Induced Expression of Cytochrome P450 1A and NAD(P) - American

Article pubs.acs.org/crt

Induced Expression of Cytochrome P450 1A and NAD(P)H:Quinone Oxidoreductase Determined at mRNA, Protein, and Enzyme Activity Levels in Rats Exposed to the Carcinogenic Azo Dye 1‑Phenylazo-2naphthol (Sudan I) Marie Stiborová,*,† Helena Dračínská,† Václav Martínek,† Dagmar Svásǩ ová,† Petr Hodek,† Jan Milichovský,† Ž aneta Hejduková,† Jaroslav Brotánek,‡ Heinz H. Schmeiser,§ and Eva Frei⊥ †

Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic Second Department of Internal Medicine, Thomayer University Hospital, Prague 4, 140 00, Czech Republic § Research Group Genetic Alteration in Carcinogenesis, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany ⊥ Division of Preventive Oncology, National Center for Tumour Diseases, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany ‡

ABSTRACT: Sudan I (1-phenylazo-2-hydroxynaphthol) is a suspected human carcinogen causing tumors in the livers and urinary bladders of rats, mice, and rabbits. Here, we investigated for the first time the influence of Sudan I exposure on the expression of several biotransformation enzymes in the livers, kidneys, and lungs of rats concomitantly at the mRNA and protein levels and assayed their enzymatic activities. We also studied its effect on the formation of Sudan I-derived DNA adducts in vitro. Sudan I increased the total amounts of cytochrome P450 (P450) in all organs tested. Western blots using antibodies raised against various P450s, NADPH:P450 reductase, and NAD(P)H:quinone oxidoreductase 1 (NQO1) showed that the expression of P450 1A1 and NQO1 was induced in the liver, kidney, and lung of rats treated with Sudan I. The higher protein levels correlated with increased enzyme activities of P450 1A1/2 and NQO1. Furthermore, 9.9-, 5.9-, and 2.8-fold increases in the formation of Sudan I oxidative metabolites catalyzed by microsomes isolated from the liver, kidney, and lung, respectively, of rats treated with Sudan I were found. The relative amounts of P450 1A and NQO1 mRNA, measured by real-time polymerase chain reaction (RT-PCR) analysis, demonstrated that Sudan I induced the expression of P450 1A1 and NQO1 mRNA in the liver, kidney, and lung, and of P450 1A2 mRNA in kidney and lung. Finally, microsomes isolated from livers, kidneys, and lungs of Sudan I exposed rats more effectively catalyzed the formation of Sudan I-DNA adducts than microsomes from organs of control rats. This was attributable to the higher P450 1A1 expression. Because P450 1A1 is playing a major role in the bioactivation of Sudan I in rat and human systems, its induction by Sudan I may have a profound effect on cancer risk by this azo dye. In addition, the induction of P450 1A1/2 and NQO1 enzymes can influence individual human susceptibility to other environmental carcinogens and have an effect on cancer risk.

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Europe.10−14 Analysis of a few market samples of turmeric, chili, and curry powders showed the presence of Sudan I (4.8− 12.1 mg/g) and related dyes.15 In addition, there is evidence that ingestion of food products contaminated with Sudan I could lead to exposure in the human gastrointestinal tract to metabolites generated by the intestinal bacteria.16,17 Therefore, the use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries.18 According to the Commission Decisions 2005/402/EC,19 all

INTRODUCTION

Sudan I [1-(phenylazo)-2-hydroxynaphthalene, C.I. Solvent Yellow 14, CAS No: 842-07-9] has been used as a food coloring but has been found to be carcinogenic causing tumors in the liver or urinary bladder of rats, mice, and rabbits, and is considered a possible weak human carcinogen.1−7 Besides its carcinogenicity, Sudan I is a potent contact allergen and sensitizer, eliciting pigmented contact dermatitis in humans.8,9 Nevertheless, it is used to color materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe and floor polishes, and gasoline.1,5 Recently, increased attention has been paid to this dye because it was found as a contaminant of several chili powder-containing foodstuffs imported to © 2013 American Chemical Society

Received: November 10, 2012 Published: January 4, 2013 290

dx.doi.org/10.1021/tx3004533 | Chem. Res. Toxicol. 2013, 26, 290−299

Chemical Research in Toxicology

Article

Chemicals. Sudan I [1-(phenylazo)-2-hydroxynaphthalene], NADPH, α-naphthoflavone (α-NF), furafylline, ellipticine, hypoxanthine, menadione (2-methyl-1,4-naphthoquinone), and calf thymus DNA were from Sigma Chemical Co. (St. Louis, MO, USA) and 7ethoxyresorufin from Fluka Chemie AG (Buchs, Switzerland). Enzymes and chemicals for the 32P-postlabeling assay were obtained from the sources described.45 All these and other chemicals were of reagent grade or better. Animal Experiments. The study was conducted in accordance with the Regulations for the Care and Use of Laboratory Animals (311/1997, Ministry of Agriculture, Czech Republic), which is in compliance with the Declaration of Helsinki. Three male Wistar rats (100−125 g) were injected i.p. with Sudan I in maize oil (20 mg/kg body weight, bw) once a day for 3 consecutive days. Three control rats received the same volume of maize oil on three days. Animals were euthanized 1 day after the last treatment by cervical dislocation. Livers, kidneys, and lungs were removed, immediately after sacrifice, frozen in liquid nitrogen, and stored at −80 °C until isolation of microsomal and cytosolic fractions. Preparation of Microsomal and Cytosolic Fractions. Microsomal and cytosolic fractions were isolated from the livers, lungs, and kidneys of rats, as described.46−48 Both subcellular preparations were analyzed for the presence of Sudan I and its metabolites by HPLC on a MN Nucleosil 100−5 C18 column (Macherey-Nagel; 4.0 × 250 mm). An isocratic flow of methanol/0.1 M NH4HCO3 (pH 8.5; 9:1, v/v) with flow rate of 0.8 mL/min was used to elute the metabolites, and detection was at 254, 333, and 480 nm. No Sudan I and its metabolites were detectable in hepatic, renal, and lung microsomal and cytosolic fractions from rats that had been treated with this compound. Total P450 in microsomes was determined by spectroscopy, following the method of Omura and Sato.49 Microsomal and Cytosolic Incubations Determining Sudan I Metabolism. Incubation mixtures, in which Sudan I oxidation was investigated, had a final volume of 750 μL and consisted of 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH, 10 mM Dglucose 6-phosphate, 1 unit/mL D-glucose 6-phosphate dehydrogenase, 10 mM MgCl2, hepatic, pulmonary, and renal microsomal samples from 3 rats (pooled samples), either control or treated with Sudan I (0.5 mg of microsomal protein), and 100 μM Sudan I dissolved in 7.5 μL of methanol. After incubation (37 °C, 20 min; Sudan I oxidation by microsomes was found to be linear up to 30 min),25,28 Sudan I metabolites were extracted into ethyl acetate, the extracts evaporated, the residues dissolved in methanol, and the products separated by HPLC as described above. The Sudan I metabolites were identified by cochromatography with authentic standards. The incubations, in which Sudan I reduction by hepatic microsomes was analyzed, were carried out under the hypoxic conditions in closed tubes. The incubation mixtures contained in a final volume of 500 μL of 50 mM potassium phosphate buffer (pH 7.4), the cofactor of P450 catalysis (1 mM NADPH), or the cofactor of NADH:cytochrome b5 reductase (1 mM NADH), pooled liver microsomal samples (1 mg of microsomal protein), and 100 μM Sudan I dissolved in 5 μL of methanol. The reaction was initiated by adding Sudan I. Each incubation mixture was purged with a stream of argon for 3 min before the addition of Sudan I and also 3 min after this addition. Although most of the oxygen was removed, we cannot exclude the negligible presence of O2 in the microsomal membranes and lumen. For the reductive metabolism of Sudan I by cytosolic NQO1 or xanthine oxidase, the incubations were as described above with only 1 mM NADH as the cofactor47 and 1 mg of cytosolic protein instead of microsomes. After the incubation of mixtures containing either microsomes or cytosols (120 min at 37 °C in closed tubes) and extraction with ethyl acetate (2 × 1 mL), the extracts were evaporated and dissolved in methanol, and the products were separated by HPLC on a CC 250/4 Nucleosil 100-5 C18 HD (Macherey-Nagel; 4.0 × 250 mm) preceded by a guard column. The column was eluted with a gradient: solvent A, 100 mM NH4HCO3, pH 8; solvent B, methanol 0−10 min 50% B, 10−16 min 50−90% B, 16−31 min 90% B, 31−36 min 90−50% B, and 36−40 min 50% B at a flow rate of 0.8 mL/min, detection at 252, 294, 315, and 475 nm. Aniline, a possible product of

chili, chili products, turmeric, and palm oil imported to the EU must be certified to be free of Sudan I, II, III, and IV. Sudan I is mutagenic to bacteria, to mammalian cells, and is a clastogenic compound.5,20 There is also evidence that this compound exhibits genotoxic effects, after its metabolic activation by hepatic P450 and peroxidase enzymes in vitro, in the rat liver and urinary bladder in vivo,5,21−31 in a human hepatoma cell line HepG2,6,7 and in human AHH-1 and MCL5 cell lines.32 C-Hydroxylated metabolites 1-(4-hydroxyphenylazo)-2-naphthol (4′-OH-Sudan I) and 1-(phenylazo)-naphthalene-2,6-diol (6-OH-Sudan I) are major products of Sudan I oxidation in vivo, and are excreted in urine,1,33 and also of its oxidation by rat and human hepatic microsomes in vitro.21,28,34 Besides the C-hydroxylated metabolites, which are considered detoxication products, the benzenediazonium ion (BDI), formed by microsome-catalyzed enzymatic splitting of the azo group of Sudan I, was found to react with DNA in vitro.21,22,25,28 The major DNA adduct formed in this reaction has been characterized and identified as the 8-(phenylazo)guanine adduct.25,28 This adduct was also found in liver DNA of rats exposed to Sudan I.29 Human P450 enzymes metabolize Sudan I to the same C-hydroxylated metabolites and DNA adducts.28,34 The P450 1A1 enzyme is the major enzyme oxidizing Sudan I in human tissues rich in this enzyme, while P450 3A4 is also active in the human liver.28,34,35 Interestingly, other P450s of the 1 family, P450 1A2 and 1B1, are more than 10-fold less effective in oxidizing Sudan I than P450 1A1.28,29 There is also evidence that even though 4′-OH-Sudan I and 6OH-Sudan I are considered detoxication metabolites, they also exhibit genotoxic effects because they form DNA adducts after their activation by peroxidases.24,26 Several studies demonstrated that Sudan I can induce P450 1A enzymes and their activities in mouse and rat livers,36−38 and also NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma (Hepa 1c1c7) cells,39,40 acting probably as a ligand of the aryl hydrocarbon receptor (AHR).36 Since P450 1A1 is the major human enzyme metabolizing Sudan I, mechanistic studies on its induction by Sudan I are needed to evaluate how chronic exposure might increase its genotoxic potential in humans. As Sudan I was also found to be reductively metabolized to aniline that may also have, to some extent, a genotoxic effect,16−18,41−44 the question whether NQO1 might participate in Sudan I reduction needs to be answered. Therefore, the objective of the present study was to evaluate whether the major enzyme oxidizing Sudan I (microsomal P450 1A1), and cytosolic NQO1, are induced by this azo dye. The expression levels of these enzymes were analyzed concomitantly at mRNA, protein, and enzyme activity levels to obtain a complete picture of the biological potency of Sudan I exposure in the three rat tissues: liver, lung, and kidneys. If indeed Sudan I increases expression levels of activating enzymes, we analyzed if this induction also leads to higher levels of its metabolites and DNA adducts when microsomes and cytosols from these organs are incubated with Sudan I.

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EXPERIMENTAL PROCEDURES

Caution: Sudan I is a potent mutagen and should be handled with care. Exposure to 32P should be avoided by working in a conf ined laboratory area, with protective clothing, plexiglass shielding, Geiger counters, and body dosimeters. Wastes must be discarded according to appropriate safety procedures. 291



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Chemical Co. (St. Louis, MO, USA). Proteins were analyzed by Western blot technique, exactly as described.55 P450 1A1/2 and NQO1 Enzyme Activity Assays. The microsomal samples were characterized for P450 1A1/2 activity using 7-ethoxyresorufin O-deethylation (EROD) activity.28,34,56 The cytosolic samples were characterized for NQO1 activity using menadione (2-methyl-1,4-naphthoquinone) as a substrate.47,55 P450 1A1, 1A2, and NQO1 mRNA Content in Rat Livers, Lungs, and Kidneys. Total RNA was isolated from frozen livers, lungs, and kidneys of three untreated rats and three rats pretreated with Sudan I and mRNA quantified by RT-PCR exactly as described.55

Sudan I reduction, eluted with a retention time of 4.8 min. Retention time of Sudan I was 25.6 min. Sudan I-DNA adduct formation was followed after the activation of Sudan I with hepatic, renal, or pulmonary microsomes (1 mg protein), or human recombinant P450 1A1 in Supersomes (150 pmol per incubation, Gentest Gentest corp., Woburn, MA, USA) under either oxidative conditions or anaerobic conditions when hepatic cytosols (1 mg protein) in the presence of 1 mM NADPH, NADH, or hypoxanthine were used as the activation system. Incubations were as described above, but also contained 0.5 mg of calf thymus DNA (2 mM dNp). After the incubation (60 min; the microsome-mediated Sudan I-derived DNA adduct formation was found to be linear up to 90 min)25,28 and extraction with ethyl acetate, DNA was isolated from the residual water phase by the phenol/chloroform extraction method as described.25,28 32 P-Postlabeling Analysis of Nucleotide Adducts Formed by Sudan I in DNA Activated with Rat Hepatic, Renal, and Pulmonary Microsomes and Cytosols. DNA adducts formed by the oxidative activation of Sudan I were quantified with the nuclease P1 version of the 32P-postlabeling assay50 as described.25,28 Adducts were separated on PEI cellulose plates with the solvent system (ii) (method B) described below. To analyze Sudan I-derived DNA adducts formed by reduction in cytosolic samples, the standard procedure of the 32P-postlabeling assay was used.51 In addition, this procedure under ATP-deficient conditions52 and the nuclease P1 enrichment version50 were also used. Labeled DNA digests were separated by two chromatographic methods on polyethylenimine (PEI)-cellulose plates. (i) Essentially as described,51 except that the D3 solvent was 3.5 M lithium formate, 8.5 M urea (pH 3.5); the D4 solvent was 0.8 M lithium chloride, 0.5 M Tris, and 8.5 M urea (pH 8.0), followed by a final wash (D5) with 1.7 M sodium phosphate (pH 6.0). D2 was omitted (method A). (ii) 32Plabeled adducts were also resolved by the modification described by Reddy et al.53 This procedure has been shown to be suitable for the resolution of DNA adducts formed by Sudan I.25,28,29 The solvents used in this case were: D1, 2.3 M sodium phosphate (pH 5.77); D2 was omitted; D3, 2.7 M lithium formate and 5.1 M urea (pH 3.5); D4, 0.36 M sodium phosphate, 0.23 M Tris-HCl, and 3.8 M urea (pH 8.0). After D4 development and brief water wash, the sheets were developed (along D4) in 1.7 M sodium phosphate (pH 6.0) (D5) to the top of the plate, followed by an additional 30−40 min development with the TLC tank partially opened to allow the radioactive impurities to concentrate in a band close to the top edge (method B).25,53 Adducts and normal nucleotides were detected and quantified by storage phosphor imaging on a Packard Instant Imager. Adduct levels were calculated in units of relative adduct labeling (RAL), which is the ratio of radioactivity counts of adducted nucleotides to those of total nucleotides in the assay. Isolation of P450s and NADPH:P450 Reductase. Recombinant rat P450 1A1 protein was purified to homogeneity from membranes of Escherichia coli transfected with a modified P450 1A1 cDNA,28 in the laboratory of H. W. Strobel (University of Texas, Medical School of Houston, Texas, USA) by P. Hodek (Charles University, Prague, Czech Republic). P450 2B4 and 2E1 enzymes were isolated from liver microsomes of rabbits induced with phenobarbital (P450 2B4) or ethanol (P450 2E1) by the procedures described.28 Human recombinant P450 3A4 was a gift of P. Anzenbacher (Palacky University, Olomouc, Czech Republic). Rabbit liver NADPH:P450 reductase was purified as described.54 Preparation of Antibodies and Estimation of P450 1A1, 1A2, 2B, 2E1, and 3A, NADPH:P450 reductase, and NQO1 Protein Content in the Microsomes and Cytosols of the Rat Liver, Lung, and Kidney. Leghorn chickens were immunized subcutaneously three times a week with antigens (rat recombinant P450 1A1, rabbit P450 2B4, rabbit P450 2E1, human recombinant P450 3A4, and rabbit NADPH:P450 reductase) (0.1 mg/animal) emulsified in complete Freund’s adjuvant for the first injection and in incomplete adjuvant for boosters. The immunoglobulin fraction was purified from pooled egg yolks using fractionation by polyethylene glycol 6000.28,34 Rabbit polyclonal antibodies against human NQO1 were from Sigma

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RESULTS Effect of Sudan I on the Expression of P450 Enzymes and NQO1 in Rat Liver, Kidney, and Lung. Sudan I treatment of rats (3 days, 20 mg/kg and day) led to significantly increased cytochrome P450 levels in the liver, kidney, and lung as shown in Table 1. The individual P450 (P450 1A1, 2B, 2E1, Table 1. Total Contents of P450 in the Liver, Kidney, and Lung Microsomes of Sudan I-Treated and Control Ratsa P450 content (nmol/mgPROTEIN) tissue

control rats

Sudan I-treated rats

fold increase over control

liver kidney lung

0.32 ± 0.07 0.17 ± 0.02 0.03 ± 0.01

1.09 ± 0.12 0.30 ± 0.06 0.1 ± 0.06

3.4** 1.8** 3.3**

The results shown are the mean ± S.D. from three rats. **, p < 0.01, values significantly different from controls (Student’s t test). a

and 3A4) and NADPH:P450 reductase were characterized by Western blots. Only the expression of P450 1A1 was stimulated in the liver, kidney, and lung by Sudan I. The expression of P450 2B, 3A, and NADPH:P450 reductase was essentially not altered by the treatment in any organ (data not shown), whereas the relative amounts of hepatic P450 2E1 decreased significantly: 2.1-fold (Figure 1). Chicken antibodies raised against the rat P450 1A1 used in this work recognized both P450 1A1 and 1A2 enzymes (see Figure 1 for hepatic P450 1A1 and 1A2) because they are highly homologous.57 The efficiency of Sudan I to induce expression of P450 1A1 protein in the liver was higher than that of P450 1A2 in this organ; 80- and 4.8-fold increases in P450 1A1 and 1A2 protein expression, respectively, were induced in the liver of rats treated with Sudan I (Figure 2). P450 1A2 was not found to be expressed in the rat kidney and lung, both in the control (untreated) rats and in rats treated with Sudan I. The induction of P450 1A1 protein expression was higher in the lung than in the kidney (11-fold versus 4.6-fold). EROD activity, which is a marker of P450 1A1 and 1A2,56,57 was induced by Sudan I in all tissues tested (Figure 3). In the liver, the increase in EROD activity was much lower than in the increase in P450 1A1 protein expression (a 7.8-fold increase in EROD activity versus an 80-fold increased protein expression; compare Figures 2 and 3). The difference between protein expression levels and EROD activity was even more conspicuous in the lung, where only a 1.6-fold increase in EROD activity was caused by Sudan I exposure. The levels of NQO1 protein were enhanced in the liver, kidney, and lung after treatment of rats with Sudan I, by 3.4-, 9.1-, and 4-fold, respectively (Figure 4). The increased expression of NQO1 was paralleled by an increased enzyme activity (Table 2). NQO1 activity was found in cytosolic fractions of all tissues tested in this study, but it was more than 292



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Figure 1. Immunoblots of hepatic microsomal P450 1A1, 1A2, and 2E1 of rats treated with Sudan I and control rats.

Figure 2. Influence of the treatment of rats with Sudan I on the expression of P450 1A1 and 1A2 protein in the liver, kidney, and lung. The values are the averages ± SEM of three parallel experiments. F, fold increase over controls, **p < 0.01, ***p < 0.001 (Student’s t test); n.d.: not detectable.

Figure 4. Expression of NQO1 protein in rats treated with Sudan I in comparison to control rats. The values are the averages ± SEM of three parallel experiments. F, fold increase over controls, *p < 0.05, **p < 0.01 (Student’s t test). The Y axis shows % band intensities of cytosols from Sudan I-treated rats relative to intensities in cytosols from control rats for each organ. Inset: immunoblots of NQO1 from untreated and Sudan I-treated rats.

Figure 3. O-Deethylation of 7-ethoxyresorufin, a marker substrate for P450 1A1/2, in hepatic, renal, and pulmonary microsomes from rats treated with Sudan I and from control rats. The values are the averages ± SEM of three parallel experiments. F, fold increase over controls, *p < 0.05, **p < 0.01 (Student’s t test).

5-times lower in kidneys than in livers (Table 2), similar to the band intensities in the Western blots (Figure 4). Changes in the mRNA levels of P450 1A1, 1A2, and NQO1 were determined by real-time PCR (RT-PCR) analysis. As shown in Table 3, treatment of rats with Sudan I markedly induced mRNA levels of P450 1A1, mainly in the liver and

lung. Although no expression of P450 1A2 protein was measurable in rat kidneys and lungs, mRNA of this enzyme was detectable in these tissues (Table 3), and expression of P450 1A2 mRNA was induced by Sudan I in the kidney and lung but not in the liver (Table 3). In the case of NQO1 293



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metabolite, 1-(3,4-dihydroxyphenylazo)-2-naphthol (3′,4′diOH-Sudan I), is also generated but only by hepatic microsomes of rats treated with Sudan I (Figure 5). A significant increase in the oxidation of Sudan I was found by microsomes isolated from rats treated with Sudan I. As shown in Figure 6, the formation of total Sudan I metabolites was

Table 2. Activity of NAD(P)H:Quinone Oxidoreductase 1 in Hepatic, Renal, and Pulmonary Cytosolic Fractions of Rats Treated with Sudan I and in Control Ratsa NQO1 activity (μmol/ min.mgPROTEIN) tissue

control rats



liver kidney lung

0.65 ± 0.01 0.12 ± 0.01 0.58 ± 0.01

3.03 ± 0.15 0.65 ± 0.01 2.53 ± 0.05

4.6** 5.4*** 4.4***

a The values shown are the averages ± S.D. of three parallel experiments. **, p < 0.01. ***, p < 0.001 values significantly different from controls (Student’s t test).

Table 3. Expression of mRNA of P450 1A1, P450 1A2, and NQO1 ΔcTa mRNA P450 1A1

P450 1A2

NQO1

tissue

control rats



liver

6.37 ± 0.07

0.58 ± 0.35

55.3**

kidney lung liver

4.45 ± 0.16 10.48 ± 0.16 −5.15 ± 0.06

4.23 ± 0.74 5.12 ± 0.31 −5.03 ± 0.21

1.2** 41.1** 0.9

kidney lung liver kidney lung

14.07 ± 0.67 13.97 ± 1.30 4.77 ± 0.49 9.38 ± 0.82 5.44 ± 0.41

9.38 ± 3.43 11.49 ± 1.33 3.97 ± 0.49 5.55 ± 0.34 3.45 ± 0.60

25.8** 5.6** 1.7** 14.1** 4.0**

Figure 6. Sum of hydroxylated Sudan I metabolites formed by hepatic, renal, and pulmonary microsomes from either control rats or rats treated with Sudan I. The values are the averages ± SEM of three parallel experiments. F, fold increase over controls, ***p < 0.001 (Student’s t test).

ΔcT values are shown relative to actin mRNA (see ref 55). The results shown are the means ± S.D. from three rats (control and treated with Sudan I). **, p < 0.01, values significantly different from controls (Student’s t test). a

increased by microsomes from the liver, kidney, and lung isolated from Sudan I-pretreated rats, which agreed well with an increase in EROD activity in these microsomes (compare Figure 3). In order to investigate the effect of the pretreatment of rats with Sudan I on the activation of Sudan I by the liver, kidney, and lung microsomes to form DNA adducts, DNA was incubated with Sudan I and microsomes, and analyzed for adducts by 32P-postlabeling. Using the nuclease P1 version of this assay,25,28,29 one major DNA adduct (the closed circle in Figure 7C) and two minor adduct spots (overlapping one another) are formed (see Figure 7A). The major adduct spot was chromatographically indistinguishable from the 3′,5′bisphospho-derivative of an 8-(phenylazo)deoxyguanosine adduct identified previously.25,28 Microsomes isolated from the liver, kidney, and lung from Sudan I-treated rats more efficiently formed Sudan I-derived DNA adducts (Figure 8).

mRNA, treatment of rats with Sudan I induced 1.7-, 14.1-, and 4-fold increases in levels of mRNA of this enzyme in the liver, kidney, and lung, respectively (Table 3). Exposure of Rats with Sudan I Enhances Microsomal Sudan I Oxidation and Its Activation to Species Forming DNA Adducts. Rat hepatic, renal, and pulmonary microsomes oxidize Sudan I to its C-hydroxylated metabolites 4′-OH-Sudan I, 6-OH-Sudan I, and 1-(4-hydroxyphenylazo)-naphthalene-2,6diol (4′,6-diOH-Sudan I) (see Figure 5 for hepatic microsomes of rats treated with Sudan I) that are the same as the metabolites generated by human P450 1A1.28,34 A low, but detectable, amount of another dihydroxylated Sudan I

Figure 5. HPLC of metabolites formed from Sudan I by hepatic microsomes of rats treated with Sudan I. 294



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Figure 7. DNA adducts formed by the incubation of DNA with Sudan I, NADPH, and hepatic microsomes of rats treated with Sudan I (A) or those with Sudan I, NADPH, and human recombinant P450 1A1 (B). (C) Schematic figure of adducts with assigned numbers and the structure of adduct 1 (closed circle). Analysis was performed by the nuclease P1 version of the 32P-postlabeling assay and the chromatographic procedure (i). Origins are located in the bottom left corners.

Table 4. Effect of Inhibitors on DNA Adduct Formation by Sudan I Activated by the Liver, Kidney, and Lung Microsomes of Either Control Rats or Rats Treated with Sudan Ia Sudan I-derived DNA adducts RALb/108 nucleotides tissue

inhibitor

control rats


liver

none α-NF ellipticine furafylline none α-NF ellipticine furafylline none α-NF ellipticine furafylline

10.2 ± 0.8 5.1 ± 0.5 (50%)* 1.8 ± 0.2 (17.6%)* 10.3 ± 1.2 (100.1%) 2.0 ± 0.2 1.1 ± 0.1 (55%)* n.d. 2.0 ± 0.2 (100%) 1.2 ± 0.1 0.7 ± 0.1 (58.3%)* n.d. 1.3 ± 0.1 (108.3%)

98.9 ± 5.3 25.0 ± 3.5 (25.3%)* 9.2 ± 1.3 (9.3%)* 99 ± 6.2 (100.1%) 12.2 ± 1.8 1.2 ± 0.1 (9.8%)* 1.0 ± 1.3 (8.2%)* 12.1 ± 1.2 (99.2%) 4.2 ± 0.4 0.4 ± 0.1 (9.5%)* n.d. 4.2 ± 0.4 (100%)

kidney

lung

Figure 8. DNA adduct formation by Sudan I activated by the liver, kidney, and lung microsomes of either control rats or rats treated with Sudan I in the presence of NADPH. Adducts in incubations without NADPH were not detectable (the detection limit of RAL was 1/1010 nucleotides). RAL, relative adduct labeling; F, fold increase over controls, **p < 0.01, ***p < 0.001 (Student’s t test).

*, p < 0.001, values significantly different from incubations without inhibitor (Student t-test). bMeans of RAL (relative adduct labeling) ± S.D. of triplicate in vitro incubations; the percentage of (activity with inhibitors relative to that without) is shown in parentheses. n.d.: not detectable (the detection limit of RAL was 1/1010 nucleotides). a

The induction levels exactly paralleled the levels of Sudan I metabolites formed in these microsomes (compare Figures 6 and 8), an activity which is attributable to P450 1A1. To further narrow down the role of P450 1A1 in Sudan I-DNA adduct formation, the effect of inhibitors was analyzed (Table 4). αNF, an inhibitor of P450 1A1/2 with the predominant inhibitory effect on P450 1A1, and ellipticine that inhibits P450 1A146 very effectively decreased the levels of Sudan Iderived-DNA adducts formed by microsomes from all organs (Table 4). On the contrary, furafylline, an inhibitor of P450 1A2,57 was ineffective. The ability of rat P450 1A1 to activate Sudan I to DNA adducts was confirmed by experiments, in which human recombinant P450 1A1 in Supersomes were used, where the same pattern of adducts was detected by 32Ppostlabeling as that found in microsomes (Figure 7B). Effect of Sudan I Pretreatment on Reduction of Sudan by Rat Hepatic Microsomes and Cytosols. Several studies indicated that Sudan I might also be reductively metabolized to produce aniline that might have a genotoxic effect.16,17,41,42,58

This anaerobic Sudan I metabolism was found to be catalyzed by bacteria of the human gastrointestinal tract16 and, to some extent, also by skin cytosolic and microsomal preparations from mice, hairless guinea pigs, and humans.59 Microsomal and cytosolic fractions contain several types of reductases (microsomal NADPH:P450 reductase, P450s, NADH:cytochrome b5 reductase, and cytosolic NQO1 or xanthine oxidase) that reductively activate several carcinogens.47,60−62 Here, we found that Sudan I is not reduced by hepatic microsomes and cytosols of both rats treated with Sudan I and control (untreated) rats; neither aniline nor 1-amino-2-naphthol, another reductive metabolite, was produced in detectable amounts by microsomes in the presence of NADPH (a cofactor of NADPH:P450 reductase in the P450-dependent system), NADH (a cofactor of NADH:cytochrome b5 reductase), or cytosols in the presence of cofactors of NQO1 or xanthine oxidase (NADPH and NADH). 295



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corresponds to findings that P450 1A2 is a constitutive enzyme in the liver, with low, if any, expression in extrahepatic tissues.57,63 The P450 1A1 and 1A2 protein expression is regulated by an aryl hydrocarbon receptor (AHR), which mediates P450 1A1 and P450 1A2 gene transcription through the xenobiotic response element (XRE), involving ligandactivated AHR.64−66 Sudan I was found to be a potent agonist for the AHR.36,38 Another P450 enzyme that is controlled by AHR, P450 1B1, has a low potency to oxidize Sudan I.28,29 Therefore, influencing of its expression by Sudan I was not investigated in this work. Nevertheless, such a study might answer the question as to whether Sudan I is also an inducer of other enzymes controlled by AHR. The induction of P450 1A1 protein expression and enzymatic activities by Sudan I corresponded to elevated mRNA levels of this enzyme. Levels of P450 1A2 mRNA in the liver were, however, unaffected, despite the increase in protein. Interestingly, the highest P450 1A2 mRNA levels were observed in the lung and kidney, while protein expression was not seen in these rat organs. The finding of our study on the inducibility of P450 1A2 by Sudan I at mRNA and protein levels, which differ in the liver and extrahepatic organs, points to mechanisms other than those of P450 1A1 regulation. Similar discrepancies between the induction of P450 1A1/2 mRNAs and protein levels were observed previously.48,65,67−69 Detailed analyses of the time dependence of the expression levels of mRNAs and proteins of the tested enzymes were not performed in this study. However, they might answer the question as to whether the transient induction of the mRNAs of P450 1A1/2 or the different half-lives for their mRNAs and proteins, and/or the effects of Sudan I on the stability of mRNAs and proteins of these enzymes are the rationale for our observation. The highest induction of NQO1 expression was found in the kidney, followed by the lung and liver, both at the transcriptional and translational levels. This finding suggests that induction of this enzyme is mediated by a different mechanism than that postulated for P450 1A1. NQO1 expression has been shown to be regulated by two distinct regulatory elements in the 5′ flanking region of the NQO1 gene, the antioxidant response element (ARE) and the XRE, involving ligand-activated AHR.70−77 ARE-mediated NQO1 gene expression is increased by a variety of antioxidants, tumor promoters, and reactive oxygen species (ROS).74 ROS have been shown to be generated in human hepatoma HepG2 cells exposed to Sudan I.6,7 ROS are also produced during the oxidation of the Sudan I metabolite, 6-OH-Sudan I, by peroxidases.26 Peroxidases are highly expressed in the kidney, where we observed the highest degree of NQO1 induction. Hence, an increased ROS formation by Sudan I might be one mechanism by which Sudan I induces NQO1. Interindividual variations in susceptibility to carcinogens, caused among others by variations in activities of drugmetabolizing enzymes in target tissues, appear to be important determinants of cancer risk.78,79 Expression levels and activities of P450 1A1/2 and NQO1 differ considerably among individuals because the enzymes are influenced by exogenous factors, including smoking, drugs, and environmental chemicals.57,67,80 The levels of enzymes can determine an individual’s risk because for both enzymes, genetic polymorphisms are known, also resulting in changed enzyme activities or substrate specificities. So far, two polymorphisms in the human NQO1 gene have been found in the general population, one of them

Effect of Sudan I Pretreatment on the Activation of Sudan I by Rat Hepatic Cytosols. Using the 32P-postlabeling technique, we also tested whether Sudan I is activated by liver cytosols to DNA binding species under anaerobic conditions. These incubation mixtures also contained the cofactors of cytosolic NQO1 (NADPH or NADH) or xanthine oxidase (NADH or hypoxanthine). For DNA-adduct analyses, three versions of the 32P-postlabeling method were applied: the standard procedure,51 the standard procedure under ATPdeficient conditions,52 and enrichment by nuclease P1,50 and two chromatographic methods (see methods A (i) and B (ii) in the Experimental Procedures section). No Sudan I-derived DNA adducts were detected by any of the 32P-postlabeling versions in hepatic cytosols, confirming the lack of metabolites formed under reducing conditions.

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DISCUSSION Sudan I induces the P450 1A1 enzyme that is responsible for oxidation metabolism of this carcinogen in rat liver, the target organ of Sudan I carcinogenicity, and in the lung and kidney. It also induces NQO1 in these organs and P450 1A2 in rat liver. This induction was found to lead to increases to various degrees in the activities of these enzymes. The rat was used as an experimental model on the basis that the same enzymes metabolize Sudan I in the livers of rats and humans.25,28,34 Therefore, the results should provide some indication of what might occur in tissues of humans exposed to this carcinogenic azo dye. The dose of Sudan I was high compared with the estimated daily intake of 30 μg/kg body weight with contaminated chili powder.2 However, the total dose of 60 mg/kg bw is in the range of the carcinogenic dose of 30 mg/kg bw. Induction of microsomal enzymes (P450s) led to higher levels of C-hydroxylated Sudan I metabolites and increased activation of this carcinogen to DNA adducts in ex vivo incubations of microsomes with DNA and Sudan I. The experiments with P450 1A1 inhibitors, in addition to the elevated levels of oxidized Sudan I metabolites, confirmed P450 1A1 to be responsible for the increased Sudan I activation to DNA adducts in Sudan I-exposed rats and in control rats in all organs tested. The NQO1 enzyme is also induced by Sudan I but is not associated with the reductive metabolism of Sudan I to aniline, nor to any other genotoxic metabolite forming DNA adducts detectable by 32P-postlabeling. Furthermore, no Sudan I reduction mediated by xanthine oxidase was found in cytosols. Hence, even though the partial contributions of cytosolic and microsomal reductions in the azoreduction of Sudan I was described previously in skin during percutaneous absorption,62 no reduction of Sudan I by rat microsomal or cytosolic enzymes was found under the conditions used in this work. Sudan I delivered i.p. is absorbed via the mesenteric veins and lymphatic systems, and passes through the liver. Thus, its concentration and effect in this tissue should be higher than that in the lung and kidney. Indeed, induction of P450 1A1 expression in the liver was more than 7- and 17-fold higher than that in lungs and kidneys, respectively. The difference in P450 1A1 induction among the organs is at the transcriptional level since the degree of the increase in P450 1A1 protein levels was paralleled by the expression of P450 1A1 mRNA. Sudan I also induces P450 1A2 protein in the liver, but no expression was found in the kidney and lung of rats. However, P450 1A2 mRNA was detectable, and even induced, in the kidneys and lung by Sudan I, whereas it was not in the liver. This 296



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being associated with an increased risk of urothelial tumors80 and leukemia in children.81 Both P450 1A1 and P450 1A2 genes are polymorphic,57,82,83 and several alleles have been found to be associated with increased risk for the development of cancers of, e.g., the lung, esophagus, breast, colon, or acute myeloid leukemia.82,84−86 Genetic polymorphisms in P450 1A1 could also be important determinants of genotoxicity and carcinogenicity of Sudan I. Furthermore, the genetic polymorphisms in P450 1A and NQO1, in addition to their induction by Sudan I, might affect the carcinogenic potency of other carcinogens (i.e., aromatic or heterocyclic amines, and nitro-aromatic compounds)47,55,87−89 in persons exposed to Sudan I. In conclusion, the results of the present study show that Sudan I induces P450 1A1 and NQO1 enzymes in the rat liver, lung, and kidney, and P450 1A2 in the liver. The mRNA, protein, and enzyme activity levels in control and Sudan Iexposed rats varied for the three enzymes and also in the liver versus extra-hepatic organs. These data are important to validate effects of other inducers and as a basis to elucidate the mechanistic aspects of enzyme induction. Microsomes from organs of exposed rats showed enhanced oxidative activation of Sudan I to species forming DNA adducts and its oxidation to C-hydroxylated metabolites. Because Sudan I is a potent genotoxin and a suspected human carcinogen, the results are important to determine the risk of Sudan I to human health. Furthermore, the induction of these biotransformation enzymes can influence an individual’s susceptibility to other carcinogens and have effects on cancer risk.

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(3) NCI (1982) Carcinogenesis Bioassay of C.I. Solvent Yellow 14 in F344/N Rats and B6C3F1 Mice, in Technical Report No. 226, US National Cancer Institute, Bethesda, MD. (4) Garner, R. C., Martin, C. N., and Clayson, D. B. (1984) Carcinogenic Aromatic Amines and Related Compounds, in Chemical Carcinogens (Searle, C., Ed.) 2nd ed., Vol. 1, CS Monograph 182, pp 175−302, American Chemical Society, Washington, DC. (5) Møller, P., and Wallin, H. (2000) Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene. Mutat. Res. 462, 13−30. (6) An, Y., Jiang, L., Cao, J., Geng, C., and Zhong, L. (2007) Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells. Mutat. Res. 627, 164−170. (7) Zhang, X., Jiang, L., Geng, C., Hu, C., Yoshimura, H., and Zhong, L. (2008) Inhibition of Sudan I genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol. Free Radical Res. 42, 189−195. (8) Kozuka, T., Tashiro, M., Sano, S., Fujimoto, K., Nakamura, Y., Hashimoto, S., and Nakaminami, G. (1980) Pigmented contact dermatitis from azo dyes. I. Cross-sensitivity in humans. Contact Dermatitis 6, 330−336. (9) Kato, S., Itagaki, H., Uchiyama, M., Kobayashi, T., and Fujiyama, Y. (1986) Role of a metabolite in allergenicity of Sudan I. Contact Dermatitis 15, 205−210. (10) Liu, Y., Song, Z., Dong, F., and Zhang, L. (2007) Flow injection chemiluminescence determination of Sudan I in hot chili sauce. J. Agric. Food Chem. 55, 614−617. (11) Wang, Y., Yang, H., Wang, B., and Deng, A. (2011) A sensitive and selective direct competitive enzyme-linked immunosorbent assay for fast detection of Sudan I in food samples. J. Sci. Food Agric. 91, 1836−1842. (12) Rebane, R., Leito, I., Yurchenko, S., and Herodes, K. (2010) A review of analytical techniques for determination of Sudan I-IV dyes in food matrixes. J. Chromatogr., A 1217, 2747−2757. (13) Oplatowska, M., Stevenson, P. J., Schulz, C., Hartig, L., and Elliott, C. T. (2011) Development of a simple gel permeation clean-up procedure coupled to a rapid disequilibrium enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I dye in spices and sauces. Anal. Bioanal. Chem. 401, 1411−1422. (14) Hu, X., Fan, Y., Zhang, Y., Dai, G., Cai, Q., Cao, Y., and Guo, C. (2012) Molecularly imprinted polymer coated solid-phase microextraction fiber prepared by surface reversible addition-fragmentation chain transfer polymerization for monitoring of Sudan dyes in chili tomato sauce and chili pepper samples. Anal. Chim. Acta 731, 40−48. (15) Dixit, S., Khanna, S. K., and Das, M. (2008) A simple 2directional high-performance thin-layer chromatographic method for the simultaneous determination of curcumin, metanil yellow, and Sudan dyes in turmeric, chili, and curry powders. J. AOAC Int. 91, 1387−1396. (16) Xu, H., Heinze, T. M., Paine, D. D., Cerniglia, C. E., and Chen, H. (2010) Sudan azo dyes and Para Red degradation by prevalent bacteria of the human gastrointestinal tract. Anaerobe 16, 114−119. (17) Pan, H., Feng, J., He, G. X., Cerniglia, C. E., and Chen, H. (2012) Evaluation of impact of exposure of Sudan azo dyes and their metabolites on human intestinal bacteria. Anaerobe 18, 445−453. (18) Federal Institute for Risk Assessment (2003) Dyes Sudan I to IV in Food, in Opinion of 10 November 2003, Federal Institute for Risk Assessment, Berlin, Germany. (19) EU (2005) Commission Decision of 23 May 2005 on emergency measures regarding chili, chili products, curcuma and palm oil (2005/402/EC). Off. J. Eur. Union L135, 34−36. (20) Zeiger, E., Andersen, B., Haworth, S., Lawlor, T., and Mortelmans, K. (1988) Salmonella mutagenicity tests. IV. Results from the testing of 300 chemicals. Environ. Mutagen. 11 (Suppl. 12), 1−158. (21) Stiborová, M., Asfaw, B., Anzenbacher, P., and Hodek, P. (1988) A new way to carcinogenicity of azo dye: the benzenediazonium ion formed from non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene

AUTHOR INFORMATION

Corresponding Author

*Tel: +420 221951285. Fax: +420 221951283. E-mail: [email protected]. Funding

This work was supported by the Grant Agency of the Czech Republic, grant P303/12/G163. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AHR, aryl hydrocarbon receptor; ARE, antioxidant response element; α-NF, α-naphthoflavone; BaP, benzo[a]pyrene; BDI, benzenediazonium ion; bw, body weight; P450, cytochrome P450; EROD, 7-ethoxyresorufin O-deethylation; HPLC, high performance liquid chromatography; 3′,4′-diOH-Sudan I, 1(3,4-dihydroxyphenylazo)-2-naphthol; 4′-OH-Sudan I, 1-(4hydroxyphenylazo)-2-naphthol; 6-OH-Sudan I, 1-(phenylazo)naphthalene-2,6-diol; 4′,6-diOH-Sudan I, 1-(4-hydroxyphenylazo)-naphthalene-2,6-diol; PEI, polyethyleneimine; RT-PCR, real-time polymerase chain reaction; RAL, relative adduct labeling; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TLC, thin-layer chromatography; XRE, xenobiotic response element

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Induced Expression of Cytochrome P450 1A and NAD(P) - American

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