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Enhancement of mutagenicity by hydroxypyrenes in Salmonella
Cancer Letters, 12 (1981) 323-327 o Elsev&r/North-Holland Scientific PublishersLtd.
ENHANCEMENT
OF MUTAGENICITY
323
BY HYDROXYPYRENES
IN
SALMONELLA
HITOSHI OKAMOTO and DAISUKE YOSHIDA Central Research Institute, The Japan Tobacco Umegaoka, Midori-ku, Yokohama 227 (Japan)
and Salt Public Corporation,
6-2,
(Received 19 January 1981) (Accepted 12 February 1981)
SUMMARY
1-Hydroxypyrene and 4,5dihydro_4,5dihydroxypyrene were isolated and identified in the metabolites of pyrene by rat liver microsomal fraction, and the presence of l&3- and 1,6-dihydroxypyrene in the metabolites was proposed. These hydroxypyrenes, as well as pyrenequinones reported previously, enhanced intensively the mutagenicity of 2-acetylaminofluorene (AAF).
INTRODUCTION
We previously reported that the pyrolysis products of carbohydrates enhanced the mutagenicities of some aromatic amines toward Salmonella typhimurium TA98 and TAlOO [ 31, and that the fractions, having high enhancement activity, were isolated from the pyrolysis product of cellulose [4]. Pyrene and 1-methylpyrene were detected in one of the fractions [H. Okamoto and D. Yoshida, unpublished data] and increased the mutagenicity of AAF several times in the presence of rat liver microsomal fraction (S-9) [ 51. Pyrne must be metabolized to expose the enhancing activity, In the previous report, 1,6- and l&pyrenequinone were isolated and identified from the metabolites of pyrene [ 51 and both showed potent enhancing activity [6]. During the course of isolation of pyrenequinones, the other fractions, having high enhancement activity, have been detected but the chemical natures remained obscure. In this paper, we report the enhancing effect of the other metabolic components of pyrene on the mutagenicity of AAF. MATERIALS AND METHODS
Incubation
Incubation mixtures contained 5 E.tmol pyrene, 40 bmol MgClz, 168 pmol 17.4 I.tmol NADPH, 0.5 mmol sodium KCl, 22.4 I.cmol glucose 6-phosphate,
324
phosphate buffer (pH 7.4) and 0.5 ml rat liver S-9 treated with polychlorinated biphenyl (PCB). Total volume was 5.2 ml and was incubated for 30 min at 37°C. The reaction mixture was extracted with approximately 80 ml of ethyl acetate. Extracts were combined from 6 incubations, dried over anhydrous sodium sulfate, evaporated to dryness and dissolved in methanol for analysis by high-pressure liquid chromatography (HPLC). High-pressure liquid chromatography The HPLC was carried out as previously described [ 51. A 25-cm Zorbax ODS column maintained at 50°C was eluted with a mixture of methanol and water (70 : 30, v/v). The effluent was monitored by UV absorption at 243 nm, divided according to the individual peak and concentrated in an evaporator to dryness. A part of each condensate was used for the mutagenesis assay and the another part was used for the identification of the metabolites. Mutagenesis assay Enhancement of mutagenicity was determined as reported previously [ 51. Mutagenicity assay was carried out according to the method of Ames et al. [l]. Mixture of tester strain of Salmonella typhimurium TA98,3 pg AAF, sample suitably diluted in dimethyl sulfoxide and 0.5 ml S-9 mix (S-9: 50 pi/plate) was poured onto an agar over layer. The number of histidine revertant colonies per plate was counted after incubation for 2 days at 37°C. S-9 was prepared from rats induced with PCB as described by Ames et al.
[Il. Identification of pyrene metabolites The individual metabolite of the effluents from HPLC was identified using some of the following measurements; the spectra of direct inlet low and high resolution mass, UV and fluorescence, and melting point. RESULTS
AND DISCUSSION
Figure 1 shows the HPLC profile of pyrene metabolites by S-9 mix. The peaks of 6 and 7 show 1,8- and 1,6-pyrenequinone, respectively, as previously reported [ 51, and the peak 9 shows the unreacted pyrene. The enhancing effects of the metabolites of these peaks on the mutagenicity of AAF are shown in Table 1. Among these metabolites, l&pyrenequinone and the metabolite of peak 8 showed the highest enhancing activity. Although the metabolites of peak 2,3,4 and 7 also enhanced the mutagenicity of AAF, the enhancing activities were comparatively weak. The enhancement of mutagenicity was slight by the metabolites of peak 1 and 5 and metabolites themselves were not mutagenic. The white crystal (compound 8) was obtained from the peak 8. Mass spectrum of the compound 8 showed a molecular ion (M’) at m/z 218 and
325
0
2
4
6
Ii--0
3 6 Retention
9 time
12 ( min)
28
Fig. 1. HPLC profile of pyrene metabolites. A 25cm Zorbax ODS column maintained at 50°C was eluted using a mixture of methanol and water (70 : 30, v/v) with 1.5 ml/min of the flow rate. The chart shows the profile monitored at 243 nm of UV wave length. Inset box shows re-injection (solvent: methanol/water, 60 : 40) of the purified materials obtained from peaks 2 and 3.
TABLE 1 ENHANCING EFFECT OF PYRENE METAROLITES Metabolite (peak no.)
1 2 3 4 5 6 7 8 9 None
ON MUTAGENICITY
His+ colonies per plate With AFF (3 rg)
Without AAF
IO /Jga
20 pgs
IO rrga
20 /.lga
161 359 258 269 181 801 536 680 543
168 572 434 426 230 511 544 799 400
26 36 44 43 41 61 49 38 46
39 43 34 34 36 67 44 53 59
150
35
-
The mutagenesis test was carried out using S. typhimurium TA98 in the presence of S-9 mix. Figures are expressed as average number of revertant colonies from duplicate plates. B Dose of metabolite.
326
fragment ion at m/z 189 (M’-29). The molecular formula of compound 8 was determined to be C16H1001 by a high resolution mass spectrometry (calculated, 218.0731; observed, 218.0744). Compound 8 showed m.p. 174-l 80°C and UV spectrum (h max at 242,268,278,347,365 and 385 nm) in methanol. The structure of compound 8 was identified as l-hydroxypyrene by mass spectrum and the comparison of m.p. [8], UV spectrum and the behavior on thin layer chromatography (TLC) [ 21. The mass spectrum of crystahine compound (compound 4) obtained from the peak 4 showed a molecular ion at m/z 236 and fragment ion at m/z 176 (M’-60). Exact mass measurement of compound 4 gave the elemental composition C16H1202 (calculated, 236.0837; observed, 236.0811). The UV spectrum of compound 4 showed h,= at 220, 257, 263, 278, 286 and 298 nm. The structure of compound 4 was identified as 4,5-dihydro-4,5dihydroxypyrene by above mentioned spectra and the properties of TLC [ 21. The direct inlet mass spectra of compounds (compound 2 and 3, respectively) contained in peaks 2 and 3 showed a molecular ion at m/z 234 and fragment ion at m/z 205 (M’-29) and m/z 176 (M’-58), respectively. The structures of compound 2 and 3 were proposed as 1,8dihydroxypyrene and 1,6-dihydroxypyrene, respectively, by the fact that the fluorescence spectra of compound 2 and 3 showed the same pattern as 1,8- and 1,6-pyrenequinone, respectively, and the mass spectra of 1,8- and 1,6-pyrenequinone gave a molecular ion at m/z 232 and fragment ion at m/z 204 (M’-28) and m/z 176 (M’-56), respectively. The structures of respective contents of peaks 1 and 5 are unknown, although the latter mass spectrum gave a molecular ion at m/z 169.However, these contents hardly contributed to enhancement activity. The metabolites of benzo[a]pyrene by rat liver microsomes have been separated using the same reversed phase column and carrier solvent system as we used for HPLC [ 7,9]. From the fact that some diol-, quinone- and phenol-forms from benzo[a]pyrene metabolites successively are eluted in turn from column, we hypothesized in a previous report [5] that dioland phenol-pyrenes would produce before and behind pyrenequinones on the chromatogram, respectively. It was demonstrated that the individual metabolite of pyrene was eluted in such order as the metabolites of benz[a] pyrene.
1,8-Pyrenequinone and l-hydroxypyrene showed the higher enhancing of mutagenicity than the other metabolites isolated from pyrene metabolites. This shows that these metabolites might mainly contribute to the exhibition of the enhancing activity of mutagenicity by pyrene. The mechanism of the enhancing action of mutagenicity by 1,8-pyrenequinone which showed the highest activity among metabolites of pyrene will be further studied. activity
ACKNOWLEDGEMENTS
We wish to thank Mr. F. Goto of this Institute
for his encouragement.
327 REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Boyland, E. and Sims, P. (1964) Metabolism of polycyclic compounds. 23. The metabolism of pyrene in rats and rabbits. Biochem. J., 90, 391-398. 3 Okamoto, H., Mizusaki, S., Yoshida, D. and Matsumoto, T. (1979) Enhancing effect of carbohydrate pyrolysates on mutagenesis in Salmonella typhimurium. Agric. Biol. Chem., 32,1433-1438. 4 Okamoto, H., Yoshida, D., Matsumoto, T. and Mizusaki, S. (1980) Enhancement of mutagenicity by the fractions of cellulose pyrolysate. Agric. Biol. Chem., 44, 563566. 5 Okamoto, H. and Yoshida, D. (1980) Metabolic formation of pyrenequinones as enhancing agents of mutagenicity in Salmonella. Cancer Letters, 11,215-220. 6 Okamoto, H. and Yoshida, D. (1980) Pyrenequinones ss mutagens and enhancing agents to other mutagens. Mutat. Res., 73,203-207. 7 Selkirk, J.K. (1980) Comparison of epoxide and free radical mechanisms for activation of benzo(a)pyrene by Sprague-Dawley rat liver microsomes. J. Natl. Cancer Inst., 64, 771-774. 8 Vollmann, H., Becker, H., Corell, M. and Streeck, H. (1937) Beitriige zur Kenntnis des Pyrens und seiner Derivate. Liebigs Annal. Chem., 531, l-159. 9 Yang, S.K., Selkirk, J.K., Plotkin, E.V. and Gelboin, H.V. (1975) Kinetic analysis of the metabolism of benzo(a)pyrene to phenols, dihydrodiols, and quinones by aryl hydrocarbon hydroxylase assay, and the effect of enzyme induction. Cancer Res., 35, 3642-3650.
ENHANCEMENT
OF MUTAGENICITY
323
BY HYDROXYPYRENES
IN
SALMONELLA
HITOSHI OKAMOTO and DAISUKE YOSHIDA Central Research Institute, The Japan Tobacco Umegaoka, Midori-ku, Yokohama 227 (Japan)
and Salt Public Corporation,
6-2,
(Received 19 January 1981) (Accepted 12 February 1981)
SUMMARY
1-Hydroxypyrene and 4,5dihydro_4,5dihydroxypyrene were isolated and identified in the metabolites of pyrene by rat liver microsomal fraction, and the presence of l&3- and 1,6-dihydroxypyrene in the metabolites was proposed. These hydroxypyrenes, as well as pyrenequinones reported previously, enhanced intensively the mutagenicity of 2-acetylaminofluorene (AAF).
INTRODUCTION
We previously reported that the pyrolysis products of carbohydrates enhanced the mutagenicities of some aromatic amines toward Salmonella typhimurium TA98 and TAlOO [ 31, and that the fractions, having high enhancement activity, were isolated from the pyrolysis product of cellulose [4]. Pyrene and 1-methylpyrene were detected in one of the fractions [H. Okamoto and D. Yoshida, unpublished data] and increased the mutagenicity of AAF several times in the presence of rat liver microsomal fraction (S-9) [ 51. Pyrne must be metabolized to expose the enhancing activity, In the previous report, 1,6- and l&pyrenequinone were isolated and identified from the metabolites of pyrene [ 51 and both showed potent enhancing activity [6]. During the course of isolation of pyrenequinones, the other fractions, having high enhancement activity, have been detected but the chemical natures remained obscure. In this paper, we report the enhancing effect of the other metabolic components of pyrene on the mutagenicity of AAF. MATERIALS AND METHODS
Incubation
Incubation mixtures contained 5 E.tmol pyrene, 40 bmol MgClz, 168 pmol 17.4 I.tmol NADPH, 0.5 mmol sodium KCl, 22.4 I.cmol glucose 6-phosphate,
324
phosphate buffer (pH 7.4) and 0.5 ml rat liver S-9 treated with polychlorinated biphenyl (PCB). Total volume was 5.2 ml and was incubated for 30 min at 37°C. The reaction mixture was extracted with approximately 80 ml of ethyl acetate. Extracts were combined from 6 incubations, dried over anhydrous sodium sulfate, evaporated to dryness and dissolved in methanol for analysis by high-pressure liquid chromatography (HPLC). High-pressure liquid chromatography The HPLC was carried out as previously described [ 51. A 25-cm Zorbax ODS column maintained at 50°C was eluted with a mixture of methanol and water (70 : 30, v/v). The effluent was monitored by UV absorption at 243 nm, divided according to the individual peak and concentrated in an evaporator to dryness. A part of each condensate was used for the mutagenesis assay and the another part was used for the identification of the metabolites. Mutagenesis assay Enhancement of mutagenicity was determined as reported previously [ 51. Mutagenicity assay was carried out according to the method of Ames et al. [l]. Mixture of tester strain of Salmonella typhimurium TA98,3 pg AAF, sample suitably diluted in dimethyl sulfoxide and 0.5 ml S-9 mix (S-9: 50 pi/plate) was poured onto an agar over layer. The number of histidine revertant colonies per plate was counted after incubation for 2 days at 37°C. S-9 was prepared from rats induced with PCB as described by Ames et al.
[Il. Identification of pyrene metabolites The individual metabolite of the effluents from HPLC was identified using some of the following measurements; the spectra of direct inlet low and high resolution mass, UV and fluorescence, and melting point. RESULTS
AND DISCUSSION
Figure 1 shows the HPLC profile of pyrene metabolites by S-9 mix. The peaks of 6 and 7 show 1,8- and 1,6-pyrenequinone, respectively, as previously reported [ 51, and the peak 9 shows the unreacted pyrene. The enhancing effects of the metabolites of these peaks on the mutagenicity of AAF are shown in Table 1. Among these metabolites, l&pyrenequinone and the metabolite of peak 8 showed the highest enhancing activity. Although the metabolites of peak 2,3,4 and 7 also enhanced the mutagenicity of AAF, the enhancing activities were comparatively weak. The enhancement of mutagenicity was slight by the metabolites of peak 1 and 5 and metabolites themselves were not mutagenic. The white crystal (compound 8) was obtained from the peak 8. Mass spectrum of the compound 8 showed a molecular ion (M’) at m/z 218 and
325
0
2
4
6
Ii--0
3 6 Retention
9 time
12 ( min)
28
Fig. 1. HPLC profile of pyrene metabolites. A 25cm Zorbax ODS column maintained at 50°C was eluted using a mixture of methanol and water (70 : 30, v/v) with 1.5 ml/min of the flow rate. The chart shows the profile monitored at 243 nm of UV wave length. Inset box shows re-injection (solvent: methanol/water, 60 : 40) of the purified materials obtained from peaks 2 and 3.
TABLE 1 ENHANCING EFFECT OF PYRENE METAROLITES Metabolite (peak no.)
1 2 3 4 5 6 7 8 9 None
ON MUTAGENICITY
His+ colonies per plate With AFF (3 rg)
Without AAF
IO /Jga
20 pgs
IO rrga
20 /.lga
161 359 258 269 181 801 536 680 543
168 572 434 426 230 511 544 799 400
26 36 44 43 41 61 49 38 46
39 43 34 34 36 67 44 53 59
150
35
-
The mutagenesis test was carried out using S. typhimurium TA98 in the presence of S-9 mix. Figures are expressed as average number of revertant colonies from duplicate plates. B Dose of metabolite.
326
fragment ion at m/z 189 (M’-29). The molecular formula of compound 8 was determined to be C16H1001 by a high resolution mass spectrometry (calculated, 218.0731; observed, 218.0744). Compound 8 showed m.p. 174-l 80°C and UV spectrum (h max at 242,268,278,347,365 and 385 nm) in methanol. The structure of compound 8 was identified as l-hydroxypyrene by mass spectrum and the comparison of m.p. [8], UV spectrum and the behavior on thin layer chromatography (TLC) [ 21. The mass spectrum of crystahine compound (compound 4) obtained from the peak 4 showed a molecular ion at m/z 236 and fragment ion at m/z 176 (M’-60). Exact mass measurement of compound 4 gave the elemental composition C16H1202 (calculated, 236.0837; observed, 236.0811). The UV spectrum of compound 4 showed h,= at 220, 257, 263, 278, 286 and 298 nm. The structure of compound 4 was identified as 4,5-dihydro-4,5dihydroxypyrene by above mentioned spectra and the properties of TLC [ 21. The direct inlet mass spectra of compounds (compound 2 and 3, respectively) contained in peaks 2 and 3 showed a molecular ion at m/z 234 and fragment ion at m/z 205 (M’-29) and m/z 176 (M’-58), respectively. The structures of compound 2 and 3 were proposed as 1,8dihydroxypyrene and 1,6-dihydroxypyrene, respectively, by the fact that the fluorescence spectra of compound 2 and 3 showed the same pattern as 1,8- and 1,6-pyrenequinone, respectively, and the mass spectra of 1,8- and 1,6-pyrenequinone gave a molecular ion at m/z 232 and fragment ion at m/z 204 (M’-28) and m/z 176 (M’-56), respectively. The structures of respective contents of peaks 1 and 5 are unknown, although the latter mass spectrum gave a molecular ion at m/z 169.However, these contents hardly contributed to enhancement activity. The metabolites of benzo[a]pyrene by rat liver microsomes have been separated using the same reversed phase column and carrier solvent system as we used for HPLC [ 7,9]. From the fact that some diol-, quinone- and phenol-forms from benzo[a]pyrene metabolites successively are eluted in turn from column, we hypothesized in a previous report [5] that dioland phenol-pyrenes would produce before and behind pyrenequinones on the chromatogram, respectively. It was demonstrated that the individual metabolite of pyrene was eluted in such order as the metabolites of benz[a] pyrene.
1,8-Pyrenequinone and l-hydroxypyrene showed the higher enhancing of mutagenicity than the other metabolites isolated from pyrene metabolites. This shows that these metabolites might mainly contribute to the exhibition of the enhancing activity of mutagenicity by pyrene. The mechanism of the enhancing action of mutagenicity by 1,8-pyrenequinone which showed the highest activity among metabolites of pyrene will be further studied. activity
ACKNOWLEDGEMENTS
We wish to thank Mr. F. Goto of this Institute
for his encouragement.
327 REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Boyland, E. and Sims, P. (1964) Metabolism of polycyclic compounds. 23. The metabolism of pyrene in rats and rabbits. Biochem. J., 90, 391-398. 3 Okamoto, H., Mizusaki, S., Yoshida, D. and Matsumoto, T. (1979) Enhancing effect of carbohydrate pyrolysates on mutagenesis in Salmonella typhimurium. Agric. Biol. Chem., 32,1433-1438. 4 Okamoto, H., Yoshida, D., Matsumoto, T. and Mizusaki, S. (1980) Enhancement of mutagenicity by the fractions of cellulose pyrolysate. Agric. Biol. Chem., 44, 563566. 5 Okamoto, H. and Yoshida, D. (1980) Metabolic formation of pyrenequinones as enhancing agents of mutagenicity in Salmonella. Cancer Letters, 11,215-220. 6 Okamoto, H. and Yoshida, D. (1980) Pyrenequinones ss mutagens and enhancing agents to other mutagens. Mutat. Res., 73,203-207. 7 Selkirk, J.K. (1980) Comparison of epoxide and free radical mechanisms for activation of benzo(a)pyrene by Sprague-Dawley rat liver microsomes. J. Natl. Cancer Inst., 64, 771-774. 8 Vollmann, H., Becker, H., Corell, M. and Streeck, H. (1937) Beitriige zur Kenntnis des Pyrens und seiner Derivate. Liebigs Annal. Chem., 531, l-159. 9 Yang, S.K., Selkirk, J.K., Plotkin, E.V. and Gelboin, H.V. (1975) Kinetic analysis of the metabolism of benzo(a)pyrene to phenols, dihydrodiols, and quinones by aryl hydrocarbon hydroxylase assay, and the effect of enzyme induction. Cancer Res., 35, 3642-3650.