein from amino acids, 2 mM L-glutamine, and 10 ␮g/l insulin. BAP was added to the each well (2 ␮g for CPR, 4 ␮g for CYP1A1) were run on 10% Bis-Tris

cultures alone or in combination with varying concentrations of NaAsO2. The polyacrylamide gels (NuPAGE; Invitrogen) together with a series of standards vehicle for BAP was dimethyl sulfoxide (DMSO), which was used at a highest consisting of either purified, recombinant human CPR (Panvera, Madison, WI) concentration of 0.1% (v/v). or human CYP1A1 (Supersomes from GENTEST). The proteins were trans- RNA Isolation and RT-PCR. Total RNA was isolated by phenol-guani- ferred to Immobilon membranes (Millipore, Bedford, MA) that were then dinium thiocyanate extraction (Chomczynski and Sacchi, 1987), and portions blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% (2.5–5 ␮g) were primed with oligo-dT, reverse-transcribed using Superscript II Tween 20 (Bio-Rad, Hercules, CA). The primary antibodies were goat anti-rat reverse transcriptase (Invitrogen, Carlsbad, CA) and treated with RNase H CPR (GENTEST) and goat anti-rat CYP1A1 (Daiichi, Tokyo, Japan), which (Invitrogen) according to the manufacturer’s protocol. Primers for the ampli- were used at dilutions of 1:1000 and 1:2000, respectively, in Tris-buffered fication of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and saline containing 0.05% Tween 20 and 0.5% nonfat dry milk. The secondary CYP1A1 cDNAs were as previously reported (Fasco et al., 1995). The primers antibody was horseradish peroxidase conjugated-donkey anti-goat IgG (Cruz for the amplification of CYP1B1 cDNA were those used previously (Spink et Marker compatible; Santa Cruz Biotechnology, Santa Cruz, CA) used at al., 1998a). Competitor DNAs were synthesized by the method of Fo¨rster 1:7500 dilution for analysis of CPR or 1:20,000 for CYP1A1. Immunoreactive (1994). Each amplified sequence spanned an exon-exon junction. PCR of the proteins were detected by using the SuperSignal West Pico Chemiluminescent CYP1A1, CYP1B1, and GAPDH cDNAs was carried out as described (Spink Substrate (Pierce Chemical), and exposed films were subjected to scanning et al., 1998a) with core reagents from PerkinElmer (Foster City, CA), but with densitometry on a Pharmacia LKB ImageMASTER densitometer (Amersham the addition of TaqStart antibody (CLONTECH, Palo Alto, CA) and Taq Biosciences, Inc., Piscataway, NJ). The resulting band intensities for the extender (Stratagene, La Jolla, CA), and were amplified with the same PCR- detected proteins fell within linear standard curves prepared over the 1.3- to plus-competitor mix used for the calibration curve at dilutions that fell within 20-fmol range for CPR and over the 0.1- to 2.0-fmol range for CYP1A1, and the linear range of the competitor curve. PCR products were resolved on a 2% were thus converted to molar amounts of CPR and CYP1A1. 3:1 NuSieve (FMC Bioproducts, Rockland, ME) agarose gel, followed by Determination of BAP Uptake. The extent of uptake of BAP into T-47D staining with 0.75 ␮g/ml ethidium bromide for photography and quantitation cells was evaluated by determining the level of BAP remaining in the medium by densitometric scanning of the photographic negative. For the determination and the amount that could be recovered from the cells at various times after

of heme oxygenase 1 (HO-1) mRNA levels, a real-time PCR technique was exposure. BAP was extracted from the media samples by use of Sep-Pak C18 used (for review, see Bustin, 2000). A 271- fragment of the HO-1 cartridges (Waters, Milford, MA), and BAP was recovered from the cDNA was amplified by using the LightCycler System (Roche Molecular trypsinized cells by sonication of the cell pellets in the presence of acetonitrile. 264 SPINK ET AL.

The BAP levels in these extracts were determined by reversed phase, high- performance liquid chromatography (Vakharia et al., 2001). Determination of Cell Viability. The effects on cell viability of exposure ␮ to 3 M BAP alone or in combination with varying concentrations of NaAsO2 for 12 h were assessed in 96-well plates, as described by Borenfreund et al. (1988), by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide (MTT). Statistical Analyses. Statistical differences among treatment groups were evaluated by analysis of variance and use of the Student-Newman-Keuls test for multiple comparisons. These evaluations were performed using the Sig- maStat software package, version 2.0 (Jandel Scientific, San Rafael, CA). For

determination of EC50 values for mRNA induction, concentration-response data were fit to the four-parameter sigmoidal function,

f ϭ y0 ϩ a/{1 ϩ exp͓ Ϫ ͑x Ϫ x0͒/b]} by using the SigmaPlot (Jandel Scientific) program.

Results The time course of BAP uptake, CYP1A1 and CYP1B1 mRNA

levels, and the rates of the 2- and 4-hydroxylation pathways of E2 metabolism in T-47D cells were determined after a single exposure to medium containing 3 ␮M BAP. There was a rapid disappearance of BAP from the medium of T-47D cultures because only 30% of the initial BAP could be recovered from the medium at 3 h (Fig. 1A). BAP was rapidly taken up in these cells and was apparently rapidly metabolized because at 3 h 20% of the initial BAP was present in the cells, but the level declined rapidly thereafter (Fig. 1B). With ex- tended incubation the cultures became depleted of BAP because by 12 h only 5% of the original BAP could be recovered from the medium, and BAP could no longer be detected in extracts of the cells. The time courses of the effects of BAP exposure on the levels of CYP1A1 and CYP1B1 mRNAs in T-47D cells were determined by using competitive RT-PCR. After exposure to 3 ␮M BAP, the levels of both the CYP1A1 (Fig. 1C) and CYP1B1 (Fig. 1D) mRNAs increased for the first 9 h, but declined steadily thereafter, returning to

near-basal levels by 48 h. The rates of 2-MeOE2 (Fig. 1E) and 4-MeOE2 (Fig. 1F) synthetic activities, reflecting the activities of CYP1A1 and CYP1B1, respectively, also showed transient elevations ␮ after exposure to 3 M BAP. Although the data for 2- and 4-MeOE2 formation are very precise, they should be seen as representing mean velocities, averaged over 6-h periods. Uncertainty of the exact nature of the time course of the induction of the activities of CYP1A1 and FIG.1.Time course of BAP uptake, CYP1A1 and CYP1B1 mRNA levels, and the

CYP1B1 arises from probable changes in metabolic velocities during rates of the 2- and 4-hydroxylation pathways of E2 metabolism in T-47D cells these 6-h assay periods. Therefore, theoretical limits for the rates of after BAP exposure. change of the metabolic velocities at the beginning and the end of Cultures of T-47D cells in six-well plates were exposed to a single application of each assay period were used to approximate ranges of possible curves medium containing 3 ␮M BAP (2 ml/well). At the indicated times, the levels of BAP in the medium (A) and in the cells (B) were determined. ND indicates that that describe the time course of induction and decline of E2 metabo- BAP was not detectable (Ͻ0.0001 nmol). RNA was isolated at the indicated times, lism. These ranges are depicted by the shaded areas within the curves and CYP1A1 (C) and CYP1B1 (D) mRNA levels were determined by competitive (Fig. 1, E and F). From these curves it is estimated that the rates of 2- RT-PCR. In E and F, media were replaced at the indicated times with media containing 1 ␮ME with 0.1% DMSO as the vehicle. After 6-h assay periods and 4-MeOE formation reached maxima by 9 to 16 h, and by 48 h, 2 2 indicated by the horizontal bars, media were recovered, conjugates were hydro- the rates of 2- and 4-MeOE synthetic activities had returned to 2 lyzed, and rates of 2-MeOE2 (E) and 4-MeOE2 (F) formation were determined near-basal levels. relative to cellular protein content as described under Materials and Methods. The The concentration-response effects of a 12-h exposure to BAP on curves bordering the shaded areas in E and F represent approximations of the theoretical limits for the time course of induction and decline of the metabolic rates the 2- and 4-hydroxylation pathways of E2 metabolism in T-47D cells as defined by these assay periods. In each panel, data are the means of triplicate were evaluated (Fig. 2). The induction curve is bell shaped, with the determinations, each replicate representing a well of cells. Standard errors are highest metabolic rates observed from exposure to 3 ␮M BAP, and shown when they are larger than the symbols. lower metabolic rates were observed with BAP concentrations lower ␮ ␮ and higher than 3 M. Because this type of concentration-response 3). At 10 M BAP, the rates of TCDD-induced 2- and 4-MeOE2 curve often reflects enzyme inhibition by the higher levels of the formation were both inhibited by more than 90%. In contrast to the

inducing agent, the effects of BAP on E2 metabolism induced by concentration-response curves for formation of 2- and 4-MeOE2, TCDD were investigated. Addition of BAP to TCDD-treated T-47D concentration-response curves for CYP1A1 and CYP1B1 mRNA

cultures during the E2 metabolism assay period markedly inhibited levels are sigmoidal, with no apparent inhibitory effects at the higher ␮ Ϯ metabolite formation when the BAP was present at 3 and 10 M (Fig. BAP levels (Fig. 4). From these data, EC50 values of 1.75 0.07 and EFFECTS OF BAP AND ARSENITE ON CYP1A1 AND CYP1B1 IN T-47D CELLS 265

FIG.2.Effect of varying BAP concentration on the 2- and 4-hydroxylation

pathways of E2 metabolism in T-47D cells. Confluent cultures of T-47D cells were exposed for 12 h to medium containing the indicated concentrations of BAP, after which media were replaced with media ␮ containing 1 ME2. After a further 6 h, media were recovered, conjugates were E F hydrolyzed, and levels of 2-MeOE2 ( ) and 4-MeOE2 ( ) were determined. Rates of metabolite formation are expressed relative to cellular protein content. Data are the means Ϯ standard error of triplicate determinations.

FIG.4.Effect of varying BAP concentrations on the levels of CYP1A1 and CYP1B1 mRNAs in T-47D cells. Confluent cultures of T-47D cells were exposed for 12 h to medium containing the indicated concentrations of BAP, after which RNA was extracted and reverse transcribed, and CYP1A1, CYP1B1, and GAPDH sequences were amplified by competitive PCR. Data are presented as the ratios of CYP1A1 (A) or CYP1B1 (B) mRNA levels to those of GAPDH mRNA and are expressed relative to the DMSO control. Data are the means Ϯ standard error of triplicate determinations.

similar effects on the CYP1A1- and CYP1B1-catalyzed activities

because the rates of 2-MeOE2 (Fig. 5D) and 4-MeOE2 (Fig. 5E) ␮ FIG.3.Effect of varying BAP concentration on TCDD-induced estrogen production were both significantly reduced at 1 M NaAsO2 and here ␮ metabolism in T-47D cells. reduced by 87 and 91%, respectively, at 10 M NaAsO2 compared ␮ Cultures were exposed to 10 nM TCDD or 0.1% (v/v) DMSO for 72 h. Media with that elicited by 3 M BAP alone. ␮ were then replaced with media containing 1 ME2 and the indicated concentration Direct effects of BAP and NaAsO2 on CYP1A1 and CYP1B1 of BAP. After a further 6 h, media were recovered, conjugates were hydrolyzed, and activities were investigated in microsomal reactions with cDNA- E F levels of 2-MeOE2 ( ) and 4-MeOE2 ( ) were determined. Rates of metabolite formation are expressed relative to cellular protein content. Data are the means Ϯ expressed enzymes. Consistent with the effects of added BAP on the standard error of triplicate determinations. activities of CYP1A1 and CYP1B1 induced by TCDD in T-47D cells (Fig. 3), BAP potently inhibited both CYP1A1 and CYP1B1. Addi- 1.88 Ϯ 0.16 ␮M BAP were determined for the induction of the tion of 10 ␮M BAP to microsomal incubations caused 96% inhibition

CYP1A1 and CYP1B1 mRNAs, respectively. of CYP1A1-catalyzed E2 2-hydroxylase activity (Fig. 6A) and 99%

To examine the effects of NaAsO2 on the induction of E2 metab- inhibition of CYP1B1-catalyzed E2 4-hydroxylase activity (Fig. 6B). olism in T-47D cells, the compound was added to cultures at various NaAsO2 also inhibited the activities of cDNA-expressed CYP1A1 and ␮ ␮ concentrations in combination with 3 M BAP. NaAsO2 at 1, 3, or 10 CYP1B1. Addition of 10 M NaAsO2 caused 21 and 43% inhibition ␮ ␮ M added in the presence of 3 M BAP did not affect cell viability of the CYP1A1 and CYP1B1 E2 hydroxylase activities, respectively.

as determined by the MTT assay (Fig. 5A). These levels of NaAsO2 Because NaAsO2 appears to both inhibit CYP1A1 and CYP1B1 in combination with BAP did not significantly affect the levels of activities and to diminish the induction of the two enzymes, an induced CYP1A1 (Fig. 5B) or CYP1B1 (Fig. 5C) mRNAs. Exposure experiment was designed to evaluate the relative importance of these ␮ of T-47D cells to NaAsO2 in combination with 3 M BAP had two effects of NaAsO2 in T-47D cells. TCDD was used as the inducer marked effects on the formation of 2- and 4-MeOE2, reflecting de- of CYP1A1 and CYP1B1 to avoid the confounding effects of enzyme creases in the activities of CYP1A1 and CYP1B1, respectively. Ex- inhibition by BAP. As previously reported (Spink et al., 1998b), ␮ ␮ posure to 10 M NaAsO2 in combination with 3 M BAP had very TCDD caused a marked elevation in the rates of formation of 266 SPINK ET AL.

FIG.6.Effects of BAP and NaAsO2 on E2 hydroxylation catalyzed by human CYP1A1 and CYP1B1. Microsomal incubations containing 10 pmol of cDNA-expressed CYP1A1 (A) or CYP1B1 (B) were preincubated without additions or with 10 ␮M BAP or 10 ␮M

NaAsO2 for 5 min before a 10-min assay of E2 hydroxylase activity as described under Materials and Methods. Data are the means Ϯ standard error of triplicate determinations and are expressed relative to control. Significant differences from .(p Ͻ 0.001 ,ءءء ;p Ͻ 0.05 ,ء) control are indicated

␣ ␣ 6 -OHE2, and 15 -OHE2, two additional products of CYP1A1-cat- alyzed E2 metabolism (Spink et al., 1992), were reduced relative to rates of cultures that received TCDD only. However, if 10 ␮M

NaAsO2 was added in the E2 metabolism phase of the experiment rather than the induction phase, no significant changes in the rates of

E2 metabolism were observed, suggesting that the effects of arsenite on E2 metabolism in T-47D cultures were primarily on the levels of TCDD-induced CYP1A1 and CYP1B1 and not as a result of inhibit-

FIG.5.Effects of BAP plus varying concentrations of NaAsO2 on cell viability, ing their activities. the induction of CYP1A1 and CYP1B1 mRNAs, and estrogen metabolism in T- Reductions in the levels of the CYP1A1 and CYP1B1 proteins after 47D cells. cotreatment with arsenite would explain the reduction in the BAP- and Confluent cultures of T-47D cells were exposed to medium containing the ␮ TCDD-induced E2 metabolism in T-47D cells. Additionally, reduc- DMSO vehicle or 3 mM BAP plus the indicated concentrations of NaAsO2 for 12 h. A, cell viability as assessed by the MTT assay. B, levels of CYP1A1 mRNA tion in the levels of NADPH-cytochrome P450 reductase could also expressed relative to those of GAPDH. C, levels of CYP1B1 mRNA expressed result in diminished rates of BAP-induced E2 metabolism. To inves- relative to those of GAPDH. D, rates of 2-MeOE2 formation, determined in a 6-h tigate the effects of cotreatments with BAP and arsenite on the levels assay, expressed relative to cellular protein content. E, rates of 4-MeOE2 formation determined in a 6-h assay, expressed relative to cellular protein content. Data are the of CPR and CYP1A1, Western immunoblot analyses of the two means Ϯ standard errors of six (A) or three (B–E) determinations. Significant proteins in cell lysates were performed. Exposure to 3 ␮M BAP did p Ͻ 0.001) and from the not significantly affect the level of CPR in T-47D cells, nor did ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) differences from control ␮ Ͻ Ͻ Ͻ 3 M BAP treatment group (†, p 0.05; ††, p 0.01; †††, p 0.001) are combined treatments of 1, 3, or 10 ␮M NaAsO together with 3 ␮M indicated. 2 BAP (Fig. 8A), but these treatments had marked effects on the levels of CYP1A1 protein (Fig. 8B). In untreated T-47D cells, CYP1A1 ␣ ␣ 2-MeOE2, 4-MeOE2,6 -OHE2, and 15 -OHE2 as a consequence of protein was not detectable. After exposure to 3 ␮M BAP, CYP1A1 the induction of CYP1A1 and CYP1B1 (Fig. 7). Addition of 10 ␮M was highly induced, and the combined treatments of 3 or 10 ␮M ␮ NaAsO2 together with 10 nM TCDD during the 12-h period for NaAsO2 together with 3 M BAP resulted in significantly reduced enzyme induction resulted in significant reductions in the rates of E2 levels of immunoreactive CYP1A1 compared with exposure to 3 ␮M metabolism determined during the assay phase. Not only were rates of BAP alone. Western immunoblot analyses of CYP1B1 expression in

2-MeOE2 and 4-MeOE2 reduced, but also rates of formation of T-47D cells were performed in a similar manner to those for CYP1A1 EFFECTS OF BAP AND ARSENITE ON CYP1A1 AND CYP1B1 IN T-47D CELLS 267

FIG.7.Effects of NaAsO2 on TCDD-induced metabolism of E2 in T-47D cells. Confluent cultures of T-47D cells were exposed to 10 nM TCDD, 10 nM TCDD ␮ plus 10 M NaAsO2, or received no additions (0.1% DMSO only) as indicated. After a 12-h period for enzyme induction, media were replaced with media con- ␮ ␮ ␮ taining 5 ME2 or 5 ME2 plus 10 M NaAsO2. After incubation for an additional 3 h, these media were recovered and E2 metabolites were determined as described under Materials and Methods. TCDD and/or NaAsO2 were added in the 12-h induction phase (I) or in the 3-h E2 metabolism phase (A) of the experiment as Ⅺ u ␣ p indicated. The rates of formation of 4-MeOE2 ( ), 2-MeOE2 ( ), 6 -OHE2 ( ), Ͻ ء f ␣ and 15 -OHE2 ( ) were as indicated. Significant differences from control ( , p p Ͻ 0.001) and from the10 nM TCDD (I) treatment group (†††, p Ͻ ,ءءء ;0.05 0.001) are indicated.

and CPR. Although these analyses were suggestive of a similar effect of arsenite in reducing the BAP-induced level of CYP1B1, the low levels of CYP1B1 expressed in T-47D cells did not allow quantitative measurements of CYP1B1 expression with the available antibody (data not shown). Thus, arsenite was shown to diminish the activities

of both CYP1A1 and CYP1B1 in cellular E2 metabolism studies FIG.8.Effects of BAP and NaAsO2 on the levels of CPR and CYP1A1 in T-47D (Figs. 5, D and E, and 7); however, the effect of arsenite on reducing cells. immunoreactive P450 protein was only clearly demonstrated for Confluent cultures of T-47D cells were exposed to medium containing the

CYP1A1 (Fig. 8B). indicated concentrations of BAP and NaAsO2 for 12 h. Cellular proteins were then Because the induction of HO-1 has been observed in response to solubilized and subjected to electrophoresis and Western immunoblotting for CPR (A) and CYP1A1 (B) with chemiluminescence detection and quantitation by scan- arsenite exposure in vivo (Falkner et al., 1993) and in vitro (Jacobs et ning densitometry. A chemiluminescence image of a representative CPR Western al., 1999), concomitant with the reduction of P450 activities, we immunoblot with 20, 13, 9.8, 6.5, 2.6, 1.3, and 0 fmol of purified human recom- investigated whether HO-1 mRNA was induced under the conditions binant human CPR analyzed in lanes 1 to 7, respectively, and samples from T-47D cultures exposed to the DMSO vehicle only in lane 8, 3 ␮M BAP in lane 9, or 3 ␮M of the experiment in Fig. 5, in which rates of CYP1A1- and CYP1B1- ␮ BAP plus 1, 3, or 10 M NaAsO2 in lanes 10 to 12, respectively, is shown (A, inset). catalyzed E2 metabolism were diminished by added NaAsO2. Real- The bar graph (A) shows mean levels (Ϯ standard error) of CPR in T-47D cell time PCR was used to quantitate the levels of HO-1 mRNA in T-47D extracts expressed relative to total cellular protein content of three separate deter- ␮ ␮ minations. A chemiluminescence image of a representative CYP1A1 Western cultures exposed to 3 M BAP or to 1, 3, or 10 M NaAsO2 together ␮ ␮ immunoblot with 2, 1, 0.75, 0.50, 0.25, 0.10, and 0 fmol of cDNA-expressed human with 3 M BAP. Exposure to 3 M BAP alone caused a 3.3-fold CYP1A1 analyzed in lanes 1 to 7, respectively, and samples from T-47D cultures elevation in HO-1 mRNA (Fig. 9). Coexposure to 1, 3, or 10 ␮M exposed to the DMSO vehicle only in lane 8, 3 ␮M BAP in lane 9, or 3 ␮M BAP ␮ NaAsO together with 3 ␮M BAP caused 8.1-, 66-, and 155-fold plus 1, 3, or 10 M NaAsO2 in lanes 10 to 12, respectively, is shown (B, inset). The 2 Ϯ elevations in the level of HO-1 mRNA in T-47D cells. bar graph (B) shows mean levels ( standard error) of CYP1A1 in T-47D cell extracts expressed relative to total cellular protein content of three separate deter- minations. ND indicates that CYP1A1 was not detected [Ͻ0.005 pmol(mg pro- Ϫ Discussion tein) 1]. Significant differences from treatment with 3 ␮M BAP alone (†, p Ͻ 0.05) are indicated. The ability of T-47D cells to rapidly metabolize BAP was initially reported by Merrick et al. (1985), who found that 95% of the BAP wise, the P450 mRNA levels were dependent on the presence of BAP, initially present at 4 ␮M was metabolized within 24 h. Dihydrodiols, suggesting a short (i.e., 1–3 h) half-life for the CYP1A1 and CYP1B1 tetraols, quinones, and sulfated compounds were detected as BAP mRNAs after metabolic removal of the BAP inducer. The rapid metabolites, as were DNA adducts (Pruess-Schwartz et al., 1986). Our turnover of the mRNAs and activities in T-47D cells after exposure to results showed a similar rapid uptake and metabolism of BAP in BAP suggests that these cells may provide a system to investigate T-47D cells concomitant with induction of CYP1A1 and CYP1B1, P450 mRNA and protein catabolism. In recent studies with HepG2 which is most likely responsible for the BAP metabolism. Comparison hepatoma cells, the CYP1A1 mRNA had a similar rapid turnover, of the time courses of the BAP levels and the levels of CYP1A1- and with a reported half-life of 2.4 h (Lekas et al., 2000). CYP1B1-catalyzed activities indicates that the activities of the two The kinetics and concentration-response relationships of BAP-me- enzymes are dependent on the presence of unmetabolized BAP. Like- diated induction of CYP1A1 and CYP1B1 are complex because PAHs 268 SPINK ET AL.

Arsenite has been previously shown to reduce the levels of hepatic P450s of the CYP1A (Falkner et al., 1993; Jacobs et al., 1999; Vakharia et al., 2001), CYP2B (Jacobs et al., 1999), and CYP3A families (Jacobs et al., 1999). The results reported here indicate that the effect of arsenite on the expression of cytochromes P450 is retained in a transformed, immortalized cell line derived from a human breast carcinoma. The fact that arsenite diminishes expression of P450s of several gene families suggests a general mechanism. One mechanism that has been offered to explain the effect is the induction of heme oxygenase by arsenite (Sardana et al., 1981; Falkner et al., 1993), which would reduce the level of active P450. Several heavy metal species are known to induce HO-1; arsenite is particularly potent in this regard. Results reported here show a marked induction of HO-1 mRNA in T-47D human breast cells coexposed to arsenite and BAP. The induction of HO-1 by arsenite and the subsequent reduction in the availability of heme as the prosthetic group for P450s may explain the reduced P450 activities in T-47D cells, hepatocyte FIG.9.Effects of BAP and NaAsO on HO-1 mRNA levels in T-47D cells. 2 cultures, and in vivo. However, recent studies by Jacobs et al. (1999) Confluent cultures of T-47D cells were exposed to medium containing the ␮ suggest that induction of HO-1 may not completely explain the effect DMSO vehicle or 3 M BAP plus the indicated concentrations of NaAsO2 for 12 h. RNA was then isolated, reverse transcribed, and aliquots of the cDNAs were of arsenite on P450 expression. HO-1 is also induced by heme in subjected to real-time PCR with primers specific for human HO-1. Levels of HO-1 hepatocyte cultures, but treatment with heme did not result in reduced Ϯ mRNA are expressed relative to total cellular RNA content. Data are the means P450 levels, whereas cotreatment of hepatocyte cultures with arsenite p Ͻ ,ء) standard error of three determinations. Significant differences from control 0.05) are indicated. and heme diminished levels of CYP2B1/2 (Jacobs et al., 1999). The authors concluded that a mechanism in addition to the induction of are inducers of, substrates for, and inhibitors of CYP1A1 and HO-1 must be involved. Further studies are underway to resolve the CYP1B1 (Shimada et al., 1998; Willett et al., 1998; Arcaro et al., role of HO-1 in the effects of arsenite reported here. 1999). The enzyme inhibitory effects result in bell-shaped concentra- The observation that the induction of CYP1A1- and CYP1B1- tion-response curves for apparent enzyme induction in vitro (Willett et catalyzed activities by BAP are markedly diminished by cotreatment al., 1998; Arcaro et al., 1999). The shapes of concentration-response with arsenite, but the levels of the mRNAs encoding CYP1A1 and curves vary markedly with time of incubation (Arcaro et al., 1999). CYP1B1 are not significantly affected indicates that the primary This has been attributed to the removal of enzyme inducers and effects of arsenite are not at the level of transcription of the two enzyme inhibitors through metabolism. These effects make estimation enzymes or mediated by effects on mRNA stability or catabolism. of toxic equivalency factors for PAHs by measurement of induced However, these results do not rule out possible effects of arsenite on enzyme activities difficult. Determination of the effects of BAP and translation. Effects of arsenite at the level of mRNA translation have other PAHs on CYP1A1 and CYP1B1 levels by measurement of been reported, although they are thought to result in increased rather enzyme activities is only of value at low concentrations of BAP, than decreased rates of translation. Arsenite and other stress inducers where its inhibitory effects are minimal. increase the level of phosphorylation of initiation factor eIF4E, a

Coexposure of T-47D cells to NaAsO2 and BAP resulted in a protein complex that binds the 7-methylguanosine cap structure of marked diminution of the activities of CYP1A1 and CYP1B1 relative mRNAs. Phosphorylation of eIF4E increases its affinity for the to those observed with BAP alone. These effects were observed at mRNA cap structure, which increases the rate of activation of trans- arsenite concentrations below those that affected cell viability. Al- lation (Wang et al., 1998). We are not aware of any reports indicating

though both CYP1A1- and CYP1B1-catalyzed E2 hydroxylase activ- that phosphorylation of eIF4E decreases the rates of translation of ities in microsomal incubations were inhibited by arsenite, the mag- specific mRNAs. nitude of inhibition was not sufficient to account for the reduction of Another point at which arsenite might affect the rates of CYP1A1- BAP-induced CYP1A1- and CYP1B1-catalyzed activities in T-47D and CYP1B1-catalyzed activities is the rate of protein turnover. Our

cells by arsenite. This was most evident for CYP1A1 because addition results show rapid decline in the rates of the BAP-induced E2 2- and ␮ of 10 M NaAsO2 inhibited CYP1A1-catalyzed E2 2-hydroxylation 4-hydroxylation pathways after metabolic removal of the BAP, sug- in microsomal assays by only 21% (Fig. 6A), whereas addition of 10 gesting rapid degradation of CYP1A1 and CYP1B1. Although there ␮ M NaAsO2 to the culture medium caused an 86% reduction in the are no data on P450 turnover in T-47D cells, rates of P450 turnover rate of the BAP-induced E2 2-hydroxylation pathway (Fig. 5D). in rat hepatocytes were found to differ among the P450s. Proteasome- Further evidence that the effect of arsenite on CYP1A1 and CYP1B1 mediated degradation was demonstrated for CYP1A2, CYP2E1, activities was on the levels of active enzyme rather than a direct CYP3A, and CYP4A (Roberts, 1997), but appears to require prior inhibitory effect or modulation of the inhibitory effect of BAP was labilization of the P450s because it can be stimulated in cell-free obtained in the experiments with TCDD. In these experiments, arsen- experiments by processes such as incubation of the P450 with a ite was only effective in the induction phase of the experiment and not suicide substrate or denaturation by freezing and thawing. Arsenite is

during the E2 metabolism assay phase (Fig. 7). The reduction in the known to react with cysteine residues of some proteins, resulting in levels of immunoreactive CYP1A1 observed on Western blots corre- loss of function (Kapahi et al., 2000). Whether proteasome-mediated

lated well with the reduction in 2-MeOE2 formation in response to degradation is involved in the turnover of human CYP1A1 and coexposure to BAP and NaAsO2. Because the levels of CPR were not CYP1B1 in extrahepatic cells and whether arsenite might affect this affected by these coexposures (Fig. 8A), the effects of NaAsO2 on process by influencing factors such as protein conformation and heme P450s do not appear to be reflective of a general effect on cellular incorporation are unknown and will be investigated in future studies. proteins. Although the underlying mechanism is not fully resolved, our EFFECTS OF BAP AND ARSENITE ON CYP1A1 AND CYP1B1 IN T-47D CELLS 269 results suggest that coexposure to arsenite in combination with PAHs ylation patterns before and after changing from high to lower concentrations of arsenic in drinking water. Environ Health Perspect 104:1200–1207. may lessen the rate of PAH metabolism by reducing the induced Jacobs JM, Nichols CE, Andrew AS, Marek DE, Wood SG, Sinclair PR, Wrighton SA, levels and thus activities of both CYP1A1 and CYP1B1. This reduc- Kostrubsky VE, and Sinclair JF (1999) Effect of arsenite on induction of CYP1A, CYP2B, and CYP3A in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol 157:51–59. tion in P450 activity may be a consequence of cross talk among Kapahi P, Takahashi T, Natoli G, Adams SR, Chen Y, Tsien RY, and Karin M (2000) Inhibition stress-response signaling pathways that are activated to limit cellular of NF-␬B activation by arsenite through reaction with a critical cysteine in the activation loop of I␬B kinase. J Biol Chem 275:36062–36066. damage caused by exposure to the toxicants. 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