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Biochemistry and Biophysics |
dlováDepartment 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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sudan I gives positive results in Salmonella typhimurium mutagenicity tests with S-9 activation (7 , 8) and is mutagenic to mouse lymphoma L5178Y TK+/- cells in vitro, with S-9 activation (8) . It is clastogenic compound, inducing micronuclei in the bone marrow of rats (3) . Whereas the metabolism of Sudan I is not understood in humans, its metabolism has been characterized in rabbits (9) , where it is metabolized primarily in the liver by oxidative or reductive reactions (9) . C-Hydroxylated metabolites 4'-OH-Sudan I and 6-OH-Sudan I were found to be the major products of Sudan I oxidation in vivo and excreted in urine (1 , 9) , and of its oxidation by rat hepatic microsomes in vitro (10) . Besides the C-hydroxylated metabolites, which are considered detoxication products, the BDI formed by microsome-dependent enzymatic splitting of the azo group of Sudan I was found to react with DNA in vitro (10, 11, 12) . The major DNA adduct formed in this reaction has been characterized and identified as the 8-(phenylazo)guanine adduct (12) . In addition to microsomal enzymes, Sudan I and its C-hydroxylated metabolites are also oxidized by peroxidases, as a consequence DNA, RNA, and protein adducts are formed (13, 14, 15) .
Because CYPs are abundant in the liver where much of the metabolism of Sudan I in experimental animals occurs (9) , CYPs were assumed to play a role in the oxidative metabolism of this carcinogen (9, 10, 11, 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 extrapolation of animal carcinogenicity data to assess human health risk, and consideration of species differences in catalytic activities of CYPs is important. In contrast to many experimental animal models, humans show large interindividual variations in the expression of CYP enzymes and catalytic activities, which may lead to different susceptibilities to carcinogens and must be considered in risk assessment (16) . To assess the human health risk of Sudan I, we have compared the capacity of livers from humans, rats, and rabbits to metabolize Sudan I. In addition, the present study was undertaken to understand which human CYP enzymes are involved in Sudan I metabolic activation and/or detoxication. This knowledge will be useful in evaluating individual susceptibility to this carcinogen.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-NF, ß-NF, NADPH, troleandomycin, ketoconazole, glucose 6-phosphate, chlorzoxazone, calf thymus DNA, coumarin, sulfaphenazole, and quinidine were from Sigma Chemical Co. (St. Louis, MO); furafylline from New England Biolabs (Beverly, MA); 6ß-hydroxytestosterone from Merck (Darmstadt, Germany); glucose 6-phosphate dehydrogenase from Serva (Heidelberg, Germany); bufuralol and its 1'-hydroxyderivative from Gentest Corp. (Woburn, MA); bicinchoninic acid from Pierce (Rockford, IL); and Sudan I from British Drug Houses (Poole, United Kingdom). 3-IPMDIA was synthesized according to Olah et al. (17)
The derivatives 4'-OH-Sudan I, 6-OH-Sudan I, 4',6-di(OH)-Sudan I and 3',4'-di(OH)-Sudan I were synthesized as described (10)
. Enzymes and chemicals for the 32P-postlabeling assay were obtained from sources described previously (12)
.
Preparation of Microsomes and Assays.
Microsomes from livers of untreated rats and rabbits were prepared as described previously (12)
. Microsomes from the livers of rats pretreated with ß-NF (12)
and Sudan I (18)
were isolated as described (12)
, those pretreated with PB, PCN, and ethanol as reported (19)
. Microsomes from human liver of eight human donors who died in a traffic accidents were isolated as described (20)
and were a gift of B. Szotáková (Faculty of Pharmacy, Charles University, Hradec Králové, The Czech Republic). The donors ranged in age from 24 to 70 years, and included five men and three women. All of the donors had no known drug history and none had a history of alcohol abuse. Microsomes from the liver of a male minipig were a gift from P. Anzenbacher (Palacky University, Olomouc, The Czech Republic) and isolated as described (20)
. Supersomes, microsomes isolated from insect cells transfected with Baculovirus constructs containing cDNA of one of the following human CYPs: CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4, with cytochrome b5 and expressing NADPH:CYP reductase were from Gentest Corp. Protein concentrations were assessed using the bicinchoninic acid protein assay (21)
. The concentration of CYP was estimated according to Omura and Sato (22)
. Rat, rabbit, and minipig liver microsomes contained 0.62, 1.82, and 0.89 nmol CYP/mg protein, respectively. Microsomes of rats induced with ß-NF, PB, PCN, and ethanol contained 1.30, 2.74, 1.55, and 1.80 nmol CYP/mg protein, respectively. The content of CYP in human hepatic microsomes is shown in Table 1
. Each human microsomal sample was analyzed for specific CYP activities by monitoring the following reactions: EROD (CYP1A1/2), coumarin 7-hydroxylation (CYP2A6), bufuralol 1'-hydroxylation (CYP2D6), tolbutamide methyl hydroxylation (CYP2C9), chlorzoxazone 6-hydroxylation (CYP2E1), and testosterone 6ß-hydroxylation (CYP3A4; Ref. 23
and references therein). These activities are shown in Table 1
.
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The bands corresponding to CYP1A1 protein of two human hepatic microsomal samples (samples 5 and 6, see Table 1
) were excised from a PVDF membrane and subjected to NH2-terminal sequencing on a Protein Sequencer LF3600D (Beckman Instruments) according to the manufacturer’s manual.
Isolation of Individual CYPs.
The CYP1A2, 2B4, 2C3, and 2E1 were isolated from liver microsomes of rabbits induced with ß-NF (CYP1A2), PB (CYP2B4), or ethanol (CYP2E1 and 2C3) by procedures described elsewhere (26
, 27)
. The CYP3A1 and 3A6 were isolated from rat and rabbit hepatic microsomes of animals induced with PCN (19)
and rifampicin (28)
, respectively. Recombinant rat CYP1A1 was purified as described (29)
from membranes of Escherichia coli transfected with a modified CYP1A1 cDNA. Recombinant human CYP1A2 was from Oxford Biomedical Research, Inc., and human recombinant CYP3A4 was a gift of P. Anzenbacher (see above). Rabbit liver NADPH:CYP reductase and cytochrome b5 were purified as described (30
, 31) .
Preparation of Antirat CYP1A1, Antirabbit CYP2E1, and Antihuman CYP3A4 Polyclonal Antibodies.
Leghorn chickens were immunized s.c. three times a week by CYP antigens (rat recombinant CYP1A1, rabbit CYP2E1, and human recombinant CYP3A4; 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 as described (32
, 33)
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Incubations.
Incubation mixtures contained the following in a final volume of 750 µl: 50 mM sodium phosphate buffer (pH 7.4), 1 mM NADPH, 10 mM D-glucose 6-phosphate, 1 unit/ml D-glucose 6-phosphate dehydrogenase, 10 mM MgCl2, microsomal fraction containing 0.05–2.4 nmol CYP, and 0.1–100 µM Sudan I dissolved in 7.5 µl methanol. Incubation mixtures, in which the efficiencies of Supersomes expressing human CYPs were tested, were the same except that 100 µM of Sudan I and only 10–50 pmol of CYP were used. Incubations using purified CYP reconstituted with NADPH:CYP reductase and cytochrome b5 (34)
contained 50–250 pmol of each CYP. After incubation (37°C, 5–140 min) the mixtures were extracted with ethyl acetate. The extracts were evaporated, dissolved in methanol, and chromatographed on a thin layer of silica gel (10)
. The BDI was detected by azo coupling with 1-phenyl-3-methyl-5-pyrazolone (10, 11, 12)
. Alternatively, the products were separated by HPLC on a MN Nucleosil 100–5 C18 column (Macherey-Nagel; 4.0 x 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. The Sudan I metabolites were identified by cochromatography with authentic standards.
Incubations in which DNA was modified by Sudan I activated with human or rat hepatic microsomes had the same composition, but contained 1 mg of calf thymus DNA and human microsomes containing 100 pmol CYP, or hepatic microsomes of rats pretreated with ß-NF (12) . DNA was isolated as described (12) .
Kinetic analyses to determine the maximum reaction rate (maximum velocity) and Michaelis constant were performed using the nonlinear least-squares method as described (35) . Incubations were the same as those described above (with microsomes) except that they contained 0.1–100 µM Sudan I, 4'-OH-Sudan I, or 6-OH-Sudan I. Mixtures were incubated at 37°C for 10 min.
Inhibition Studies.
The following chemicals were used to inhibit the metabolism of Sudan I (specific CYPs known to be inhibited): -NF (CYP1A1/2); furafylline (CYP1A2); 3-IPMDIA (CYP2B; 36
); DDTC (CYP2A6 and 2E1); sulfaphenazole (CYP2C); quinidine (CYP2D); and troleandomycin and ketoconazole (CYP3A). Inhibitors were dissolved in 7.5 µl of methanol to yield final concentrations of 1–400 µM in the incubation mixtures. An equal volume of methanol alone was added to the control incubations.
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 using version 6.12 Statistical Analysis System software. Spearman correlation coefficients were based on a sample size of 8. All of the Ps are two-tailed and considered significant at the 0.05 level.
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RESULTS |
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. On the basis of cochromatography with the synthetic standards, the major metabolites produced from Sudan I by all of the tested microsomes were identified as 4'-OH-Sudan I and 6-OH-Sudan I. Additional minor products were 4',6-di(OH)-Sudan I and 3',4'di(OH)-Sudan I (Fig. 1)
. Another metabolite was a colorless product, which was identified previously as BDI (Refs. 10
, 12
; not shown in the chromatogram of HPLC in Fig. 1
). Whereas in microsomes of rabbit and minipig 100 µM Sudan I was prefertially oxidized to the 6-hydroxy-naphthol derivative of Sudan I, those of human and rat predominantly produced 4'-OH-Sudan I (Fig. 2)
. The ratios of metabolites were the same at lower Sudan I concentrations down to 10 µM.
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Involvement of Rat CYP Enzymes in Oxidation of Sudan I.
Individual CYP enzymes were induced in rats. Incubations of Sudan I with microsomes from ß-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 concentration of -NF and Sudan I inhibited its oxidation by 70%. Inhibitors of other CYP enzymes caused either weak (ketoconazole, troleandomycin, and 3-IPMDIA) or no inhibition (furafylline, sulfaphenazole, quinidine, and DDTC). The formation of Sudan I metabolites with ß-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, 4'OH-Sudan I, and 6-OH-Sudan I, in ß-NF microsomes are 1.7, 4.6, and 2.8 nmol/min per nmol total CYP and 21, 79, and 40 µM, respectively.
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. -NF inhibited Sudan I oxidation as in microsomes (Fig. 4)
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. All of the above results indicate that rat CYP1A1 and human recombinant CYP1A1 are the most efficient enzymes metabolizing Sudan I.
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. 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)
.
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Using two independent methods, we were able to detect and quantify CYP1A1 in human hepatic microsomes. A polyclonal antibody raised against rat recombinant CYP1A1, which highly cross-reacts with recombinant human CYP1A1 and only poorly with CYP1A2, was used in the first method (Fig. 6A)
. The detection sensitivity was as low as 0.005 pmol CYP1A1 per lane. In immunoblots (Fig. 6B)
, this polyclonal antibody reacted with one and/or two immunoreactive bands in most analyzed human hepatic microsomes. The high and low mobility bands (Fig. 6B)
were assumed to be CYP1A1 and 1A2, respectively, based on the reported electrophoretic mobilities of these proteins in microsomes from human tissues (44
, 45)
. To confirm that the band with lower molecular weight really corresponds to human CYP1A1, NH2-terminal sequencing was carried out with this protein band. The bands of microsomal samples 5 and 6 were excised from a PVDF membrane and subjected to automated Edman degradation. The sequence of nine amino acids, LFPISMSAT, was identical to the residues 2–10 of the NH2-terminal sequence of CYP1A1 (MLFPISMSAT; Ref. 46
). NH2-terminal methionine was not found in the CYP1A1 protein band by NH2-terminal sequencing.
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, being present at <0.6% of total hepatic CYP. With the same antibody, we also estimated the expression levels of CYP1A2 in all of the human microsomal samples. The CYP1A2 content ranged from 5 to 35 pmol per mg of microsomal protein (data not shown). To resolve which of these two CYPs is the predominant enzyme oxidizing Sudan I, correlations between the CYP1A1 or 1A2 protein levels and Sudan I oxidation were used. A significant correlation was seen between hepatic CYP1A1 content and Sudan I oxidation (r = 0.810; P = 0.010), but not between the content of CYP1A2 and oxidation of this carcinogen (r = 0.405; P = 0.320). Because the EROD activity highly correlated with CYP1A1 content (r = 0.762; P < 0.05) but not with the content of CYP1A2 protein (r = -0.309; P = 0.456), O-deethylation of ethoxyresorufin seems to be catalyzed mainly by CYP1A1 in human hepatic microsomes used in the study.
Whereas catalytic activities of CYP2A6, 2C9, 2D6, and 3A4 did not exhibit significant correlation with the levels of Sudan I metabolites formed by the same human hepatic samples, a significant correlation was seen with the CYP2E1 activity (Table 2)
. However, there is a cross-correlation between EROD and chlorzoxazone 6-hydroxylation activity (r = 0.783; P = 0.038) within these liver samples. To additionally clarify this correlation, multivariate analysis was used to investigate the dependence of the Sudan I oxidation on these two isoform activities. The two activities (CYP1A and 2E1) in each microsomal sample were combined in pairs to see if a combination of two activities gave an improvement in the correlation with Sudan I oxidation, i.e., an increase in the correlation coefficient when compared with the correlation with the individual activities. The inclusion of the CYP2E1 activity produced no improvement in the correlation coefficient. Multivariate analysis was also used to examine the dependence of the Sudan I oxidation on activities of CYP3A4 and 2C9. Although the activities of these CYPs did not exhibit significant correlations with Sudan I oxidation, these activities showed certain correlation tendencies (Table 2)
and recombinant CYP3A4 was active with Sudan I (Fig. 5)
. The inclusion of the CYP3A4 or 2C9 activities with CYP1A in multivariate analysis produced no improvement in the correlation coefficient.
To confirm the role of individual human hepatic CYP enzymes in metabolism of Sudan I, two human microsomal samples with high CYP1A, 2E1, and 3A4 activities, samples 5 and 8, were selected, and incubations were carried out in the absence and presence of specific inhibitors of CYP1A1/2, 1A2, 2E1, and 3A4, -NF, furafylline, DDTC, and ketoconazole, respectively. A substrate of CYP2E1, chlorzoxazone, was used as additional inhibitor. -NF inhibited Sudan I metabolism to 50%, whereas no effect of furafylline, DDTC, or chlorzoxazone was observed. Ketoconazole weakly inhibited the 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 DNA Adducts.
Using the nuclease P1 version of the 32P-postlabeling assay we found that during oxidation of Sudan I by human hepatic microsomes DNA adducts are formed. One major (the closed circle in Fig. 7D
) and two minor adduct spots, overlapping the major adduct, were detected in autoradiographs of DNA reacted with Sudan I activated by human microsomes (Fig. 7A)
. The major adduct spot exhibited similar chromatographic properties as the major adduct formed in DNA by Sudan I activated with rat microsomes (Fig. 7B)
, which corresponds to the 3',5'-bisphospho-derivative of an 8-(phenylazo)deoxyguanosine adduct identified previously (12)
. The identity of the major adduct in human and rat microsomes was confirmed by cochromatography on PEI-cellulose plates in two different solvent systems (not shown).
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. A highly significant correlation was found between the EROD activity and the formation of Sudan I-DNA adducts (r = 0.905; P = 0.002) in human microsomes (Table 2)
. In addition, Sudan I-DNA adduct formation highly correlated with levels of the CYP1A1 protein determined in microsomes by Western blot analysis (Table 2)
. A weaker but significant correlation was determined between CYP2E1 activities and formation of Sudan I-DNA adducts (Table 2)
. Again, a cross-correlation between the CYP1A and 2E1 activities in these liver samples might explain these results (see above). The binding of Sudan I to DNA catalyzed by one microsomal sample with high CYP1A and 2E1 activities (sample 8) was inhibited by -NF, but not by furafylline, a selective inhibitor of human CYP1A2, or DDTC, an inhibitor of CYP2E1. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, 40, 41, 42, 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 oxidation was supported by strong correlation coefficients between the levels of CYP1A1 protein expression (or the rates of EROD), and the levels of Sudan I metabolites and/or Sudan I-derived DNA adducts in the eight human hepatic microsomal samples. The participation of CYP1A1 in Sudan I metabolism was confirmed also by inhibition of 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 inhibitors is sometimes difficult, because one inhibitor may be more effective with one substrate than another. Nevertheless, the utilization of pure CYP1A1 as well as microsomes containing human recombinant CYP1A1 fully corroborated the major role of CYP1A1 in the metabolism of Sudan I. Interestingly, the highly homologous human CYP1A1 and 1A2 with 73% amino acid sequence identity exhibit extremely different potency to oxidize Sudan I. CYP1A2 is almost ineffective in Sudan I oxidation. Besides the CYP1A1, the CYP3A4 enzyme might also participate in Sudan I oxidation in human hepatic microsomes, because human recombinant CYP3A4 oxidizes Sudan I. The efficiency of this CYP to oxidize Sudan I is 
10-fold lower than that of CYP1A1. However, because of high expression levels of CYP3A4 in human livers, its contribution to Sudan I metabolism might be relevant, although the correlation studies showed only correlation tendencies with levels of Sudan I metabolites and DNA adducts.
Human CYP1A1 seems to be induced by planar aromatic compounds binding to the aryl hydrocarbon receptor, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (42) and/or by polycyclic hydrocarbons present in cigarette smoke (40) . The CYP1A1 enzyme is strongly induced by Sudan I itself in rats by this mechanism (47) . Hence, long-term occupational exposure of humans to Sudan I might be an important risk 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 Sudan I. This is highly significant in view of the prediction of Sudan I carcinogenicity to humans. Whereas Sudan I is carcinogenic to rats (1, 2, 3, 4, 5) , its carcinogenicity to humans has not yet been proven. Sudan I was evaluated to be still unclassifiable as carcinogenic to humans by IARC (5) .4 In a meeting March 3–5, 1999, a European Union commission working group for classification, packaging, and labeling of dangerous substances recommended that Sudan I should be considered of "concern for man owing to possible carcinogenic effects" (Cat. Carc. 3; Ref. 5 ) and of "concern for man because of possible mutagenic effects" (Muta. Cat. 3; Ref. 5 ). We fully support the recommendation of this working group. Our results, showing for the first time an analogy in the Sudan I metabolism by human and rat enzymes, strongly suggest a carcinogenic potential of this rat carcinogen for humans. An increased cancer risk should be taken into account mainly for individuals working in the dye industry and exposed to Sudan I, its derivatives, and to other compounds inducing CYP1A1. Furthermore, caution is highly recommended in using this dye and its derivatives to color materials, which are used by humans in their daily use.
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FOOTNOTES |
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1 Supported in part by the Grant Agency of Charles University (Grant 204/2001/B/CH/PrF), the Grant Agency of the Czech Republic (Grant 203/01/0996), and the Ministry of Education of the Czech Republic (Grant MSM 113100001). 
2 To whom requests for reprints should be addressed, at Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, The Czech Republic. Phone: 420-2-2195-2333; Fax: 420-2-2195-2331; E-mail: stiborov{at}natur.cuni.cz.
3 The abbreviations used are: Sudan I, 1-(phenylazo)-2-naphthol (C.I. Solvent Yellow 14); -NF, -naphthoflavone; ß-NF, ß-naphthoflavone; BDI, benzenediazonium ion; CYP, cytochrome P450; DDTC, diethyldithiocarbamate; EROD, 7-ethoxyresorufin O-deethylation; 3-IPMDIA, 3-isopropenyl-3-methyldiamantane; 4'-OH-Sudan I, 1-(4-hydroxyphenylazo)-2-hydroxynaphthol; 6-OH-Sudan I, 1-(phenylazo)-naphthalene-2,6-diol; 4',6-di(OH)-Sudan I, 1-(4-hydroxyphenylazo)-naphthalene-2,6-diol; 3',4'-di(OH)-PB, phenobarbital; PCN, pregnenolone-16-carbonitrile; PEI, polyethylenimine; PVDF, polyvinylidene difluoride; RAL, relative adduct labeling; HPLC, high-performance liquid chromatography.
4 Internet address: http://www.iarc.fr for lists of IARC evaluations, November 1998.
Received 5/23/02. Accepted 8/ 8/02.
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etick L., Hodek P. The first identification of the benzenediazonium ion formation from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomes of rat livers. Cancer Lett., 40: 319-326, 1988.[Medline]
K., Svoboda Z., Kvetina J. Presence and activity of cytochrome P450 isoforms in minipig liver microsomes. Comparison with human liver samples. Drug Metab. Dispos., 26: 56-59, 1998.
ulc M., Stiborová M. -Naphthoflavone acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6. Chem. Biol. Interact., 138: 85-106, 2001.[Medline]
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 R. P. Gupta, Y. A. He, K. S. Patrick, J. R. Halpert, and N. H. Bell CYP3A4 Is a Vitamin D-24- and 25-Hydroxylase: Analysis of Structure Function by Site-Directed Mutagenesis J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1210 - 1219. [Abstract] [Full Text] [PDF]
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M. Stiborova, J. Sejbal, L. Borek-Dohalska, D. Aimova, J. Poljakova, K. Forsterova, M. Rupertova, J. Wiesner, J. Hudecek, M. Wiessler, et al. The Anticancer Drug Ellipticine Forms Covalent DNA Adducts, Mediated by Human Cytochromes P450, through Metabolism to 13-Hydroxyellipticine and Ellipticine N2-Oxide Cancer Res., November 15, 2004; 64(22): 8374 - 8380. [Abstract] [Full Text] [PDF]
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M. Stiborova, M. Miksanova, S. Smrcek, C. A. Bieler, A. Breuer, K. A. Klokow, H. H. Schmeiser, and E. Frei Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans Carcinogenesis, May 1, 2004; 25(5): 833 - 840. [Abstract] [Full Text] [PDF]
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V. M. Arlt, M. Stiborova, A. Hewer, H. H. Schmeiser, and D. H. Phillips Human Enzymes Involved in the Metabolic Activation of the Environmental Contaminant 3-Nitrobenzanthrone: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase Cancer Res., June 1, 2003; 63(11): 2752 - 2761. [Abstract] [Full Text] [PDF]
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