Sudan I Is a Potential Carcinogen for Humans: Evidence for Its Metabolic Activation and Detoxication by Human Recombinant Cytochrome P450 1A1 and Liver Microsomes1

[CANCER RESEARCH 62, 5678–5684, October 15, 2002] Sudan I Is a Potential Carcinogen for Humans: Evidence for Its Metabolic Activation and Detoxication by Human Recombinant Cytochrome P450 1A1 and Liver Microsomes1

Marie Stiborova´,2 Va´clav Martı´nek, Helena Ry´dlova´, Petr Hodek, and Eva Frei Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague 2, The Czech Republic [M. S., V. M., H. R., P. H.], and Division of Molecular Toxicology, German Cancer Research Center, 69120 Heidelberg, Germany [E. F.]

ABSTRACT liver or urinary bladder in rats, mice, and rabbits, and is considered a possible carcinogen and mutagen for humans (1–5). Besides its car- 1-Phenylazo-2-hydroxynaphthol (Sudan I, C.I. Solvent Yellow 14) is a liver cinogenicity, Sudan I is a potent contact allergen and sensitizer, and urinary bladder carcinogen in mammals. We compared the ability of hepatic microsomal samples from different species including human to me- eliciting pigmented contact dermatitis in human (6). Nevertheless, it is tabolize Sudan I. Comparison between experimental animals and human widely used to color materials such as hydrocarbon solvents, oils, fats, cytochromes P450 (CYP) is essential for the extrapolation of animal carcino- waxes, plastics, printing inks, and shoe and floor polishes (1, 5). genicity data to assess human health risk. Human microsomes generated the Moreover, Sudan I is an important compound, not because it is still pattern of Sudan I metabolites reproducing that formed by hepatic micro- widely used, but because it is the simplest in a series of dyes and somes of rats. Using hepatic microsomes of rats pretreated with specific CYP pigments that are used in very great quantities and occur everywhere inducers, microsomes from Baculovirus-transfected insect cells expressing in red- and-orange colored consumer products, foods, and printed recombinant human CYP enzymes, purified CYP enzymes, and selective CYP inhibitors, we found that rat CYP1A1 and recombinant human matter. Such a wide use of these azo dyes could result in a consider- CYP1A1 are the most efficient enzymes metabolizing Sudan I. Microsomes able exposure. from livers (the target of Sudan I carcinogenicity) of different human donors Sudan I gives positive results in Salmonella typhimurium muta- were used to estimate whether authentic human CYPs oxidize Sudan I. Using genicity tests with S-9 activation (7, 8) and is mutagenic to mouse ϩ/Ϫ Western blot analysis and NH2-terminal sequencing, we were able to detect lymphoma L5178Y TK cells in vitro, with S-9 activation (8). and quantify CYP1A1 in human hepatic microsomes. The sequence of nine It is clastogenic compound, inducing micronuclei in the bone amino acids of the protein band cross-reacting with antirat CYP1A1 in marrow of rats (3). Whereas the metabolism of Sudan I is not human microsomes, LFPISMSAT, matched the sequence of human CYP1A1 perfectly (residues 2–10). CYP1A1 expression levels varied significantly understood in humans, its metabolism has been characterized in among the different human microsomes (0.04–2.4 pmol/mg protein), and rabbits (9), where it is metabolized primarily in the liver by constituted <0.6% of the total hepatic CYP complement. All of the human oxidative or reductive reactions (9). C-Hydroxylated metabolites hepatic microsomal samples oxidized Sudan I to C-hydroxymetabolites. 4Ј-OH-Sudan I and 6-OH-Sudan I were found to be the major Moreover, using the nuclease P1-enhanced version of the 32P-postlabeling products of Sudan I oxidation in vivo and excreted in urine (1, 9), assay, we found that human microsomes were competent in activating Sudan and of its oxidation by rat hepatic microsomes in vitro (10). I to form adducts with DNA. The role of specific CYP enzymes in the human Besides the C-hydroxylated metabolites, which are considered hepatic microsomal metabolism was investigated by correlating the CYP- catalytic activities (or CYP contents) in each microsomal sample with the detoxication products, the BDI formed by microsome-dependent levels of individual metabolites and/or Sudan I-DNA adducts formed by the enzymatic splitting of the azo group of Sudan I was found to react same microsomes, and by examining the effects of agents that can inhibit with DNA in vitro (10–12). The major DNA adduct formed in this specific CYP in Sudan I metabolism. On the basis of these studies, we reaction has been characterized and identified as the 8-(phenyla- attribute most of Sudan I metabolism in human microsomes to CYP1A1, but zo)guanine adduct (12). In addition to microsomal enzymes, Sudan participation of CYP3A4 cannot be ruled out. These results, the first report I and its C-hydroxylated metabolites are also oxidized by peroxi- on the metabolism of Sudan I by human CYP enzymes, strongly suggest a dases, as a consequence DNA, RNA, and protein adducts are carcinogenic potency of this rodent carcinogen for humans. formed (13–15). Because CYPs are abundant in the liver where much of the INTRODUCTION metabolism of Sudan I in experimental animals occurs (9), CYPs were assumed to play a role in the oxidative metabolism of this Sudan I3 was used as a food coloring in several countries (1), but it has been recommended as unsafe, because it causes tumors in the carcinogen (9–12), but as yet no data are available on the participation of human CYP enzymes in its metabolism. Comparison between experimental animals and human CYPs is essential for the Received 5/23/02; accepted 8/8/02. The costs of publication of this article were defrayed in part by the payment of page extrapolation of animal carcinogenicity data to assess human charges. This article must therefore be hereby marked advertisement in accordance with health risk, and consideration of species differences in catalytic 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by the Grant Agency of Charles University (Grant 204/2001/B/ activities of CYPs is important. In contrast to many experimental CH/PrF), the Grant Agency of the Czech Republic (Grant 203/01/0996), and the Ministry animal models, humans show large interindividual variations in the of Education of the Czech Republic (Grant MSM 113100001). 2 To whom requests for reprints should be addressed, at Department of Biochemistry, expression of CYP enzymes and catalytic activities, which may Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, The Czech Republic. lead to different susceptibilities to carcinogens and must be con- Phone: 420-2-2195-2333; Fax: 420-2-2195-2331; E-mail: [email protected]. sidered in risk assessment (16). To assess the human health risk of 3 The abbreviations used are: Sudan I, 1-(phenylazo)-2-naphthol (C.I. Solvent Yellow 14); ␣-NF, ␣-naphthoflavone; ␤-NF, ␤-naphthoflavone; BDI, benzenediazonium ion; Sudan I, we have compared the capacity of livers from humans, CYP, cytochrome P450; DDTC, diethyldithiocarbamate; EROD, 7-ethoxyresorufin rats, and rabbits to metabolize Sudan I. In addition, the present O-deethylation; 3-IPMDIA, 3-isopropenyl-3-methyldiamantane; 4Ј-OH-Sudan I, 1-(4- hydroxyphenylazo)-2-hydroxynaphthol; 6-OH-Sudan I, 1-(phenylazo)-naphthalene-2,6- study was undertaken to understand which human CYP enzymes diol; 4Ј,6-di(OH)-Sudan I, 1-(4-hydroxyphenylazo)-naphthalene-2,6-diol; 3Ј,4Ј-di(OH)- are involved in Sudan I metabolic activation and/or detoxication. PB, phenobarbital; PCN, pregnenolone-16␣-carbonitrile; PEI, polyethylenimine; PVDF, polyvinylidene difluoride; RAL, relative adduct labeling; HPLC, high-performance liquid This knowledge will be useful in evaluating individual suscepti- chromatography. bility to this carcinogen. 5678

MATERIALS AND METHODS two distinct bands. Visualization was with an alkaline phosphatase-conjugated antichicken IgG rabbit antibody and 5-bromo-4-chloro-3-indolylphosphate/ ␣ ␤ Chemicals. -NF, -NF, NADPH, troleandomycin, ketoconazole, glucose nitrobluetetrazolium as dye. CYP contents were read from a standard curve 6-phosphate, chlorzoxazone, calf thymus DNA, coumarin, sulfaphenazole, and with either recombinant human CYP1A1 or 1A2 (in Supersomes) over the quinidine were from Sigma Chemical Co. (St. Louis, MO); furafylline from linear portion of the response curve (generally 0.01–1.5 pmol of CYPs) ␤ New England Biolabs (Beverly, MA); 6 -hydroxytestosterone from Merck generated by scanning the membrane with a computerized image-analyzing (Darmstadt, Germany); glucose 6-phosphate dehydrogenase from Serva (Hei- system (Imstar). This system consistently demonstrated a detection sensitivity Ј delberg, Germany); bufuralol and its 1 -hydroxyderivative from Gentest Corp. as low as 0.005 pmol CYP1A1 per lane. Reference samples (0.01–1.5 pmol of (Woburn, MA); bicinchoninic acid from Pierce (Rockford, IL); and Sudan I human recombinant CYP1A1 and 1A2) were routinely incorporated into each from British Drug Houses (Poole, United Kingdom). 3-IPMDIA was synthe- electrophoresis to standardize determinations. Human microsomal CYP2E1 Ј sized according to Olah et al. (17) The derivatives 4 -OH-Sudan I, 6-OH- and 3A4 proteins were probed with chicken polyclonal antibody raised against Ј Ј Ј Sudan I, 4 ,6-di(OH)-Sudan I and 3 ,4 -di(OH)-Sudan I were synthesized as rabbit CYP2E1 and human CYP3A4, and visualized as described above. described (10). Enzymes and chemicals for the 32P-postlabeling assay were The bands corresponding to CYP1A1 protein of two human hepatic micro- obtained from sources described previously (12). somal samples (samples 5 and 6, see Table 1) were excised from a PVDF Preparation of Microsomes and Assays. Microsomes from livers of un- membrane and subjected to NH -terminal sequencing on a Protein Sequencer treated rats and rabbits were prepared as described previously (12). Micro- 2 LF3600D (Beckman Instruments) according to the manufacturer’s manual. somes from the livers of rats pretreated with ␤-NF (12) and Sudan I (18) were Isolation of Individual CYPs. The CYP1A2, 2B4, 2C3, and 2E1 were isolated as described (12), those pretreated with PB, PCN, and ethanol as isolated from liver microsomes of rabbits induced with ␤-NF (CYP1A2), PB reported (19). Microsomes from human liver of eight human donors who died (CYP2B4), or ethanol (CYP2E1 and 2C3) by procedures described elsewhere in a traffic accidents were isolated as described (20) and were a gift of B. (26, 27). The CYP3A1 and 3A6 were isolated from rat and rabbit hepatic Szota´kova´ (Faculty of Pharmacy, Charles University, Hradec Kra´love´, The microsomes of animals induced with PCN (19) and rifampicin (28), respec- Czech Republic). The donors ranged in age from 24 to 70 years, and included tively. Recombinant rat CYP1A1 was purified as described (29) from mem- five men and three women. All of the donors had no known drug history and branes of Escherichia coli transfected with a modified CYP1A1 cDNA. Re- none had a history of alcohol abuse. Microsomes from the liver of a male combinant human CYP1A2 was from Oxford Biomedical Research, Inc., and minipig were a gift from P. Anzenbacher (Palacky University, Olomouc, The Czech Republic) and isolated as described (20). Supersomes, microsomes human recombinant CYP3A4 was a gift of P. Anzenbacher (see above). Rabbit isolated from insect cells transfected with Baculovirus constructs containing liver NADPH:CYP reductase and cytochrome b5 were purified as described cDNA of one of the following human CYPs: CYP1A1, 1A2, 1B1, 2A6, 2B6, (30, 31). Preparation of Antirat CYP1A1, Antirabbit CYP2E1, and Antihuman 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4, with cytochrome b5 and expressing NADPH:CYP reductase were from Gentest Corp. Protein concentrations were CYP3A4 Polyclonal Antibodies. Leghorn chickens were immunized s.c. assessed using the bicinchoninic acid protein assay (21). The concentration of three times a week by CYP antigens (rat recombinant CYP1A1, rabbit CYP was estimated according to Omura and Sato (22). Rat, rabbit, and minipig CYP2E1, and human recombinant CYP3A4; 0.1 mg/animal) emulsified in liver microsomes contained 0.62, 1.82, and 0.89 nmol CYP/mg protein, re- complete Freund’s adjuvant for the first injection and in incomplete adjuvant spectively. Microsomes of rats induced with ␤-NF, PB, PCN, and ethanol for boosters. The immunoglobulin fraction was purified from pooled egg yolks contained 1.30, 2.74, 1.55, and 1.80 nmol CYP/mg protein, respectively. The as described (32, 33). content of CYP in human hepatic microsomes is shown in Table 1. Each Incubations. Incubation mixtures contained the following in a final vol- human microsomal sample was analyzed for specific CYP activities by mon- ume of 750 ␮l: 50 mM sodium phosphate buffer (pH 7.4), 1 mM NADPH, 10 itoring the following reactions: EROD (CYP1A1/2), coumarin 7-hydroxyla- mMD-glucose 6-phosphate, 1 unit/ml D-glucose 6-phosphate dehydrogenase, tion (CYP2A6), bufuralol 1Ј-hydroxylation (CYP2D6), tolbutamide methyl 10 mM MgCl2, microsomal fraction containing 0.05–2.4 nmol CYP, and hydroxylation (CYP2C9), chlorzoxazone 6-hydroxylation (CYP2E1), and tes- 0.1–100 ␮M Sudan I dissolved in 7.5 ␮l methanol. Incubation mixtures, in tosterone 6␤-hydroxylation (CYP3A4; Ref. 23 and references therein). These which the efficiencies of Supersomes expressing human CYPs were tested, activities are shown in Table 1. were the same except that 100 ␮M of Sudan I and only 10–50 pmol of CYP CYP Content in Human Hepatic Microsomes by Western Blot. Immu- were used. Incubations using purified CYP reconstituted with NADPH:CYP noquantitation of human liver microsomal CYP 1A1, 1A2, 2E1, and 3A4 was reductase and cytochrome b5 (34) contained 50–250 pmol of each CYP. After estimated by SDS-PAGE. Samples containing 75-␮g microsomal proteins incubation (37°C, 5–140 min) the mixtures were extracted with ethyl acetate. were solubilized and subjected to electrophoresis on SDS/10% polyacrylamide The extracts were evaporated, dissolved in methanol, and chromatographed on gels (24). After migration, proteins were transferred onto PVDF membranes. a thin layer of silica gel (10). The BDI was detected by azo coupling with Human microsomal CYP1A1 and 1A2 proteins were probed with a chicken 1-phenyl-3-methyl-5-pyrazolone (10–12). Alternatively, the products were polyclonal antibody raised against rat recombinant CYP1A1 as reported else- separated by HPLC on a MN Nucleosil 100–5 C18 column (Macherey-Nagel; ϫ where (25). This antibody recognized both CYP1A1 and 1A2 in rat liver 4.0 250 mm). An isocratic flow of methanol: 0.1 M NH4HCO3 (pH 8.5; 9:1, microsomes, as well as human CYP1A1 and 1A2 expressed in Supersomes as v/v) with flow rate of 0.8 ml/min was used to elute the metabolites, and

Table 1 CYP-dependent catalytic activities, CYP1A1 levels, amounts of ring-hydroxylated Sudan I metabolites, and DNA adducts formed by Sudan I in human hepatic microsomal samples All results are presented as means of duplicate experiments. CYP1A1 content was determined by Western blot as described in “Materials and Methods.” Assays for CYP activities were carried out as described elsewhere (23). Tolbutamide Human hepatic pmol CYP Coumarin 7- methyl Bufuralol 1Ј- Chlorzoxazone Estosterone 6␤ microsomal per mg pmol CYP1A1 EROD hydroxylation hydroxylation hydroxylation 6-hydroxylation hydroxylation Sudan I Sudan I-DNA samples protein per mg protein (CYP1A1/2)a (CYP2A6)a (CYP2C9)a (CYP2D6)a (CYP2E1)a (CYP3A4)a metabolitesb adductsc 1 80 0.080 5.65 0.93 0.76 2.46 1.80 8.44 0.19 1.16 2 90 0.081 4.09 1.42 0.06 0.34 0.69 2.22 0.13 0.77 3 270 0.347 6.34 2.03 0.18 5.55 2.09 6.31 0.24 1.99 4 60 0.187 7.19 0.40 0.06 3.71 1.83 14.39 0.22 2.13 5 220 1.280 10.82 0.84 0.17 2.53 2.45 10.19 0.32 2.29 6 140 0.600 11.73 0.98 0.26 0.92 2.61 6.94 0.40 3.28 7 460 0.040 6.86 0.43 0.11 4.11 1.37 3.96 0.21 1.07 8 400 2.400 12.08 0.58 0.15 3.13 1.51 6.86 0.25 3.29 a CYP activities in nmol/min/nmol CYP, except for EROD activity, which is in pmol/min/nmol CYP. b nmol total C-hydroxylated metabolites/min/nmol CYP. c RAL/107 nucleotides per nmol CYP. 5679

Fig. 1. HPLC chromatogram of Sudan I metabolites formed by human microsomes. Incubations [1 mM NADPH, 10 mMD-glucose 6-phosphate, and1 units/ml D-glucose 6-phosphate dehydrogenase, human microsomal sample no. 2 containing 0.1 nmol CYP, 100 ␮M Sudan I dissolved in 7.5 ␮l methanol in 50 mM potassium phosphate buffer (pH 7.4), and a final volume of 750 ␮l] were stopped after 20 min by extraction with ethyl acetate and analyzed by HPLC (see “Materials and Methods”).

detection was at 254, 333, and 480 nm. The Sudan I metabolites were RESULTS identified by cochromatography with authentic standards. Incubations in which DNA was modified by Sudan I activated with human Metabolism of Sudan I by Rat, Rabbit, Minipig, and Human or rat hepatic microsomes had the same composition, but contained 1 mg of Hepatic Microsomes. When Sudan I was incubated with rat, rabbit, calf thymus DNA and human microsomes containing 100 pmol CYP, or minipig, or human hepatic microsomes in the presence of NADPH, hepatic microsomes of rats pretreated with ␤-NF (12). DNA was isolated as several product peaks were observed by HPLC analysis (Fig. 1). On described (12). the basis of cochromatography with the synthetic standards, the major Kinetic analyses to determine the maximum reaction rate (maximum ve- metabolites produced from Sudan I by all of the tested microsomes locity) and Michaelis constant were performed using the nonlinear least- were identified as 4Ј-OH-Sudan I and 6-OH-Sudan I. Additional squares method as described (35). Incubations were the same as those de- minor products were 4Ј,6-di(OH)-Sudan I and 3Ј,4Јdi(OH)-Sudan I scribed above (with microsomes) except that they contained 0.1–100 ␮M (Fig. 1). Another metabolite was a colorless product, which was Sudan I, 4Ј-OH-Sudan I, or 6-OH-Sudan I. Mixtures were incubated at 37°C identified previously as BDI (Refs. 10, 12; not shown in the chromat- for 10 min. ogram of HPLC in Fig. 1). Whereas in microsomes of rabbit and Inhibition Studies. The following chemicals were used to inhibit the minipig 100 ␮M Sudan I was prefertially oxidized to the 6-hydroxy- ␣ metabolism of Sudan I (specific CYPs known to be inhibited): -NF naphthol derivative of Sudan I, those of human and rat predominantly (CYP1A1/2); furafylline (CYP1A2); 3-IPMDIA (CYP2B; 36); DDTC produced 4Ј-OH-Sudan I (Fig. 2). The ratios of metabolites were the (CYP2A6 and 2E1); sulfaphenazole (CYP2C); quinidine (CYP2D); and trole- same at lower Sudan I concentrations down to 10 ␮M. andomycin and ketoconazole (CYP3A). Inhibitors were dissolved in 7.5 ␮lof To resolve which CYPs are able to oxidize Sudan I, five experi- methanol to yield final concentrations of 1–400 ␮M in the incubation mixtures. An equal volume of methanol alone was added to the control incubations. mental approaches were used: (a) induction of specific CYPs; 32P Postlabeling and Recovery of Individual Nucleotide Adducts. For DNA modified with activated Sudan I, the nuclease P1 version of the 32P- postlabeling assay (37) was used (12). The labeled digests were chromatographed on thin layer plates of PEI cellulose as described previously (12). Adducts and normal nucleotides were detected and quantified by storage phosphor imaging on a Packard Instant Imager. Adduct levels were calculated in units of RAL, which is the ratio of cpm of adducted nucleotides to cpm of total nucleotides in the assay. Cochromatography on PEI Cellulose. Adduct spot 1 of DNA modified by Sudan I activated with human hepatic microsomes detected by the 32P- postlabeling assay and that generated by hepatic microsomes of rats were excised from chromatograms and extracted (12). For cochromatographic analyses, the extracts were dissolved in water so that equal amounts of radioactivity could be applied for each sample. Developments of these adducts were carried out in D3 and D4 directions (12) using two different solvents systems: (a)D3 solvent was 2.7 M lithium formate, 5.1 M urea (pH 3.5) and D4, 0.36 M sodium phosphate, 0.23 M Tris-HCl, 3.8 M urea (pH 8.0); and (b) D3 solvent was 2.7 M lithium formate, 5.1 M urea (pH 3.5) and D4, 4 N ammonium hydroxide/ isopropanol (1:1). Statistical Analyses. Statistical association between CYP-linked catalytic activities (or CYP protein levels) in human hepatic microsomal samples and levels of individual Sudan I metabolites or Sudan I-DNA adducts formed by the same microsomes were determined by the Spearman correlation coefficient Fig. 2. Oxidation of Sudan I to ring-hydroxylated metabolites by rat, rabbit, minipig, ␮ using version 6.12 Statistical Analysis System software. Spearman correlation and human hepatic microsomes. Microsomes containing 1 nmol CYP and 100 M Sudan I were used in all of the experiments. Human hepatic microsomal sample no. 2 was used. coefficients were based on a sample size of 8. All of the Ps are two-tailed and Other conditions were as in Fig. 1. Values of Sudan I metabolites are averages of triplicate considered significant at the 0.05 level. incubations. SDs were Յ10%. 5680

CYP1A1 and human recombinant CYP1A1 are the most efficient enzymes metabolizing Sudan I. Estimation of CYP1A1 in Human Hepatic Microsomes and Its Involvement in Sudan I Oxidation. To identify authentic human CYPs capable of oxidizing Sudan I, microsomal samples from livers of eight different human donors were used in additional experiments. All of the human microsomal preparations metabolized Sudan I (Ta- ble 1). Correlations between the CYP catalytic activities (Table 1) and the amounts of each of the C-hydroxylated Sudan I metabolites in each microsomal sample were used to examine the role of specific human CYP enzymes in the metabolism of Sudan I. The formation of all of the Sudan I C-hydroxylated metabolites was highly correlated with EROD activity, a marker for CYP1A1/2 (Table 2). Whereas CYP1A2 protein is constitutively expressed in the human

Fig. 3. Oxidation of Sudan I to ring-hydroxylated metabolites by hepatic microsomes from rats pretreated with selective CYP inducers. Microsomes containing 1 nmol CYP and 100 ␮M Sudan I were used in all of the experiments. Other conditions were as in Fig. 1. Values of Sudan I metabolites are averages of triplicate incubations. SDs were Յ10%.

(b) selective inhibition of CYPs; (c) utilization of the purified CYPs reconstituted with NADPH:CYP reductase; (d) heterologous expression systems (Supersomes); and (e) correlation of the efficiencies of microsomal samples to oxidize Sudan I with known marker activities of CYPs or with amounts of expressed CYP proteins. Involvement of Rat CYP Enzymes in Oxidation of Sudan I. Individual CYP enzymes were induced in rats. Incubations of Sudan I with microsomes from ␤-NF- or Sudan I-treated rats led to a 10-fold increase in Sudan I metabolism, although induction with PB resulted in a 2-fold increase (Fig. 3). An inhibitor of CYP1A1/2, ␣-NF, was highly effective in inhibiting Sudan I oxidation; an equimolar con- Fig. 4. Oxidation of Sudan I to ring-hydroxylated metabolites by purified rat and rabbit centration of ␣-NF and Sudan I inhibited its oxidation by 70%. or recombinant human CYPs reconstituted with rabbit NADPH:CYP reductase and the ␣ Inhibitors of other CYP enzymes caused either weak (ketoconazole, effect of -NF on Sudan I oxidation by CYP1A1. One-hundred pmol reconstituted CYP/incubation and 100 ␮M Sudan I were used in all of the experiments. Other conditions troleandomycin, and 3-IPMDIA) or no inhibition (furafylline, sulfa- were as in Fig. 1. Values of Sudan I metabolites are averages of triplicate incubations. SDs phenazole, quinidine, and DDTC). The formation of Sudan I metab- were Յ10%. olites with ␤-NF microsomes was time-dependent and linear up to 20 min. Not only Sudan I, but its first hydroxylated products are substrates for additional oxidation by CYP. The values of maximum velocity and apparent Michaelis constant for the oxidation of these three substrates, Sudan I, 4ЈOH-Sudan I, and 6-OH-Sudan I, in ␤-NF microsomes are 1.7, 4.6, and 2.8 nmol/min per nmol total CYP and 21, 79, and 40 ␮M, respectively. Oxidation of Sudan I by Purified CYP Enzymes. To identify the role of individual CYPs in oxidation of Sudan I, several CYP enzymes were purified, reconstituted with NADPH:CYP reductase and cytochrome b5 (34), and used as the oxidation system. All of the CYPs reconstituted with reductase were active with their typical substrates. Of the CYP enzymes tested, rat recombinant CYP1A1 was the most efficient enzyme oxidizing 100 ␮M Sudan I (Fig. 4). ␣-NF inhibited Sudan I oxidation as in microsomes (Fig. 4). Oxidation of Sudan I by Recombinant Human CYP Enzymes. To investigate whether human recombinant CYPs oxidize Sudan I, we used microsomes of Baculovirus-transfected insect cells containing recombinantly expressed human CYPs and NADPH:CYP reductase. The recombinant human CYPs used in the experiments efficiently oxidized their typical substrates. Human CYP1A1, and to a much Fig. 5. Oxidation of Sudan I to ring-hydroxylated metabolites by human recombinant CYPs. Twenty-five pmol human recombinant CYP/incubation and 100 ␮M Sudan I were lesser extent, CYP3A4, metabolized Sudan I. Other CYPs were al- used in all of the experiments. Values of Sudan I metabolites are averages of triplicate most ineffective (Fig. 5). All of the above results indicate that rat incubations. SDs were Յ10%. 5681

Table 2 Spearman correlation coefficients (r) among CYP-linked catalytic activities or CYP1A1 contents and levels of ring-hydroxylated metabolites of Sudan I or Sudan I-DNA adducts formed by human hepatic microsomes (n ϭ 8) Coumarin 7- Tolbutamide methyl Bufuralol 1Ј- Chlorzoxazone Testosterone CYP CYP1A1 EROD hydroxylation hydroxylation hydroxylation 6-hydroxylation 6␤-hydroxylation content content (CYP1A1/2) (CYP2A6) (CYP2C9) (CYP2D6) (CYP2E1) (CYP3A4) Total C-hydroxymetabolites 0.286 0.810b 0.903b Ϫ0.024 0.405 0.095 0.810b 0.405 4Ј-OH-Sudan I 0.286 0.810b 0.903b Ϫ0.024 0.405 0.095 0.810b 0.405 6-OH-Sudan I 0.167 0.810b 0.752c Ϫ0.142 0.667 Ϫ0.071 0.833b 0.452 4Ј,6-diOH-Sudan I 0.5 0.714c 0.813b 0 0.214 0.119 0.643 0.191 DNA adducts 0.071 0.952a 0.905b Ϫ0.143 0.405 Ϫ0.024 0.738c 0.548 a P Ͻ 0.001. b P Ͻ 0.01. c P Ͻ 0.05. liver, the content of CYP1A1 enzyme in this organ is low; so low that oxidation of this carcinogen (r ϭ 0.405; P ϭ 0.320). Because the it has been discussed whether it is expressed in this organ at all or only EROD activity highly correlated with CYP1A1 content (r ϭ 0.762; in extrahepatic tissues (38, 39), and readily induced by ligands of the P Ͻ 0.05) but not with the content of CYP1A2 protein (r ϭϪ0.309; aryl hydrocarbon receptor (40). In contrast, composite of results P ϭ 0.456), O-deethylation of ethoxyresorufin seems to be catalyzed obtained with mRNA, protein, and activity measurements indicates mainly by CYP1A1 in human hepatic microsomes used in the study. that low expression levels of CYP1A1 occur in human livers (41–43) Whereas catalytic activities of CYP2A6, 2C9, 2D6, and 3A4 did at Ͻ1% of total hepatic CYP (42, 43). not exhibit significant correlation with the levels of Sudan I metabo- Using two independent methods, we were able to detect and quan- lites formed by the same human hepatic samples, a significant corre- tify CYP1A1 in human hepatic microsomes. A polyclonal antibody lation was seen with the CYP2E1 activity (Table 2). However, there raised against rat recombinant CYP1A1, which highly cross-reacts is a cross-correlation between EROD and chlorzoxazone 6-hydroxy- with recombinant human CYP1A1 and only poorly with CYP1A2, lation activity (r ϭ 0.783; P ϭ 0.038) within these liver samples. To was used in the first method (Fig. 6A). The detection sensitivity was additionally clarify this correlation, multivariate analysis was used to as low as 0.005 pmol CYP1A1 per lane. In immunoblots (Fig. 6B), investigate the dependence of the Sudan I oxidation on these two this polyclonal antibody reacted with one and/or two immunoreactive isoform activities. The two activities (CYP1A and 2E1) in each bands in most analyzed human hepatic microsomes. The high and low microsomal sample were combined in pairs to see if a combination of mobility bands (Fig. 6B) were assumed to be CYP1A1 and 1A2, two activities gave an improvement in the correlation with Sudan I respectively, based on the reported electrophoretic mobilities of these oxidation, i.e., an increase in the correlation coefficient when com- proteins in microsomes from human tissues (44, 45). To confirm that pared with the correlation with the individual activities. The inclusion the band with lower molecular weight really corresponds to human of the CYP2E1 activity produced no improvement in the correlation CYP1A1, NH2-terminal sequencing was carried out with this protein coefficient. Multivariate analysis was also used to examine the de- band. The bands of microsomal samples 5 and 6 were excised from a pendence of the Sudan I oxidation on activities of CYP3A4 and 2C9. PVDF membrane and subjected to automated Edman degradation. Although the activities of these CYPs did not exhibit significant The sequence of nine amino acids, LFPISMSAT, was identical to the correlations with Sudan I oxidation, these activities showed certain residues 2–10 of the NH2-terminal sequence of CYP1A1 (MLF- correlation tendencies (Table 2) and recombinant CYP3A4 was active PISMSAT; Ref. 46). NH2-terminal methionine was not found in the with Sudan I (Fig. 5). The inclusion of the CYP3A4 or 2C9 activities CYP1A1 protein band by NH2-terminal sequencing. with CYP1A in multivariate analysis produced no improvement in the The CYP1A1 expression levels varied greatly among the different correlation coefficient. Ͻ human microsomal samples (Table 1), being present at 0.6% of total To confirm the role of individual human hepatic CYP enzymes in hepatic CYP. With the same antibody, we also estimated the expres- metabolism of Sudan I, two human microsomal samples with high sion levels of CYP1A2 in all of the human microsomal samples. The CYP1A, 2E1, and 3A4 activities, samples 5 and 8, were selected, and CYP1A2 content ranged from 5 to 35 pmol per mg of microsomal incubations were carried out in the absence and presence of specific protein (data not shown). inhibitors of CYP1A1/2, 1A2, 2E1, and 3A4, ␣-NF, furafylline, To resolve which of these two CYPs is the predominant enzyme DDTC, and ketoconazole, respectively. A substrate of CYP2E1, oxidizing Sudan I, correlations between the CYP1A1 or 1A2 protein chlorzoxazone, was used as additional inhibitor. ␣-NF inhibited levels and Sudan I oxidation were used. A significant correlation was Sudan I metabolism to 50%, whereas no effect of furafylline, DDTC, seen between hepatic CYP1A1 content and Sudan I oxidation or chlorzoxazone was observed. Ketoconazole weakly inhibited the ϭ ϭ (r 0.810; P 0.010), but not between the content of CYP1A2 and oxidation of Sudan I by these human microsome samples by 15%. All of these results strongly suggest that Sudan I oxidation in human hepatic microsomes is mediated mainly by CYP1A1, as in the systems using the isolated rat recombinant and human CYP1A1 enzymes (see Figs. 4 and 5). Nevertheless, although CYP3A4 activities showed poor correlation with Sudan I oxidation (Table 2), the inhibition of Sudan I oxidation by ketoconazole indicated that the participation of CYP3A4 in Sudan I oxidation in human hepatic microsomes cannot be excluded. Sudan I Is Activated by Human Hepatic Microsomes to Form 32 Fig. 6. Immunoblots of human recombinant CYP1A1 and 1A2 expressed in Super- DNA Adducts. Using the nuclease P1 version of the P-postlabeling somes (A) and human liver microsomes (B). Human recombinant CYP1A1 and 1A2 (A; assay we found that during oxidation of Sudan I by human hepatic 0.1, 0.5, 1.0, and 1.5 pmol), and 75 ␮g of microsomal proteins were separated on SDS/10% polyacrylamide gel, transferred onto a PVDF membrane, and probed with a microsomes DNA adducts are formed. One major (the closed circle in chicken antirat CYP1A1 polyclonal antibody. Fig. 7D) and two minor adduct spots, overlapping the major adduct, 5682

Sudan I by the human enzymatic system is analogous to that observed in rats. Human microsomes generated the same pattern of Sudan I metabolites as hepatic microsomes of rats. In addition, the present study documents the role of specific human CYP enzymes in metabolic pathways of Sudan I. CYP1A1 seems to be the principal enzyme responsible for the metabolism of Sudan I. There is still conflicting evidence for the expression or inducibility of CYP1A1 protein in human livers (39–43). Using a highly efficient chicken polyclonal antibody raised against rat CYP1A1, strongly cross-reacting with human recombinant CYP1A1, we were able to detect and quantify the CYP1A1 protein content in human hepatic samples used in the study by Western blot analysis with a detection sensitivity of 0.005 pmol CYP1A1 per lane. Moreover, we sequenced

for the first time the nine NH2-terminal amino acids of the CYP1A1 protein band, separated from other human hepatic microsomal proteins by SDS-PAGE. This amino acid sequence was identical with that of CYP1A1 cDNA (46). The successful immunodetection of CYP1A1 shown in our study may be explained by the use of a highly sensitive and selective anti-CYP1A1 antibody. The range of CYP1A1 expression levels in our eight human livers (see Table 2) is comparable with values reported recently (42, 43). The role of CYP1A1 in Sudan I Fig. 7. Autoradiographs of PEI-cellulose TLC maps of 32P-labeled digests of calf oxidation was supported by strong correlation coefficients between thymus DNA reacted with Sudan I, NADPH, and human hepatic microsomes (sample no. the levels of CYP1A1 protein expression (or the rates of EROD), and 5; A), with the same system, but with rat hepatic ␤-NF microsomes (B), and with the same system, but without Sudan I (C). D, schematic figure of adducts with assigned numbers. the levels of Sudan I metabolites and/or Sudan I-derived DNA adducts The major adduct is represented by the closed circle. Analysis was performed by the in the eight human hepatic microsomal samples. The participation of nuclease P1 version of the assay. Chromatographic conditions are described (12). Auto- radiography was at Ϫ80°Cfor4(A),1(B),and8h(C). Origins are located in the bottom CYP1A1 in Sudan I metabolism was confirmed also by inhibition of left corners (D3 from bottom to top and D4 from left to right). Sudan I oxidation by ␣-NF, an inhibitor of CYP1A1/2, whereas furafylline, a specific inhibitor of CYP1A2, did not inhibit Sudan I oxidation. It should be noted that the interpretation of the results of were detected in autoradiographs of DNA reacted with Sudan I inhibitors is sometimes difficult, because one inhibitor may be more activated by human microsomes (Fig. 7A). The major adduct spot effective with one substrate than another. Nevertheless, the utilization of exhibited similar chromatographic properties as the major adduct pure CYP1A1 as well as microsomes containing human recombinant formed in DNA by Sudan I activated with rat microsomes (Fig. 7B), CYP1A1 fully corroborated the major role of CYP1A1 in the metabolism which corresponds to the 3Ј,5Ј-bisphospho-derivative of an 8-(pheny- of Sudan I. Interestingly, the highly homologous human CYP1A1 and lazo)deoxyguanosine adduct identified previously (12). The identity 1A2 with 73% amino acid sequence identity exhibit extremely different of the major adduct in human and rat microsomes was confirmed by potency to oxidize Sudan I. CYP1A2 is almost ineffective in Sudan I cochromatography on PEI-cellulose plates in two different solvent oxidation. Besides the CYP1A1, the CYP3A4 enzyme might also par- systems (not shown). ticipate in Sudan I oxidation in human hepatic microsomes, because The adducts were quantified and expressed as RALs (Table 1). A human recombinant CYP3A4 oxidizes Sudan I. The efficiency of this highly significant correlation was found between the EROD activity CYP to oxidize Sudan I is ϳ10-fold lower than that of CYP1A1. and the formation of Sudan I-DNA adducts (r ϭ 0.905; P ϭ 0.002) in However, because of high expression levels of CYP3A4 in human livers, human microsomes (Table 2). In addition, Sudan I-DNA adduct its contribution to Sudan I metabolism might be relevant, although the formation highly correlated with levels of the CYP1A1 protein deter- correlation studies showed only correlation tendencies with levels of mined in microsomes by Western blot analysis (Table 2). A weaker Sudan I metabolites and DNA adducts. but significant correlation was determined between CYP2E1 activities and formation of Sudan I-DNA adducts (Table 2). Again, a cross- Human CYP1A1 seems to be induced by planar aromatic com- correlation between the CYP1A and 2E1 activities in these liver pounds binding to the aryl hydrocarbon receptor, e.g., 2,3,7,8- samples might explain these results (see above). The binding of Sudan tetrachlorodibenzo-p-dioxin (42) and/or by polycyclic hydrocarbons I to DNA catalyzed by one microsomal sample with high CYP1A and present in cigarette smoke (40). The CYP1A1 enzyme is strongly induced 2E1 activities (sample 8) was inhibited by ␣-NF, but not by furafyl- by Sudan I itself in rats by this mechanism (47). Hence, long-term line, a selective inhibitor of human CYP1A2, or DDTC, an inhibitor occupational exposure of humans to Sudan I might be an important risk of CYP2E1. factor for individuals, improving Sudan I metabolism and binding to DNA, thereby increasing its toxicological relevance. Our results suggest that rats may predict human susceptibility to DISCUSSION Sudan I. This is highly significant in view of the prediction of Sudan We present for the first time data that show that human hepatic I carcinogenicity to humans. Whereas Sudan I is carcinogenic to rats microsomes metabolize carcinogenic Sudan I. Human microsomes (1–5), its carcinogenicity to humans has not yet been proven. Sudan I oxidize Sudan I to ring hydroxylated metabolites and are capable of was evaluated to be still unclassifiable as carcinogenic to humans by activating this carcinogen to species binding to DNA. The major DNA IARC (5).4 In a meeting March 3–5, 1999, a European Union com- adduct generated by Sudan I activated by human microsomes exhibits mission working group for classification, packaging, and labeling of the same chromatographic properties as the 8-(phenylazo)deoxy- dangerous substances recommended that Sudan I should be consid- guanosine adduct identified in rat microsomal systems. One of the most important results of our study is the finding that metabolism of 4 Internet address: http://www.iarc.fr for lists of IARC evaluations, November 1998. 5683

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2002 American Association for Cancer Research. METABOLISM OF SUDAN I BY HUMAN CYP1A1 ered of “concern for man owing to possible carcinogenic effects” (Cat. microsomes. Comparison with human liver samples. Drug Metab. Dispos., 26: Carc. 3; Ref. 5) and of “concern for man because of possible muta- 56Ð59, 1998. 21. Wiechelman, K. J., Braun, R. D., and Fitzpatrick, J. D. Investigation of the bicin- genic effects” (Muta. Cat. 3; Ref. 5). We fully support the recom- choninic acid protein assay: identification of the groups responsible for color forma- mendation of this working group. Our results, showing for the first tion. Anal. Biochem., 175: 231Ð237, 1988. time an analogy in the Sudan I metabolism by human and rat enzymes, 22. Omura, T., and Sato, R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239: 2370Ð2378, 1964. strongly suggest a carcinogenic potential of this rat carcinogen for 23. Guengerich, F. P., and Shimada, T. Oxidation of toxic and carcinogenic chemicals by humans. An increased cancer risk should be taken into account mainly human cytochrome P450 enzymes. Chem. Res. Toxicol., 4: 391Ð407, 1991. 24. Guengerich, F. P., Wang, P., and Davidson, N. K. Estimation of isozymes of for individuals working in the dye industry and exposed to Sudan I, its microsomal cytochrome P-450 in rats, rabbits, ad humans using immunochemical derivatives, and to other compounds inducing CYP1A1. Furthermore, staining coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. caution is highly recommended in using this dye and its derivatives to Biochemistry, 21: 1698Ð1706, 1982. 25. Sonner, M., and Crestiel, T. Delayed ontogenesis of CYP1A2 in the human liver. Eur. color materials, which are used by humans in their daily use. J. Biochem., 251: 893Ð898, 1998. 26. Haugen, D. A., and Coon, M. J. Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microso- REFERENCES mal cytochrome P-450. J. Biol. Chem., 251: 7929Ð7939, 1976. 1. Sudan I. IARC Monographs, Vol. 8, pp. 225–231. Lyon, France: IARC, 1975. 27. Yang, C. S., Tu, Y. Y., Koop, D. R., and Coon, M. J. Metabolism of nitrosamines by 2. Garner, R. C., Martin, C. N., and Clayson, D. B. Carcinogenic aromatic amines and purified rabbit liver cytochrome P-450 isozymes. Cancer Res., 45: 1140Ð1145, 1985. ˇ ␣ related compounds. In: C. E. Searle, (ed.). Chemical Carcinogens. 3rd Ed., ACN 28. Borek-Dohalska«, L., Hodek, P., Sulc, M., and Stiborova«, M. -Naphthoflavone acts Monograph 182., Vol. 1, pp. 175–302. Washington, DC.: American Chemical Soci- as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6. ety, 1984. Chem. Biol. Interact., 138: 85Ð106, 2001. 3. Westmoreland, C., and Gatehouse, D. G. The differential clastogenicity of Solvent 29. Saito, T., and Strobel, H. W. Purification to homogeneity and characterization of a Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test form of cytochrome P-450 with high specificity for benzo[a]pyrene from ␤-naph- (observation of species and tissues specificity). Carcinogenesis (Lond.), 12: 1403– thoflavone-pretreated rat liver microsomes. J. Biol. Chem., 256: 984Ð988, 1981. 1407, 1991. 30. Yasukochi, Y., Peterson, J. A., and Masters, B. NADPH-cytochrome c (P450) 4. NCI Carcinogenesis Bioassay of C. I. Solvent Yellow 14 in F344/N Rats and B6C3F1 reductase: spectrophotometric and stopped flow kinetic studies on the formation of Mice. Technical Report No. 226. Bethesda, MD: United States National Cancer reduced flavoprotein intermediates. J. Biol. Chem., 254: 7097Ð7104, 1979. Institute, 1982. 31. Strittmatter, P., Fleming, P., Connors, M., and Corcoran, D. Purification of cyto- 5. Moller, P., and Wallin, H. Genotoxic hazards of azo pigments and other colorants chrome b5. Methods Enzymol., 52: 97Ð101, 1978. related to 1-phenylazo-2-hydroxynaphthalene. Mutat. Res., 462: 13–30, 2000. 32. Polson, A., von Wechmar, M. B., and van Regenmortel M. H. Isolation of viral IgY 6. Kozuka, T., Tashiro, M., Sano, S., Fujimoto, K., Nakamura, Y., Hashimoto, S., and antibodies from yolks of immunized hens. Immmunol. Commun., 9: 475Ð493, 1980. Nakaminami, G. Pigmented contact dermatitis from azo dyes. I. Cross-sensitivity in 33. Polson, A., von Wechmar, M. B., and Fazakerley, G. Antibodies to proteins from yolk humans. Contact Dermatitis, 6: 330–336, 1980. of immunized hens. Immmunol. Commun., 9: 494Ð514, 1980. 7. Zeiger, E., Andersen, B., Haworth, S., Lawlor, T., and Mortelmans, K. Salmonella 34. Yamazaki, H., Shimada, T., Martin, M. V., and Guengerich, P. F. Stimulation of

mutagenicity tests. IV. Results from the testing of 300 chemicals. Environ. Mutagen., cytochrome P450 reactions by apo-cytochrome b5. Evidence against transfer of heme 12(Suppl. 11): 1–158, 1988. from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase. J. Biol. Chem., 8. Cameron, T. P., Hughes, T. J., Kirby, P. E., Fung, V. A., and Dunkel, V. C. Mutagenic 276: 30885Ð30891, 2001. activity of 27 dyes and related chemicals in the Salmonella/microsome and mouse 35. Cleland, W. W. Statistical analysis of the enzyme kinetic data. Methods Enzymol., ϩ Ϫ lymphoma TK / assays. Mutat. Res., 189: 223–261, 1987. 63: 103Ð138, 1983. 9. Childs, J. J., and Clayson, D. S. The metabolism of 1-phenylazo-2-naphthol in the 36. Stiborova«, M., Borek-Dohalska«, L., Hodek, P., Mra«z, J., and Frei, E. New selective rabbit. Biochem. Pharmacol., 15: 1247–1258, 1966. inhibitors of cytochromes P450 2B and their application to antimutagenesis of 10. Stiborova´, M., Asfaw, B., Anzenbacher, P., Lesˇeticky« L., and Hodek, P. The first tamoxifen. Arch. Biochem. Biophys., 403: 41Ð49, 2002. identification of the benzenediazonium ion formation from a non-aminoazo dye, 37. Reddy, M. V., and Randerath, K. Nuclease P1-mediated enhancement of sensitivity 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomes of rat livers. Cancer of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis (Lond.), Lett., 40: 319Ð326, 1988. 7: 1543Ð1551, 1986. 11. Stiborova«, M., Asfaw, B., Anzenbacher, P., and Hodek, P. A new way to carcino- 38. Murray, B. P., Edwards, R. J., Murray, S., Singleton, A. M., Davies, D. S., and genicity of azo dye: the benzenediazonium ion formed from non-aminoazo dye, Boobis, A. R. Human hepatic CYP1A1 and CYP1A2 content, determined with 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomal enzymes binds to specific anti- peptide antibodies, correlates with the mutagenic activation of PhIP. deoxyguanosine residues of DNA. Cancer Lett., 40: 327Ð333, 1988. Carcinogenesis (Lond.), 14: 585Ð592, 1993. 12. Stiborova«, M., Asfaw, B., Frei, E., Schmeiser, H. H., and Wiessler, M. Benzenedia- 39. Edwards, R. J., Adams, D. A., Watts, P. S., Davies, D. S., and Boobis, A. R. zonium ion derived from Sudan I forms an 8-(phenylazo)guanine adduct. Chem. Res. Development of a comprehensive panel of antibodies against the major xenobiotic Toxicol., 8: 489Ð498, 1995. metabolising forms of cytochrome P450 in humans. Biochem. Pharmacol., 56: 13. Stiborova«, M., Asfaw, B., and Anzenbacher, P. Activation of carcinogens by perox- 377Ð387, 1998. idase. Horseradish peroxidase-mediated formation of benzenediazonium ion from a 40. Brockmoller, J., Cascorbi, I., Henning, S., Meisel, C., and Roots, I. Molecular non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) and its binding to genetics of cancer susceptibility. Pharmacology (Basel), 61: 212Ð227, 2000. DNA. FEBS Lett., 232: 387Ð390, 1988. 41. Guengerich, F. P. Human cytochrome P450 enzymes. In: P. R. Ortiz de Montellano, 14. Stiborova«, M., Frei, E., Schmeiser, H. H., Wiessler, M., and Hradec, J. Mechanism of (ed.). Cytochrome P450, pp. 473Ð535. New York: Plenum Press, 1995. formation and 32P-postlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I). 42. Drahushuk, A. T., McGarrigle, B. P., Larsen, K. E., Stegeman, J. J., and Olson, J. R. Carcinogenesis (Lond.), 11: 1843Ð1848, 1990. Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut 15. Stiborova«, M., Schmeiser, H. H., and Frei, E. Prostaglandin H synthase mediated liver slices incubated in dynamic organ culture. Carcinogenesis (Lond.), 19: 1361Ð oxidation and binding to DNA of a detoxication metabolite of carcinogenic Sudan I 1368, 1998. 1-(phenylazo) 2, 6-dihydroxynaphthalene. Cancer Lett., 142: 53Ð60, 1999. 43. Sy, S. K. B., Tang, B. K., Pastrakuljic, A., Roberts, E. A., and Kalow, W. Detailed 16. Guengerich, F. P. Comparisons of catalytic selectivity of cytochrome P450 subfamily characterization of experimentally derived human hepatic CYP1A1 activity and members from different species. Chem. Biol. Interact., 106: 161Ð182, 1997. expression using differential inhibition of ethoxyresorufin O-deethylation by fluvox- 17. Olah, G. A., Wu, A., Farooq, O., and Prakash, G. K. S. Olefins from crowded amine. Eur. J. Clin. Pharmacol., 57: 377Ð386, 2001. carbonyl compounds with tert-butyllithium (tert-butylmagnesium chloride)/thionyl 44. Shimada, T., Yun, C-H., Yamazaki, H., Gautier, J-C., Beaume, P. H., and chloride. Study of carbocationic reaction intermediates and rearrangement-cleavage Guengerich, F. P. Characterization of human lung microsomal cytochrome P-450 1A1 under stable ion conditions using 13C NMR spectroscopy. J. Org. Chem., 55: and its role in he oxidation f chemical carcinogens. Mol. Pharmacol., 41: 856Ð864, 1792Ð1796, 1990. 1992. 18. Stiborova«, M., Frei, E., Sopko, B., Wiessler, M., and Schmeiser, H. H. Carcinogenic 45. Wheeler, C. W., Park, S. S., and Guenthner, T. M. Immunochemical analysis of a aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of cytochrome P-450IA1 homologue in human lung microsomes. Mol. Pharmacol., 38: patients with Chinese herbs nephropathy. Carcinogenesis (Lond.), 23: 617Ð625, 634Ð643, 1990. 2002. 46. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. Human P1-450 gene sequence and 19. Gut, I. Nedelcheva, V., Soucek, P., Stopka, P., Vodicka, P., Gelboin, H. V., and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism. Nucleic Ingelman-Sundberg M. The role of CYP2E1 and 2B1 in metabolic activation of Acids Res., 12: 4503Ð4520, 1985. benzene derivatives. Arch. Toxicol., 71: 45Ð56, 1996. 47. Lubet, R. A., Connolly, G., Kouri, R. E., Nebert, D. W., and Bigelow, S. W. 20. Anzenbacher, P., Soucek, P., Anzenbacherova«, E., Gut, I., Hruby«, K., Svoboda, Z., Biological effects of the Sudan dyes. Role of the Ah cytosolic receptor. Biochem. and Kvetina, J. Presence and activity of cytochrome P450 isoforms in minipig liver Pharmacol., 32: 3053Ð3058, 1983.

5684

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2002 American Association for Cancer Research. Sudan I Is a Potential Carcinogen for Humans: Evidence for Its Metabolic Activation and Detoxication by Human Recombinant Cytochrome P450 1A1 and Liver Microsomes

Marie Stiborová, Václav Martínek, Helena Rýdlová, et al.

Cancer Res 2002;62:5678-5684.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/62/20/5678

Cited articles This article cites 42 articles, 9 of which you can access for free at: http://cancerres.aacrjournals.org/content/62/20/5678.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/62/20/5678.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/62/20/5678. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.