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Detoxication products of the carcinogenic azodye Sudan I (Solvent Yellow 14) bind to nucleic acids after activation by peroxidase
Cancer Letters, 68 (1993)
43
43 - 47
Elsevier Scientific Publishers Ireland Ltd.
Detoxication products of the carcinogenic azodye Sudan I (Solvent Yellow 14) bind to nucleic acids after activation by peroxidase Marie Stiborovba,
Eva Freib, Heinz H. Schtneiserb,
Manfred Wiesslerb and Jan Hradecc
of Biochemistry, Facul@ of Natural Sciences, Charles Uniuersity, Albertou 2030, 128 40 Prague 2 (Czechoslooakia), blnstitute of Toxicology and Chemotherapy, German Cancer Research Center, Im Neuenheimer Fe/d 280, 6900 Heideberg {Germany) and ‘Department of Experimental Oncology, Institute of Radiotherapy, 180 00 Prague 8 (Czechoslovakia) ‘Department
(Received 14 April 1992) (Accepted 23 October 1992)
Summary
Introduction
The C-hydroxyderiuatiues of the carcinogenic dye Sudan 1, 1 -phenylazo-2,6-dihydroxynaphthalene and 7-(4-hydroxyphenylazo)Z-hydroxynaphthaiene, which are considered to be detoxication products of this dye bind to DNA or tRNA after oxidation into actiue metabolites by peroxidase and H202 in oitro. The 3ZP-postlabeling analysis of DNA modi-
It was reported by IARC that the nonaminoazo dye l-phenylazo-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14) was used as food colouring in several countries. This dye has, however, been recommended as unsafe for use in food. Nevertheless, it is widely used to colour other materials [ 11. The compound is carcinogenic leading to tumors in the liver and/or urinary bladder in rats, mice and rabbits [ 1 - 31. In vivo studies on the metabolism of Sudan I in rabbits revealed that this compound is oxidized in the liver by cytochrome P-450 to derivatives hydroxylated in the aromatic rings. The resulting products 1-(4-hydroxyphenyIazo)-2-hydroxynaphthalene (4’OH-Sudan I), 1-phenylazo-2,6-dihydroxynaphthalene (6-OH-Sudan I) and 1-(4-hydroxyphenyIazo)-2,6_dihydroxynaphthalene were detected unconjugated or conjugated with glucuronic acid in both bile and urine of animals exposed to this dye [4]. 4 ’ -OH-Sudan I and 6-OH-Sudan I were found to be the major products of Sudan I oxidation by microsomal cytochrome P-450 in vitro and in vivo [1,4,5]. Hydroxylation of aromatic rings is considered to be a common detoxication pathway characteristic for carcinogenic
fied by active metabolites of both Sudan I derivatives prouides evidence that the covalent binding to DNA is the principal type of DNA modification. Since the urinary bladder is rich in peroxidases, the participation of these enzymes in actiuation of detoxicating products of Sudan I may be inuolued in the inition of Sudan I-carcinogenesis in this organ.
Keywords: azo dye; detoxication and activation of cacinogens; peroxidase; DNA- and tRNA-binding
Correspondence
to: M. Stiborovl,
Department
Faculty of Natural Sciences, Charles University, 128 40 Prague 2, Czechoslovakia.
0304~3835/92/$05.00
0
1992
Printed and Published in Ireland
of Biochemistry, Albertov 2030,
Elsevier Scientific Publishers Ireland Ltd.
44
compounds [Z]. The question, however, arises whether the detoxicating metabolites of Sudan I could not be the substrate of other enzymes, which would activate these detoxicating products to ultimate carcinogens. Besides cytochrome P-450 which both detoxicates and activates Sudan I in livers [4 - 71, peroxidase is also supposed to be implicated in the Sudan I activation in the tissues rich in this enzyme [6,7]. Moreover, since C-hydroxyderivatives of Sudan I were found as a major metabolic excreting products in urines we focus our attention to find whether these compounds are activated by peroxidase, which is abundant in the urinary bladder. Material and Methods
Radiochemicals 14C-Sudan I (0.54 mCi/mmol) was synthesized as described earlier [5] and converted to its C-hydroxyderivatives by incubation with rat liver microsomal enzymes and NADPH [5]. Both 4’-OH-Sudan I and 6-OH-Sudan I were separated from the reaction mixture by thin layer chromatography on silica gel G (Woelm) [5] and purified by rechromatography under the same conditions, Incubations Incubations for the binding experiments of both metabolites to nucleic acids were composed as described earlier [6,7] and contained 0.2 mM of the 14C-labeled compound (r*CSudan I or its 14C-labeled hydroxyderivatives) dissolved in methanol (40 ~I/250 ~1 incubation) , horseradish peroxidase (Boehringer) and H202. They were incubated, unless otherwise stated, for 60 min and 37OC. Reaction mixtures were extracted with ethyl acetate, DNA or tRNA were isolated and its radioactivity determined as described earlier [6,7]. The ethylacetate extracts from the reaction mixtures containing radioactive 14Clabeled hydroxyderivatives of Sudan I and their products were evaporated under a stream of nitrogen, dissolved in a minimal volume of methanol and chromatographed on thin layer
of silica gel and eluted with diethyl ether/nhexan (3: 1, v/v). The residual [t4C]4’-OHSudan I or [r4C]6-OH Sudan I and their products were mechanically separated from plates, placed in a scintillation vial and the radioactivity was counted. 32P-Labeling of the DNAdigest and recovery of individual nucleotide adducts. Chemicals and materials for DNA digestion and 32P-postIabeling were from sources reported previously [8,9]. DNA modified by hydroxyderivatives of Sudan I used for the 32P-labeling of digest was prepared by incubation with peroxidase system and isolated as described above. Control incubations were performed without the addition of the compound (methanol was used instead of the compounds). DNA was digested to deoxyribonucleoside 3-monophosphates using micrococcal nuclease and spleen phosphodiesterase and then with nuclease Pl as described [8,9]. The digested DNA was then 32P-labeled using polynucleotide kinase and [y-32P]ATP (200 &i) synthesized in the laboratory from ADP and [32P]orthophosphate also as described previously [8]. Labeled digests were chromatographed on thin layers of PEI-celulose in a similar way to that described previously [8], except that 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, 8.5 M urea, pH 8.0 and followed a final wash with 1.7 sodium phosphate, pH 6.0 D2 was omitted. Results and Discussion
Like the parent compound Sudan I, both its C-hydroxyderivatives did not bind to nucleic acids when incubated with DNA or tRNA in the absence of peroxidase, H202 or both. However, efficient blinding of metabolites of both these derivatives occurred in complete incubations. When compared to Sudan I, the oxidation of 6-OH-Sudan I was also effective whereas significantly lower amounts of 4’-OHSudan I were oxidized by the peroxidase system (Table I). The products of peroxidase activation of both hydroxymetabolites bound
45
Table1. Binding of Sudan I, [ 14C] 4’-OH-Sudan calf thymus
I and [r4C] 6-OH-Sudan
I activated
by the peroxidase
system to
DNA and rat liver tRNA in vitro
Compound
DNA binding nmol/mg
tRNA binding
% of converted Sudan I and its hydroxyderivatives
I 14C]Sudan I [ “kZ]4 ’ -OH-Sudan I [ “C]6-OH-Sudan 1
4.2 f 0.3 2.1 f 0.2 3.3 f 0.2
14.8 f 0.9 1.9 f 0.1 5.9 f 0.5
85.3 60.1 93.3
f 0.9 + 0.6 f 0.9
The average binding levels and standard deviations were obtained from triplicate determinations. The DNA- and tRNAbinding was assayed as described [6,7]. The binding levels of the control samples (without H202 or without peroxidase) for 4’-OH-Sudan I were 0.3 and 0 nmol/mg, respectively, for 6-OH-Sudan I 0.4 and 0 nmol/mg, respectively.
less efficiently to DNA and in particular, to tRNA than the products formed from Sudan I by peroxidase (Table I) [6,7]. Unlike the the preferential binding of parent dye, metabolites of both C-hydroxyderivatives to tRNA was far less pronounced. The binding of C-hydroxyderivatives of Sudan I to nucleic acids after activation by peroxidase was measured at pH 4.6, 7.46 and 8.4. The highest levels of binding were detected at pH 8.4 (results not shown). The nucleic acids modified by metabolites of both compounds studied were isolated from the reaction mixtures by the procedures which included phenol/chloroform extraction [6,7]. This suggested that a covalent binding of ultimate form(s) of compounds is the predominant modification. The covalent binding of metabolites was confirmed by the 32Ppostlabeling analysis. Several adducts of 4’OH-Sudan I and of 6-OH Sudan I were detected on the autoradiograms shown in Fig. 1. The chromatographic conditions chosen were suitable for separation of adducts formed from aromatic carcinogens with a pronounced hydrophobicity [8]. The adducts obtained from both C-hydroxyderivatives and DNA showed similar chromatographic properties as those formed from carcinogens like aristolochic acid, benzo[a]pyrene or Sudan I with DNA [lo]. Although the exact nature of adducts has not yet been elucidated their pronounced liphophilicity indicates that a polycyclic aromatic
system is covalently linked to the nucleic acid. The chromatographic behaviour of adducts of both Sudan I derivatives is different from that of parent Sudan I [7] and also the metabolites of both derivatives tested differ from each other in this respect. In addition to detoxication products (Chydroxyderivatives), Sudan I is in vivo also metabolized by reductive reactions to l-amino2-naphthol and p-aminophenol [ 111. The former compound is not carcinogenic [2,12]. The latter compound is mutagenic, but its carcinogenicity is questionable [13]. Both these products occur also in the urine either free or as glucuronides or sulphates [ 111. Activation of Sudan I and of its C-hydroxyderivatives is a prerequisite for their binding to DNA. This activation may be catalyzed by microsomal cytochrome P-450 [2,5,14] but also by peroxidases as reported in this paper. The urinary bladder contains low quantities of cytochrome P-450 but is rich in peroxidase [12,15,16]. In animals exposed to Sudan I, the urine contains only low quantities of the parent Sudan I [l] but is abundant in its Chydroxy derivatives resulting from the hydroxylation of the parent dye in the liver [ 111. Thus the products of detoxication of Sudan I may be converted to ultimate carcinogens by peroxidase and may form DNA adducts in the urinary bladder. Binding to DNA is characteristic for ultimate carcinogens [Z] and this interaction is believed to represent a fundamen-
46
tal step in the initiation of chemical carcinogenesis [7]. Since not only active metabolites of Sudan I [7] but also those of its detoxication products form DNA-adducts, C-hydroxyderivatives of this dye may be considered to be potential procarcinogens. This implies that the combination of detoxicating and activating reactions catalyzed by several enzymes (i.e. cytochrome P-450, peroxidases) seems to be crucial for the final formation of DNA adducts from carcinogens. The mechanism of organ specificity of Sudan I (and/or other carcinogens) has not yet been fully explained. Besides other mechanisms (the efficiencies of conjugating reactions for carcinogens in different organs, the stability of conjugates) the abilities of several enzymes (as well as their levels) to activate carcinogens in different organs is considered to be one of the most important mechanisms [7,18]. Because of findings presented in this paper, peroxidase could be supposed as one enzyme which may participate in formation of ultimate carcinogens and DNA adducts in the urinary bladder and, hence, it may contribute to the organ specificity of Sudan I. Nevertheless, it should be noted that cancer in a specific organ may also be a function of promotional pressures on initiated cells in target organs and not only the levels of DNA adducts [ 191. The function of promotional pressures in a Sudan Imediated tumorigenesis remains to be explained. References
Fig. 1. Autoradiograms of PEI-cellulose TLC maps of 32P-labeled digests of DNA treated with peroxidase, H202 and 4’-OH-Sudan I (A), 6-OH-Sudan I (B) and with the same system, but without derivatives of Sudan I (C). Analysis was performed by the nuclease Pl version Material and Methods). (see of the assay Autoradiography was at ambient temperature for 1 h. Origins are located at the bottom left corners (D3 from bottom
to top
and
D4 from
left to right).
IARC (1975) IARC Monographs. Sudan I. IARC, Lyon, Vol. 8, pp. 225-231. Garner, R.C., Martin, C.N. and Clayson, D.B. (1984) Carcinogenic aromatic amines and related compounds. In: Chemical Carcinogens. 3rd edition, Vol. 1, ACS Monograph No. 182, pp. 175-302, Editor: C.E. Searle. American Chemical Society, Washington DC. Westmoreland, C. and Gatehouse, D.G. (1991) The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observation of species and tissues specificity. Carcinogenesis, 12, 1403 - 1407. Childs, J.J. and Clayson, D.S. (1966) The metabolism of l-phenylazo-2-naphthol in the rabbit. Biochem. Pharmacol., 15, 1247- 1258.
47
5
Stiborova, M., Asfaw, 6.. Anzenbacher, P., LeJetickjr, 1. and Hodek, P. (1988) The first identification of the benzenediazonium ion formation from a non-aminoazodye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomes of rat livers. Cancer Lett., 40, 319-326. 6 Stiborova, M., Frei, E., Klokow, K., Wiessler, M., SafamC, L., Anzenbacher, P. and Hradec, J. (1990) Peroxidasemediated reaction of the carcinogenic anon-aminoazo dye 1-phenylazo-2-hydroxynaphthalene with transfer ribonucleic acid. Carcinogenesis, 11, 1789 - 1794. 7 Stiborova, M., Frei, E., Schmeiser, H.H., Wiessler, M. and Hradec, J. (1990) Mechanism of formation and 32Ppostlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye I-phenylazo-2hydroxynaphthalene (Sudan I). Carcinogenesis, 11, 1843 - 1848. 8 Reddy, M.V. and Randerath, K. (1986) Nuclease Plmediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543 - 1551. 9 Schmeiser, H., Diple, A., Schurdak, M.E., Randerath, E. and Randerath, K. (1988) Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz(a)anthracene-DNA adducts. Carcinogenesis, 9, 633 - 638. 10 Schmeiser, H.H., Schoepe, K.-B. and Wiessler, M. (1988) DNA adduct formation of aristolochic acid I and II in vitro and in vivo. Carcinogenesis, 9, 297-303. 11 Daniel, J.W. (1962) The excretion and metabolism of edible food colours. Toxicol. Appl. Pharmacol., 4, 572 - 594.
12
13
14
15
16
17
18
Yamazoe, Y., Miller, D.W., Wies, G.C., Dooley, K.L., Zenser, T.V., &land, F.A. and Kadlubar, F.F. (1985) DNA adducts formed by ring-oxidation of the carcinogen P-naphthylamine with prostaglandin H-synthase in vitro and in the dog urothelium in vivo. Carcinogenesis, 6, 1379 - 1389. Josephy, P.D., Eling, T.E. and Mason, R.P. (1983) Oxidation of p-aminophenol catalyzed by horseradish peroxidase and prostaglandin synthase. Mol. Pharmacol., 23, 461-466. Stiborovi, M., Asfaw, B., Anzenbacher, P. and Hodek, P. (1988) A new way to carcinogenicity of azo dyes: the benzenediazonium ion formed from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomal enzymes binds to deoxyguanosine residues of DNA. Cancer Lett., 40, 327-333. Yamazoe, Y., Zenser, T.V., Miller, D.W. and Kadlubar, F.F. (1988) Mechanism of formation and structural characterization of DNA adducts derived from peroxidase activation of benztdine. Carcinogenesis, 9, 1635 - 1641. O’Brien, 0. (1988) Radical formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics: the reactivity of these radicals with GSH, DNA and unsaturated lipid. Free Rad. Biol. Med., 4, 169- 183. Wogan, G.N. and Gorelick, N.J. (1985) Chemical and biochemical dosimetry of exposure to genotoxic chemicals. Environ. Health Perspect., 62, 5- 18. Barrett, J.C. and Wiseman, R.W. (1987) Cellular and molecular
mechanisms
relevance
to carcinogen
Perspect.,
76, 65-
70.
of
multistep
risk assesmant.
carcinogenesis: Environ.
Health
43
43 - 47
Elsevier Scientific Publishers Ireland Ltd.
Detoxication products of the carcinogenic azodye Sudan I (Solvent Yellow 14) bind to nucleic acids after activation by peroxidase Marie Stiborovba,
Eva Freib, Heinz H. Schtneiserb,
Manfred Wiesslerb and Jan Hradecc
of Biochemistry, Facul@ of Natural Sciences, Charles Uniuersity, Albertou 2030, 128 40 Prague 2 (Czechoslooakia), blnstitute of Toxicology and Chemotherapy, German Cancer Research Center, Im Neuenheimer Fe/d 280, 6900 Heideberg {Germany) and ‘Department of Experimental Oncology, Institute of Radiotherapy, 180 00 Prague 8 (Czechoslovakia) ‘Department
(Received 14 April 1992) (Accepted 23 October 1992)
Summary
Introduction
The C-hydroxyderiuatiues of the carcinogenic dye Sudan 1, 1 -phenylazo-2,6-dihydroxynaphthalene and 7-(4-hydroxyphenylazo)Z-hydroxynaphthaiene, which are considered to be detoxication products of this dye bind to DNA or tRNA after oxidation into actiue metabolites by peroxidase and H202 in oitro. The 3ZP-postlabeling analysis of DNA modi-
It was reported by IARC that the nonaminoazo dye l-phenylazo-2-hydroxynaphthalene (Sudan I, Solvent Yellow 14) was used as food colouring in several countries. This dye has, however, been recommended as unsafe for use in food. Nevertheless, it is widely used to colour other materials [ 11. The compound is carcinogenic leading to tumors in the liver and/or urinary bladder in rats, mice and rabbits [ 1 - 31. In vivo studies on the metabolism of Sudan I in rabbits revealed that this compound is oxidized in the liver by cytochrome P-450 to derivatives hydroxylated in the aromatic rings. The resulting products 1-(4-hydroxyphenyIazo)-2-hydroxynaphthalene (4’OH-Sudan I), 1-phenylazo-2,6-dihydroxynaphthalene (6-OH-Sudan I) and 1-(4-hydroxyphenyIazo)-2,6_dihydroxynaphthalene were detected unconjugated or conjugated with glucuronic acid in both bile and urine of animals exposed to this dye [4]. 4 ’ -OH-Sudan I and 6-OH-Sudan I were found to be the major products of Sudan I oxidation by microsomal cytochrome P-450 in vitro and in vivo [1,4,5]. Hydroxylation of aromatic rings is considered to be a common detoxication pathway characteristic for carcinogenic
fied by active metabolites of both Sudan I derivatives prouides evidence that the covalent binding to DNA is the principal type of DNA modification. Since the urinary bladder is rich in peroxidases, the participation of these enzymes in actiuation of detoxicating products of Sudan I may be inuolued in the inition of Sudan I-carcinogenesis in this organ.
Keywords: azo dye; detoxication and activation of cacinogens; peroxidase; DNA- and tRNA-binding
Correspondence
to: M. Stiborovl,
Department
Faculty of Natural Sciences, Charles University, 128 40 Prague 2, Czechoslovakia.
0304~3835/92/$05.00
0
1992
Printed and Published in Ireland
of Biochemistry, Albertov 2030,
Elsevier Scientific Publishers Ireland Ltd.
44
compounds [Z]. The question, however, arises whether the detoxicating metabolites of Sudan I could not be the substrate of other enzymes, which would activate these detoxicating products to ultimate carcinogens. Besides cytochrome P-450 which both detoxicates and activates Sudan I in livers [4 - 71, peroxidase is also supposed to be implicated in the Sudan I activation in the tissues rich in this enzyme [6,7]. Moreover, since C-hydroxyderivatives of Sudan I were found as a major metabolic excreting products in urines we focus our attention to find whether these compounds are activated by peroxidase, which is abundant in the urinary bladder. Material and Methods
Radiochemicals 14C-Sudan I (0.54 mCi/mmol) was synthesized as described earlier [5] and converted to its C-hydroxyderivatives by incubation with rat liver microsomal enzymes and NADPH [5]. Both 4’-OH-Sudan I and 6-OH-Sudan I were separated from the reaction mixture by thin layer chromatography on silica gel G (Woelm) [5] and purified by rechromatography under the same conditions, Incubations Incubations for the binding experiments of both metabolites to nucleic acids were composed as described earlier [6,7] and contained 0.2 mM of the 14C-labeled compound (r*CSudan I or its 14C-labeled hydroxyderivatives) dissolved in methanol (40 ~I/250 ~1 incubation) , horseradish peroxidase (Boehringer) and H202. They were incubated, unless otherwise stated, for 60 min and 37OC. Reaction mixtures were extracted with ethyl acetate, DNA or tRNA were isolated and its radioactivity determined as described earlier [6,7]. The ethylacetate extracts from the reaction mixtures containing radioactive 14Clabeled hydroxyderivatives of Sudan I and their products were evaporated under a stream of nitrogen, dissolved in a minimal volume of methanol and chromatographed on thin layer
of silica gel and eluted with diethyl ether/nhexan (3: 1, v/v). The residual [t4C]4’-OHSudan I or [r4C]6-OH Sudan I and their products were mechanically separated from plates, placed in a scintillation vial and the radioactivity was counted. 32P-Labeling of the DNAdigest and recovery of individual nucleotide adducts. Chemicals and materials for DNA digestion and 32P-postIabeling were from sources reported previously [8,9]. DNA modified by hydroxyderivatives of Sudan I used for the 32P-labeling of digest was prepared by incubation with peroxidase system and isolated as described above. Control incubations were performed without the addition of the compound (methanol was used instead of the compounds). DNA was digested to deoxyribonucleoside 3-monophosphates using micrococcal nuclease and spleen phosphodiesterase and then with nuclease Pl as described [8,9]. The digested DNA was then 32P-labeled using polynucleotide kinase and [y-32P]ATP (200 &i) synthesized in the laboratory from ADP and [32P]orthophosphate also as described previously [8]. Labeled digests were chromatographed on thin layers of PEI-celulose in a similar way to that described previously [8], except that 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, 8.5 M urea, pH 8.0 and followed a final wash with 1.7 sodium phosphate, pH 6.0 D2 was omitted. Results and Discussion
Like the parent compound Sudan I, both its C-hydroxyderivatives did not bind to nucleic acids when incubated with DNA or tRNA in the absence of peroxidase, H202 or both. However, efficient blinding of metabolites of both these derivatives occurred in complete incubations. When compared to Sudan I, the oxidation of 6-OH-Sudan I was also effective whereas significantly lower amounts of 4’-OHSudan I were oxidized by the peroxidase system (Table I). The products of peroxidase activation of both hydroxymetabolites bound
45
Table1. Binding of Sudan I, [ 14C] 4’-OH-Sudan calf thymus
I and [r4C] 6-OH-Sudan
I activated
by the peroxidase
system to
DNA and rat liver tRNA in vitro
Compound
DNA binding nmol/mg
tRNA binding
% of converted Sudan I and its hydroxyderivatives
I 14C]Sudan I [ “kZ]4 ’ -OH-Sudan I [ “C]6-OH-Sudan 1
4.2 f 0.3 2.1 f 0.2 3.3 f 0.2
14.8 f 0.9 1.9 f 0.1 5.9 f 0.5
85.3 60.1 93.3
f 0.9 + 0.6 f 0.9
The average binding levels and standard deviations were obtained from triplicate determinations. The DNA- and tRNAbinding was assayed as described [6,7]. The binding levels of the control samples (without H202 or without peroxidase) for 4’-OH-Sudan I were 0.3 and 0 nmol/mg, respectively, for 6-OH-Sudan I 0.4 and 0 nmol/mg, respectively.
less efficiently to DNA and in particular, to tRNA than the products formed from Sudan I by peroxidase (Table I) [6,7]. Unlike the the preferential binding of parent dye, metabolites of both C-hydroxyderivatives to tRNA was far less pronounced. The binding of C-hydroxyderivatives of Sudan I to nucleic acids after activation by peroxidase was measured at pH 4.6, 7.46 and 8.4. The highest levels of binding were detected at pH 8.4 (results not shown). The nucleic acids modified by metabolites of both compounds studied were isolated from the reaction mixtures by the procedures which included phenol/chloroform extraction [6,7]. This suggested that a covalent binding of ultimate form(s) of compounds is the predominant modification. The covalent binding of metabolites was confirmed by the 32Ppostlabeling analysis. Several adducts of 4’OH-Sudan I and of 6-OH Sudan I were detected on the autoradiograms shown in Fig. 1. The chromatographic conditions chosen were suitable for separation of adducts formed from aromatic carcinogens with a pronounced hydrophobicity [8]. The adducts obtained from both C-hydroxyderivatives and DNA showed similar chromatographic properties as those formed from carcinogens like aristolochic acid, benzo[a]pyrene or Sudan I with DNA [lo]. Although the exact nature of adducts has not yet been elucidated their pronounced liphophilicity indicates that a polycyclic aromatic
system is covalently linked to the nucleic acid. The chromatographic behaviour of adducts of both Sudan I derivatives is different from that of parent Sudan I [7] and also the metabolites of both derivatives tested differ from each other in this respect. In addition to detoxication products (Chydroxyderivatives), Sudan I is in vivo also metabolized by reductive reactions to l-amino2-naphthol and p-aminophenol [ 111. The former compound is not carcinogenic [2,12]. The latter compound is mutagenic, but its carcinogenicity is questionable [13]. Both these products occur also in the urine either free or as glucuronides or sulphates [ 111. Activation of Sudan I and of its C-hydroxyderivatives is a prerequisite for their binding to DNA. This activation may be catalyzed by microsomal cytochrome P-450 [2,5,14] but also by peroxidases as reported in this paper. The urinary bladder contains low quantities of cytochrome P-450 but is rich in peroxidase [12,15,16]. In animals exposed to Sudan I, the urine contains only low quantities of the parent Sudan I [l] but is abundant in its Chydroxy derivatives resulting from the hydroxylation of the parent dye in the liver [ 111. Thus the products of detoxication of Sudan I may be converted to ultimate carcinogens by peroxidase and may form DNA adducts in the urinary bladder. Binding to DNA is characteristic for ultimate carcinogens [Z] and this interaction is believed to represent a fundamen-
46
tal step in the initiation of chemical carcinogenesis [7]. Since not only active metabolites of Sudan I [7] but also those of its detoxication products form DNA-adducts, C-hydroxyderivatives of this dye may be considered to be potential procarcinogens. This implies that the combination of detoxicating and activating reactions catalyzed by several enzymes (i.e. cytochrome P-450, peroxidases) seems to be crucial for the final formation of DNA adducts from carcinogens. The mechanism of organ specificity of Sudan I (and/or other carcinogens) has not yet been fully explained. Besides other mechanisms (the efficiencies of conjugating reactions for carcinogens in different organs, the stability of conjugates) the abilities of several enzymes (as well as their levels) to activate carcinogens in different organs is considered to be one of the most important mechanisms [7,18]. Because of findings presented in this paper, peroxidase could be supposed as one enzyme which may participate in formation of ultimate carcinogens and DNA adducts in the urinary bladder and, hence, it may contribute to the organ specificity of Sudan I. Nevertheless, it should be noted that cancer in a specific organ may also be a function of promotional pressures on initiated cells in target organs and not only the levels of DNA adducts [ 191. The function of promotional pressures in a Sudan Imediated tumorigenesis remains to be explained. References
Fig. 1. Autoradiograms of PEI-cellulose TLC maps of 32P-labeled digests of DNA treated with peroxidase, H202 and 4’-OH-Sudan I (A), 6-OH-Sudan I (B) and with the same system, but without derivatives of Sudan I (C). Analysis was performed by the nuclease Pl version Material and Methods). (see of the assay Autoradiography was at ambient temperature for 1 h. Origins are located at the bottom left corners (D3 from bottom
to top
and
D4 from
left to right).
IARC (1975) IARC Monographs. Sudan I. IARC, Lyon, Vol. 8, pp. 225-231. Garner, R.C., Martin, C.N. and Clayson, D.B. (1984) Carcinogenic aromatic amines and related compounds. In: Chemical Carcinogens. 3rd edition, Vol. 1, ACS Monograph No. 182, pp. 175-302, Editor: C.E. Searle. American Chemical Society, Washington DC. Westmoreland, C. and Gatehouse, D.G. (1991) The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observation of species and tissues specificity. Carcinogenesis, 12, 1403 - 1407. Childs, J.J. and Clayson, D.S. (1966) The metabolism of l-phenylazo-2-naphthol in the rabbit. Biochem. Pharmacol., 15, 1247- 1258.
47
5
Stiborova, M., Asfaw, 6.. Anzenbacher, P., LeJetickjr, 1. and Hodek, P. (1988) The first identification of the benzenediazonium ion formation from a non-aminoazodye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomes of rat livers. Cancer Lett., 40, 319-326. 6 Stiborova, M., Frei, E., Klokow, K., Wiessler, M., SafamC, L., Anzenbacher, P. and Hradec, J. (1990) Peroxidasemediated reaction of the carcinogenic anon-aminoazo dye 1-phenylazo-2-hydroxynaphthalene with transfer ribonucleic acid. Carcinogenesis, 11, 1789 - 1794. 7 Stiborova, M., Frei, E., Schmeiser, H.H., Wiessler, M. and Hradec, J. (1990) Mechanism of formation and 32Ppostlabeling of DNA adducts derived from peroxidative activation of carcinogenic non-aminoazo dye I-phenylazo-2hydroxynaphthalene (Sudan I). Carcinogenesis, 11, 1843 - 1848. 8 Reddy, M.V. and Randerath, K. (1986) Nuclease Plmediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543 - 1551. 9 Schmeiser, H., Diple, A., Schurdak, M.E., Randerath, E. and Randerath, K. (1988) Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz(a)anthracene-DNA adducts. Carcinogenesis, 9, 633 - 638. 10 Schmeiser, H.H., Schoepe, K.-B. and Wiessler, M. (1988) DNA adduct formation of aristolochic acid I and II in vitro and in vivo. Carcinogenesis, 9, 297-303. 11 Daniel, J.W. (1962) The excretion and metabolism of edible food colours. Toxicol. Appl. Pharmacol., 4, 572 - 594.
12
13
14
15
16
17
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