Carcinogenesisvol.il no.11 pp. 1915-1919, 1990
In vitro prostoglaedm HI symtlhiase- and moiiooxygeiniase-medtaited toimdiinig of afflatoxim IBj to DNA nm gmmea-pig tissue microsomes
Ling Liu1, Jonathan M.Daniels1, Richard K.Stewart1 and Thomas E.Massey1'2-3 Departments of 'Pharmacology and Toxicology and 2Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 3
To whom correspondence should be addressed
Introduction Aflatoxin B, (AFB,*) is a mycotoxin produced by some strains of Aspergillus flavus and A.parasiticus which grow on grains and some other foodstuffs in warm and humid conditions (1). AFB, is a potent hepatocarcinogen in a wide number of species (2,3), and is also likely to be an important etiological agent in human liver cancer (1,4). Oxidative metabolism of AFB, is required to form an unstable 2,3-epoxide metabolite which can bind preferentially to the N7 position of guanine in cellular DNA (5,6). •Abbreviations: AF, aflatoxin; P450, cytochrome P450 monooxygenase; AA, arachidonic acid; B[a]P-7,8-diol, benzo[a]pyrene-7,8-dihydrodiol; PHS, prostaglandin H synthase; BPB, bromophenacyl bromide, CIP, chloroform/isoamyl alcohol/phenol; ANOVA, analysis of variance; [ 3 H]AFB|-DNA binding, covalent binding of [•'HJAFB, to DNA molecules.
© Oxford University Press
1915
Downloaded from http://carcin.oxfordjournals.org/ at University of California, San Fransisco on April 9, 2015
In order to study the mechanism of cancer production by aflatoxin B, (AFB,) in extrahepatic tissues which have relatively low cytochrome P450 monooxygenase (P450) activity, we have examined prostaglandin H synthase (PHS)mediated AFB, activation ([ 3 H]AFB,-DNA binding). [ 3 H]AFB, was activated by both purified PHS and microsomal PHS from guinea-pig kidney and liver, as well as by P450 in lung, kidney and liver microsomes, though P450-mediated [ 3 H]AFB,-DNA binding in lung and liver was much higher than that catalyzed by PHS. Arachidonic acid (AA)-dependent [ 3 H]AFB,-DNA binding could be inhibited by the PHS inhibitor indomethacin (0.1 mM), but was enhanced by the P450 inhibitor SKF-525A (3 mM), confirming that the reaction was independent of P450. Pulmonary PHS-mediated [ 3 H]AFB,-DNA binding was < 0 . 1 pmol [ 3 H]AFB,/mg protein/min. HPLC analysis showed only minimal formation of [3H]AFM, and [3H]AFQ, by PHS, confirming that the low rate of PHS-dependent [ % ] A F B i - D N A adduct formation was not due to conversion of AFB, to other metabolites by PHS. The omission of AA did not diminish [3H]AFB, —DNA binding. In AA-free incubates, indomethacin inhibited, and SKF-525A enhanced, [3H]AFB, -DNA binding to a similar degree as in complete incubates, indicating that DNA binding in AA-free incubates was catalyzed by PHS. This reaction was also inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor, by 92%. These data are consistent with previous reports indicating the ability of AFB, to stimulate the release of endogenous AA from membranes, presumably by stimulating phospholipase A2 activity, which may lead to enhanced bioactivation of AFB, by PHS in vivo.
Although the liver is normally considered the principal target organ, AFB|-induced tumors in lungs and kidneys of experimental animals have also been reported (7,8). Futhermore, AFB, has been found in pulmonary tissues of some lung cancer patients who had been exposed to A^ZavMS-contaminated dusts by inhalation (9). An epidemiological study also showed that peanut-processing workers exposed to AFB,-contaminated dusts had increased mortalities due to respiratory cancer, compared to unexposed cohorts (10). Agricultural surveys have demonstrated that AFB| is frequently present in respirable dusts in grain terminals at levels higher than those found in contaminated food products (11), and intravenously administered AFB, and metabolites are excreted in the urine of monkeys and rats (12). Thus, both the lung and the kidney may be exposed to elevated concentrations of AFB, under certain conditions. In addition, AFB, can be activated to a DNA-binding metabolite(s) in cultured animal and human lung epithelial cells (13—15), and AFB, macromolecular adducts are formed in kidney tissue (16). Most investigations into the mechanism of AFB, activation have focused on cytochrome P450 monooxygenase (P450), a pivotal enzyme system in hepatic AFB, metabolism. However, during the synthesis of prostaglandins from arachidonic acid (AA), some xenobiotics, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol) and AFB,, can be oxidized by peroxyl radicals generated via interactions of lipid hydroperoxides with the heme group of prostaglandin H synthase (PHS), and thereby undergo bioactivation (17). There is evidence that AFB, can be activated by PHS in mouse embryo fibroblasts (18) and ram seminal vesicle microsomes (19). In ram seminal vesicle microsome-mediated activation, the major DNA adduct was identified as 2,3-dihydro-2-(jV7-guanyl)-3-hydroxy-AFB,, the same adduct produced by P450-dependent activation (19). Also, AFB| has been found to stimulate the production of prostaglandins E2 and F ^ in canine kidney endothelial cells (20). Moreover, relatively high PHS activity occurs in kidney and lung, so PHS may serve as an alternative enzyme for xenobiotic activation, particularly in tissues with low P450 activity. To our knowledge, there are no in vivo data available on the susceptibility of guinea-pigs to AFB, -induced pulmonary and renal carcinogenesis, or in vitro data regarding AFB, bioactivation in guinea-pig lung and kidney preparations. However, guinea-pigs have been shown to be more sensitive to the acute hepatotoxicity of AFB] than either rats or mice (21), and cultured renal cells from guinea-pig were more susceptible to cytotoxicity than were those from rats (22). Furthermore, following in vivo administration of radiolabelled AFB, to various species, guinea-pig liver had relatively high levels of AFB, binding to macromolecules (23). Moreover, guinea-pig lung and kidney have been reported to have high PHS activity (24), and its lung is capable of catalyzing the co-oxidation of B[a]P-7,8-diol (25,26). So far as we know, the activation of AFB, by pulmonary and renal PHS has not been examined. Therefore, the present study, focusing on the role of PHS and P450 in catalyzing the covalent binding of AFB, to DNA in guinea-pig lung,
L.Liu et al.
kidney and liver microsomes, provides new data regarding the mechanism(s) of AFB) activation in this species. Materials and methods Chemicals Chemicals were obtained as follows: [3H]AFB| from Moravek Biochemicals, Inc., Brea, CA, USA (99.2% purity by TLC); AA from Nu-Chek-Prep, Inc., Elysian, MN, USA; purified PHS from Oxford Biomedical Research, Inc., Oxford, MI, USA;. SKF-525A from Smith, Kline and French, Montreal, Canada, calf thymus DNA, hematin, NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, indomethacin, 4-bromophenacyl bromide (BPB), protease XXIV, ribonuclease A, ribonuclease Tl, and non-radioactive AFB|, aflatoxins M, (AFM,) and Q, (AFQ,) from Sigma Chemical Co., St Louis, MO, USA. All other materials were of reagent grade and were obtained from common commercial suppliers.
Data analysis and statistics The results are expressed as mean ± SD. Statistically significant differences were determined by repeated measures one-way analysis of variance (ANOVA), or by one-way ANOVA, followed by Newman-Keuls test. When Cochran's test
o o
rn
° 4
ND AA
AA+ID
ND -AA
3
Fig. 1. [ H]AFB,-DNA binding catalyzed by purified PHS. Purified PHS (300 U) was incubated with 0.2 mM AA, 2.2 /*M hematin, 1 mg/ml calf thymus DNA, 50 ^M [3H]AFB, (10 mCi/mmol) in pH 7.8 phosphate buffer for 60 min. Results are expressed as mean ± SD from three or four separate experiments, in which assays were performed in duplicate. Values for boiled controls (42.2 ± 25.7 pmol [3H]AFB,/3OO U) were subtracted Determination of microsomal PHS- and P450-mediated [3H]AFB/—DNA binding from all activities ID, indomethacin (0.1 mM) ND, not detectable (
In vitro prostoglaedm HI symtlhiase- and moiiooxygeiniase-medtaited toimdiinig of afflatoxim IBj to DNA nm gmmea-pig tissue microsomes
Ling Liu1, Jonathan M.Daniels1, Richard K.Stewart1 and Thomas E.Massey1'2-3 Departments of 'Pharmacology and Toxicology and 2Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6 3
To whom correspondence should be addressed
Introduction Aflatoxin B, (AFB,*) is a mycotoxin produced by some strains of Aspergillus flavus and A.parasiticus which grow on grains and some other foodstuffs in warm and humid conditions (1). AFB, is a potent hepatocarcinogen in a wide number of species (2,3), and is also likely to be an important etiological agent in human liver cancer (1,4). Oxidative metabolism of AFB, is required to form an unstable 2,3-epoxide metabolite which can bind preferentially to the N7 position of guanine in cellular DNA (5,6). •Abbreviations: AF, aflatoxin; P450, cytochrome P450 monooxygenase; AA, arachidonic acid; B[a]P-7,8-diol, benzo[a]pyrene-7,8-dihydrodiol; PHS, prostaglandin H synthase; BPB, bromophenacyl bromide, CIP, chloroform/isoamyl alcohol/phenol; ANOVA, analysis of variance; [ 3 H]AFB|-DNA binding, covalent binding of [•'HJAFB, to DNA molecules.
© Oxford University Press
1915
Downloaded from http://carcin.oxfordjournals.org/ at University of California, San Fransisco on April 9, 2015
In order to study the mechanism of cancer production by aflatoxin B, (AFB,) in extrahepatic tissues which have relatively low cytochrome P450 monooxygenase (P450) activity, we have examined prostaglandin H synthase (PHS)mediated AFB, activation ([ 3 H]AFB,-DNA binding). [ 3 H]AFB, was activated by both purified PHS and microsomal PHS from guinea-pig kidney and liver, as well as by P450 in lung, kidney and liver microsomes, though P450-mediated [ 3 H]AFB,-DNA binding in lung and liver was much higher than that catalyzed by PHS. Arachidonic acid (AA)-dependent [ 3 H]AFB,-DNA binding could be inhibited by the PHS inhibitor indomethacin (0.1 mM), but was enhanced by the P450 inhibitor SKF-525A (3 mM), confirming that the reaction was independent of P450. Pulmonary PHS-mediated [ 3 H]AFB,-DNA binding was < 0 . 1 pmol [ 3 H]AFB,/mg protein/min. HPLC analysis showed only minimal formation of [3H]AFM, and [3H]AFQ, by PHS, confirming that the low rate of PHS-dependent [ % ] A F B i - D N A adduct formation was not due to conversion of AFB, to other metabolites by PHS. The omission of AA did not diminish [3H]AFB, —DNA binding. In AA-free incubates, indomethacin inhibited, and SKF-525A enhanced, [3H]AFB, -DNA binding to a similar degree as in complete incubates, indicating that DNA binding in AA-free incubates was catalyzed by PHS. This reaction was also inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor, by 92%. These data are consistent with previous reports indicating the ability of AFB, to stimulate the release of endogenous AA from membranes, presumably by stimulating phospholipase A2 activity, which may lead to enhanced bioactivation of AFB, by PHS in vivo.
Although the liver is normally considered the principal target organ, AFB|-induced tumors in lungs and kidneys of experimental animals have also been reported (7,8). Futhermore, AFB, has been found in pulmonary tissues of some lung cancer patients who had been exposed to A^ZavMS-contaminated dusts by inhalation (9). An epidemiological study also showed that peanut-processing workers exposed to AFB,-contaminated dusts had increased mortalities due to respiratory cancer, compared to unexposed cohorts (10). Agricultural surveys have demonstrated that AFB| is frequently present in respirable dusts in grain terminals at levels higher than those found in contaminated food products (11), and intravenously administered AFB, and metabolites are excreted in the urine of monkeys and rats (12). Thus, both the lung and the kidney may be exposed to elevated concentrations of AFB, under certain conditions. In addition, AFB, can be activated to a DNA-binding metabolite(s) in cultured animal and human lung epithelial cells (13—15), and AFB, macromolecular adducts are formed in kidney tissue (16). Most investigations into the mechanism of AFB, activation have focused on cytochrome P450 monooxygenase (P450), a pivotal enzyme system in hepatic AFB, metabolism. However, during the synthesis of prostaglandins from arachidonic acid (AA), some xenobiotics, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol) and AFB,, can be oxidized by peroxyl radicals generated via interactions of lipid hydroperoxides with the heme group of prostaglandin H synthase (PHS), and thereby undergo bioactivation (17). There is evidence that AFB, can be activated by PHS in mouse embryo fibroblasts (18) and ram seminal vesicle microsomes (19). In ram seminal vesicle microsome-mediated activation, the major DNA adduct was identified as 2,3-dihydro-2-(jV7-guanyl)-3-hydroxy-AFB,, the same adduct produced by P450-dependent activation (19). Also, AFB| has been found to stimulate the production of prostaglandins E2 and F ^ in canine kidney endothelial cells (20). Moreover, relatively high PHS activity occurs in kidney and lung, so PHS may serve as an alternative enzyme for xenobiotic activation, particularly in tissues with low P450 activity. To our knowledge, there are no in vivo data available on the susceptibility of guinea-pigs to AFB, -induced pulmonary and renal carcinogenesis, or in vitro data regarding AFB, bioactivation in guinea-pig lung and kidney preparations. However, guinea-pigs have been shown to be more sensitive to the acute hepatotoxicity of AFB] than either rats or mice (21), and cultured renal cells from guinea-pig were more susceptible to cytotoxicity than were those from rats (22). Furthermore, following in vivo administration of radiolabelled AFB, to various species, guinea-pig liver had relatively high levels of AFB, binding to macromolecules (23). Moreover, guinea-pig lung and kidney have been reported to have high PHS activity (24), and its lung is capable of catalyzing the co-oxidation of B[a]P-7,8-diol (25,26). So far as we know, the activation of AFB, by pulmonary and renal PHS has not been examined. Therefore, the present study, focusing on the role of PHS and P450 in catalyzing the covalent binding of AFB, to DNA in guinea-pig lung,
L.Liu et al.
kidney and liver microsomes, provides new data regarding the mechanism(s) of AFB) activation in this species. Materials and methods Chemicals Chemicals were obtained as follows: [3H]AFB| from Moravek Biochemicals, Inc., Brea, CA, USA (99.2% purity by TLC); AA from Nu-Chek-Prep, Inc., Elysian, MN, USA; purified PHS from Oxford Biomedical Research, Inc., Oxford, MI, USA;. SKF-525A from Smith, Kline and French, Montreal, Canada, calf thymus DNA, hematin, NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, indomethacin, 4-bromophenacyl bromide (BPB), protease XXIV, ribonuclease A, ribonuclease Tl, and non-radioactive AFB|, aflatoxins M, (AFM,) and Q, (AFQ,) from Sigma Chemical Co., St Louis, MO, USA. All other materials were of reagent grade and were obtained from common commercial suppliers.
Data analysis and statistics The results are expressed as mean ± SD. Statistically significant differences were determined by repeated measures one-way analysis of variance (ANOVA), or by one-way ANOVA, followed by Newman-Keuls test. When Cochran's test
o o
rn
° 4
ND AA
AA+ID
ND -AA
3
Fig. 1. [ H]AFB,-DNA binding catalyzed by purified PHS. Purified PHS (300 U) was incubated with 0.2 mM AA, 2.2 /*M hematin, 1 mg/ml calf thymus DNA, 50 ^M [3H]AFB, (10 mCi/mmol) in pH 7.8 phosphate buffer for 60 min. Results are expressed as mean ± SD from three or four separate experiments, in which assays were performed in duplicate. Values for boiled controls (42.2 ± 25.7 pmol [3H]AFB,/3OO U) were subtracted Determination of microsomal PHS- and P450-mediated [3H]AFB/—DNA binding from all activities ID, indomethacin (0.1 mM) ND, not detectable (
