Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes Sugiyanto, C. E. Scharping, M. E. McManus, D. J. Birkett, G. M. Holder & A. J. Ryan To cite this article: Sugiyanto, C. E. Scharping, M. E. McManus, D. J. Birkett, G. M. Holder & A. J. Ryan (1992) The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes, Xenobiotica, 22:11, 1299-1307 To link to this article: http://dx.doi.org/10.3109/00498259209053158
Published online: 23 Apr 2010.
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Date: 09 December 2015, At: 04:18
XENOBIOTICA,
1992, VOL. 22,
NO.
11, 1299-1 307
The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes
Downloaded by [University of California, San Diego] at 04:18 09 December 2015
SUGIYANTOTS, C. E. SCHARPING?, M. E. McMANUS9, D. J. BIRKETTg, G. M. HOLDER*? and A. J. RYAN? Department of Pharmacy, University of Sydney, NSW 2006, Australia Clinical Pharmacology, Flinders Medical School, Bedford Park, South Australia 5042
5 Department of
Received 18 December 1991; accepted 30 April 1992
1. The metabolism of 3H-benzo[a]pyrene (BP), 3H-7-methylbenz[c]acridine (7MBAC) and 3H-dibenz[a,~lacridine(DBAJAC) have been studied in human liver microsomes from 13 subjects. 2. When the metabolism of these carcinogens to more polar ethyl acetate-soluble metabolites were compared, the activities towards the nitrogenous carcinogens were twice that determined for BP. 3. The specific rates of formation of the three proximate carcinogens, BP-7,Sdihydrodiol, 7MBAC-3,4-dihydrodioI and DBAJAC-3,4-dihydrodiol per nmol cytochrome P-450 for 12 subjects were positively correlated. 4. These dihydrodiols constituted 5.9 *0.7% (mean _+ SEM), 57.8 2.6%and 3.0 +0.4% of the total metabolites identified by cochromatography with standards, 7MBAC. DBAJAC and BP respectively.
Introduction Interest in the mechanism of initiation of cancer has extended to all aspects of investigations into the behaviour of carcinogens. This has included both the metabolic activation and detoxication of carcinogens, the mutagenic and carcinogenic potential of the parent carcinogen and its metabolites, and their reactivity with DNA (Jeffrey 1987, Yang and Silverman 1988). Among the different classes of chemical carcinogens the polycyclic aromatic hydrocarbons have probably received the greatest attention. T h e metabolic profiles of many of these compounds have been determined and the ultimate carcinogenic species identified (Yang and Silverman 1988, Thakker et al. 1985a). In addition, the stereochemistry associated with the formation of the carcinogenically active and inactive diolepoxides has been well characterized. T o date most of these studies have been carried out in vkvo in animals or in vitro using subcellular animal tissue fractions, cells and purified enzymes. Recently considerable attention has also been focused on the ability of human systems to metabolize benzo[a]pyrene (BP) (Ekstroem et al. 1982, Hall et al. 1989, Kremers et al. 1981, Prough et al. 1979, Souhali-El Amri et al. 1986). While polycyclic aromatic hydrocarbons such as BP have been extensively investigated, polycyclic aza-aromatic compounds which occur environmentally (Sawicki et al. 1965, van Duuren et al. 1960) have been essentially ignored. In the present study we report on *To whom correspondence should be addressed.
1Current address: Faculty of Pharmacy,
Gadjah Mada University, Yogyakarta 55281, Indonesia.
0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd
1300
Sugiyanto et al.
12
CH3
B
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7MBFlC
6
OBFlJRC
BP Figure 1. Structures and numbering of 7-methylbenz[c]acridine (7MBAC), dibenz[a, jlacridine (DBAJAC), and benzo[a]pyrene (BP).
the ability of human liver microsomes to metabolize 7-methylbenz[c]acridine (7MBAC) and dibenz[a, ilacridine (DBAJAC) (figure 1). For comparison we have also determined the ability of the same microsomal preparations to metabolize BP.
Methods Radiochemicals 3H-BP was purchased from Amersham Australia (17.4340 Ci/mmol), and 7-3H-7MBAC (2.6Cilrnmol) (Cheung et al. 1980) and 14-3H-DBAJAC (22.1 Ci/mmol) (Rosario et 01. 1987a) were available from their previous synthesis. All radioisotopically labelled chemicals were purified by extraction of impurities from a toluene solution with 0.1 5 M NaOH in 85% dimethyl sulphoxide (DMSO). Subsequent h.p.1.c. on the reverse-phase systems used to separate metabolites afforded materials of 97-99.5% radiochemical purity with no radioactive impurity peaks. Chemicals BP and NADPH were purchased from Sigma Chemical Co., St Louis, MO, USA, and BP metabolite standards were obtaimed from the NCI Chemical Carcinogen Repository at the Midwest Research Institute, Kansas City, MO, USA. These were trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene(BP4,5-DHD), trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-DHD), trans-9.10-dihydroxy9,10-dihydrobenzo[o]pyrene (BP-9,10-DHD), benzo[a]pyrene-1,6-quinone and its 6,12-, and 3,6isomers (BP-l,6-Q, BP-6,12-Q and BP-3, 6-Q respectively), and 3-hydroxybenzo[a]pyrene and the 9phenol (3-hydroxy-BP and 9-hydroxy-BP respectively). Four isomeric benzo[a]pyrene-7,8,9,10tetrahydrotetraols were also available these being the hydrolysis products of the two vicinal7,8-dioI9,10epoxides derived from BP-7,8-DHD. 7MBAC and standards, trans-3,4-dihydroxy-7-methy1-3,4-dihydrobenz[c]acridine(7MBAC-3,4DHD), trans-l,2-dihydroxy-7-methyl-l,2-dihydrobenz[c]acridine(7MBAC-1,2-DHD), trans-5,6dihydroxy-7-methyl-8,9-dihydrobenz[c]acridine (7MBAC-8,9-DHD), 7-hydroxymethylbenz[c]acridine (7-hydroxy-MBAQ 7MBAC-S,6-oxide, phenols 5-hydroxy-7-MBAC and 9-hydroxy-7MBAC and 7-hydroxymethylbenz[c]acridine-5,6-oxide(7HOMBAC-5,6-oxide) were available from previous syntheses (Boux et al. 1980,Duke et al. 1984, Lap et al. 1983). DBAJAC and its metabolites, trans-5,6-dihydroxy-5,6-dihydrodibenz[a, j ] acridine (DBAJAC-5,6DHD), trans-3,4-dihydroxy-3,4-dihydrodibenz[a,~~acridine (DBAJAC-3,4-DHD), the phenols 3hydroxy dibenz[a, jlacridine (3-hydroxy-DBAJAC) and 4-hydroxydibenz[a,~]acridine (4-hydroxyDBAJAC), dibenz[a,j]acridine-5,6-oxide (DBAJAC-5,6-oxide) and dibenz[a,jlacridine-N-oxide) had heen prepared previously (Rosario et al. 1987 b).
H.p.1.c. Separations of metabolites from each other and the parent compound were performed using methanol-water gradients on a twin pump Beckman 421 system with a Jasco Uvidec 100 V U.V.detector at 254nm for BP metabolites of 275nm for 7MBAC and DBAJAC metabolites. Separations of BP metabolites were performed at ambient temperature, while those of the nitrogenous compounds were performed at 40°C. T h e column was either a 10pm Merck Hibar RP-8 (25 x 0.46cm int. diam.) usingml/min and 1 min fractions for the radioactivity assay (system 1) o r a Brownlee 3 pm Velosep RP8
Human hepatic metabolism of two aza-arenes
1301
Table 1. Clinical data for liver donor
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Subject
Age Sex Smoking history
Alcohol consumption
H5
62
M Unknown
Unknown
H6 H7 H8 H9 H10
18 44 24 19 67
M F M M F
Social Social Social Social Social
H11
23
M None
Moderate
H12 H13 H14* H15
66 61 48 22
M None F None for 3 years F Unknown M Unknown
Minimal Minimal Unknown Unknown
sv 1
72
F None
Unknown
sv2
66
M Smoker
Unknown
Unknown None 20 cig/day None None
Drug treatment Phenytoin, dexamethasone, gentamicin, rinatidine, cotrimoxazole Penicillin, cotrirnoxazole Dexamethasone Dopamine Dopamine Dopamine, morphine, frusemide, rnidazolam Phenytoin, carbamazepine, fluphenazine Insulin Metoprolol, methyclothiazide Unknown Phenobarbitone, phenytoin, dexamethasone, methylprednisolone, morphine, dopamine, frusemide, atenolol, hydralazine, amoxil, gentamicin, vit. K, folic acid Metformin, propranolol, digoxin, hydrochlorthiazide, amiloride disopyramide Prazosin, pindolol
P-450 (nmol/mg) 0.77 0.30 0.32 0.2 1 0.33 0.22 0.26
0.53 0.3 3 0.22 033
0.73
0.22
*All liver donors were Caucasians except subject H14 who was of Asian origin.
(10x0.32cm int. diam.) using 0.7ml/min and 10 drop fractions (system 2). They were used interchangeably (Sugiyanto et al. 1990). T h e reverse-phase fractions of the 7MBAC metabolite which cochromatographed with the 3,4-dihydrodiol were combined, stripped of solvent, redissolved in about 2Opl CHCL, and further chromatographed on a Brownlee 10pm silica column (25 x 0.46cm int. diam.) using 2% ethanol in cyclohexane.
Human liver microsomes Thirteen samples of human liver microsomes were obtained principally from a liver bank stored at -80°C. Clinical details are shown in table 1. Most were obtained from organ donors with the exception of H12, H13 and SV2 which were taken at the time of liver resection for cancer, and SV1 which was taken from a patient with undiagnosed liver disease. T h e liver used in the last four samples was normal tissue. Microsomes were prepared and stored in 20% glycerol at -80°C until required (up to 4 years after collection), and no significant losses of cytochrome P-450 were seen between samples freshly assayed and at times of metabolic incubation in the current study. Cytochrorne P-450 was measured according to the method of Guengerich (1984). Metabolism Incubations (1.0ml) contained potassium phosphate buffer (100pmol) p H 7.4, NADP (0.5 pmol), glucose 6-phosphate (1.5 pmol), MgCI, (4.5 pmol), all added in buffer (100p1), and glucose 6- phosphate dehydrogenase (1 IU). Substrates, BP (60nmol), 7MBAC (40 nmol), or dibenz[a,jlacridine (60nmol) were added in acetone ( 5 0 ~ 1 )to mixtures containing 0.6rng, 0.4mg and 0.5mg microsomal protein respectively for the three substrates. In some cases 1,1,1-trichloropropene 2,3-oxide (TCPO) (1.5 mM) was added in ethanol (1Op1) to inhibit epoxide hydrase. Following a 2 min preincubation in the absence of cofactor, the reaction was started by the addition of NADP and proceeded at 37°C for 15 min, 20min and 20min for the three substrates respectively, Usually about 0.5 pCi of tritium was employed except with 7MBAC as a substrate when the assay of 7MBAC-3,4-DHD required rechromatography on a normalphase system. In this case about 5 pCi was used. Linear reaction conditions were determined with the assay previously described (Ireland et al. 1981) in which unreacted substrate was extracted into hexane
1302 Table 2.
Sugiyanto et al. Total mixed function oxidase activities of human liver microsomes towards PAH. Substrate Subject H5 H6 H7 H8
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H9 H10 H11 H12 H13 H14 H15 sv1 sv2 Meana kSEM
7MBAC 097' 1.32 1.88 238 078 2.50 098 019 030 036 035 020 1.20 1.03 +021d
DBAJAC
BP
0.7Sb 0.40 0.60 0.50 0.26 0.55 0.26 0.10 0.10 0.08 0.12 0.15 0.26
0.93" 1.38 0.66 1.46 0.85 1.50 1.73 0.32 0.36 0.77 0.62
0.72' 0.41 0.21 0.31 0.28 0.33 0.45 0.17 0.12 0.17 0.21 -
-E
-
0.82
0.18
0.36
0.08
0.33 +O-Obd
0.95 +0.13d
0.30 f0.05d
0.50 k0.07
0.15 +@05
-'
0.23" 040 060 1.08 0.53 0.55 051 0.15 0.38 073 042
0.18' 0.12 0.19 0.23 0.18 0.12 0.13 0.08 0.13 0.16 0.14
a Expressed as the mean of duplicate incubations as nmol substrate oxidized/nmol cytochrome P-450 per min. Expressed as nmol substrate oxidized/mg protein per min ' Insufficient liver was available to assay for M F O activity towards BP and DBAJAC Significantly different from BP metabolism ( P i0.05 by Student's t-test).
and metabolites were determined by counting aliquots of the aqueous alkaline DMSO phase. Using liver microsomes from subject H9 and substrate concentrations previously shown to be saturating for control and induced rat liver microsomes (Gill et al. 1987, Ireland et al. 1981) metabolism was shown to be proportional to time up to 30 min for 7MBAC and DBAJAC and 20 min for BP, and to be proportional to protein concentrations up to 0.4mg/ml for 7MBAC, 06mg/ml for DBAJAC and 0.8mg/ml for BP. Using the h.p.1.c. assay for H12, and H15, and the extraction assay for HIS, no inhibition of metabolism by acetone (1@5Oyl) used to introduce the substrate was seen for BP. Metabolite distributions were determined by extraction of the metabolites and unreacted substrate into ethyl acetate (3 x 1.5 ml) and evaporation of the organic phase. Metabolite markers were added, and the total dissolved in dimethyl formamide (20 pl) before chromatography on a reverse-phase system. Fractions were assayed for radioactivity. Total metabolic activities were determined from the fraction of radioactivity which chromatographed before the unchanged substrate. No more than 15% of substrate was consumed. T h e proportion of total metabolites cochromatographing with standards was greater than 88% for 7MBAC, 77% for DBAJAC and 76% for BP, and product distributions were determined as the percentage of total metabolites which cochromatographed with each standard. These were expressed also as specific enzyme activities. Because amounts of microsomal samples were limited, assays were performed only in duplicate. Results, expressed as total activities, agreed to within less than f 15%, and product profiles generally agreed within less than +20% of their means (Sugiyanto et al. 1990).
Results Comparison of aza-aromatic polycyclic compounds with benzo[alpyrene Total cytochrome P-4 50 levels determined ranged from 0.21-0.77 nmol/mg microsomal protein. Metabolism results are presented as total metabolism of the three substrates (table 2) and as mean of metabolite distributions for 7MBAC, DBAJAC and BP metabolites (tables 3, 4 and 5). BP was included in the study as a reference polycyclic aromatic hydrocarbon because it has been studied previously with human hepatic microsomes. Under conditions of limited substrate consumption, secondary more polar metabolites were minor (less than 3%) and the tables exclude tetraols and secondary metabolites of dihydrodiols. Activity towards the
Human hepatic metabolism of two aza-arenes Table 3.
Formation of individual metabolites" during incubation of 3H-7MBAC with human liver microsomes. Product
Specific activity
Relative amount
7-Hydroxy-MBAC-5,6-oxide
94 i37 285 f72 64+15 30k6
19.3 1.0 24.9 k 1.6 5.9 k 0.7 46k1.0
7-Hydroxy-MBAC & 7MBAC-10,ll-DHD 7MBAC-S,6-oxide Phenolsb
152230 90 f25 112+22
22.0 I 1.7 8.7k 1.1 11.0f07
7MBAC-5.6-DHD 7MBAC-8,9-DHD 7MBAC-3,4-DHD
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1303
*Expressed as the means fSEM (n = 13) as pmol metabolite/nmol cytochrome P-450 per rnin and as the percentage of total metabolites. The 9- and 5-hydroxy-7-methylbenz[c]acridinewere determined together because their separation was not complete. Table 4. Formation of individual metabolites" during incubation of 3H-DBAJAC with human fiver microsomes. Product DBAJAC-5,6-DHD DBAJAC-3,4-DHD Phenolic 5,6-oxide DBAJAC-5,6-oxide 3-Hydroxy-DBAJAC 4-Hydroxy-DBAJAC
Specific activity
Relative amount
68f3 532 k 74 41 +6 54f7 37k16 74 f 20
6.8f1.0 57.8 f2.6 4.5 _+ 0.5 6.0 f0.4 3.3f 1.1 7.4 & 1.o
'See table 3: n=12.
Table 5. Formation of individual BP metabolitesa during incubations of 3H-BP with human liver microsomes. Product BP-9,lO-DHD BP-4,S-DHD BP-7,S-DHD Phenols and quinonesb
Specific activity
Relative amount
21 k 3 77k 10 15f4 307 f46
4.6 f0.6 2 4 f 1.7 3.0 f0.4 60.9 f 1.7
See table 3; n = 12. bBecause separations of the 3-, and 9-phenol from each other and the quinones were not consistent over the study all are determined together. a
nitrogenous substrates was significantly greater than that expressed towards BP being about twice that of the latter. When the donors listed in table 1 are sorted according to age, two groups may be discerned, one aged between 18 and 48 years and the other aged between 61 and 77 years. There was a significant difference ( P < 0.05) between the younger ( n = 7) and older ( n = 5) group for BP metabolism (0.61 f0.24 (mean fSD) nmol/nmol cytochrome P-450/min; 0.33 0.15 nmol/nmol cytochrome P-450/min respectively). While the means of the group data for 7MBAC and DBAJAC metabolism were greater for younger donors (1.1 5 k0.76 and 1.07f0.45 respectively) than older donors (0.89 f0.90 and 0.79 f0.48 respectively), these differences were not as great as for BP metabolism and did not reach statistical significance (P>OO5).
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Sugiyanto et al.
Oxidation of 7-methylbenz[c]acridine Human liver microsomes have the capacity to oxidize the nitrogenous carcinogen 7MBAC to a variety of products found as rat liver metabolites (table 3 ) (Boux et al. 1983, Gill et al. 1986a). T h e metabolite distributions indicated that the 8,9dihydrodiol and the K-region dihydrodiol (5,6-dihydrodiol) were the major metabolites. While the 7-hydroxy MBAC fraction and 5,6-oxide were both formed in substantial proportions, phenols and the 3,4-dihydrodiol were quantitatively minor. The extent of K- region oxidation was comparable with the fraction of 8,9-dihydrodiol found when the sum of the K-region dihydrodiol and oxide is considered. Coincubation of 7MBAC, cofactors, and the epoxide hydrase inhibitor T C P O (Oesch 1973) with microsomes from subjects H7, H 8 and H 9 decreased dihydrodiol formation expressed as a fraction of total metabolites by 35%, 40% and 92%, respectively. A concomitant increase in phenol and 5,6-oxide formation was seen. Oxidation of dibenz [a, jlacridine Human liver microsomal metabolite distributions for DBA JAC (table 4) showed considerable similarities to those of rat liver microsomes (Gill et al. 1986b; 1987). T h e 3,4-dihydrodiol with a vicinal double bond in the bay-region was the single dominant metabolite and the extent of its formation was considerably greater in human preparations at 57.8% compared to about 40% for rat preparations. T h e sum of the dihydrodiols reached about 65% of the total ethyl acetate-soluble metabolites. About 13% of oxidation occurred at the K-region. Incubation of H7, H8 and H 9 in the presence of T C P O resulted in 97%, 96% and 98% inhibition of dihydrodiol formation respectively, and fractions corresponding to phenols and the 5,6-oxide increased. Benzo[a]pyrene metabolism T h e amount of trans-BP-7,8-dihydrodiolformed was 3% of total extractable metabolites, while the relative amounts of the 9,lO- and K-region or 5,6dihydrodiols were greater. T h e total proportion of dihydrodiols formed in the absence of epoxide hydrase inhibition was about 20%. Incubations in the presence of T C P O reduced the relative amounts of the dihydrodiols formed by 83% and 72% for H 7 and H8 respectively. Phenols and BP-quinones were major metabolites even in the absence of epoxide hydrase inhibition.
Discussion Human hepatic cytochrome P-450 levels were similar to those found by several other groups using liver from renal transplant donors and other trauma victims (Beaune et al. 1986, Kremers et al. 1981, Souhali-El Amri et al. 1986, von Bahr et al. 1980), and measured rates of BP oxidation were either similar or less than literature values, namely, 0.5 (Prough et al. 1979), 0-11 (Kremers et al. 1981), 0 1 4 (Ekstroem et al. 1982), and 0.18 nmol product/min per mg protein (Beaune et al. 1986). T h e amount of BP-7,8-DHD formed, about 3% of total extractable metabolites in the present study, is less than reported data of 13% (Ekstroem et al. 1982), 8% (Hall et al. 1989), and 6% (Prough et al. 1979, Kremers et al. 1981). T h e nitrogenous polycyclic aromatic compounds, 7MBAC and DBAJAC, were preferred to BP, as substrates by human liver enzymes. Similar observations with BP and 7MBAC were made for rat liver microsomal metabolism (Ireland et al. 1981).
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Human hepatic metabolism of two aza-arenes
1305
However, activities of liver microsomal preparations obtained from untreated rats towards nitrogenous compounds were about 5-6 times greater than those of human preparations when expressed per mg protein. Human hepatic metabolite distributions followed patterns similar to those found for rodent liver preparations (Boux et al. 1983, Gill et al. 1986a) except for the greater total of dihydrodiols at more than 50% for the human liver. Using correlation analysis (Beaune et al. 1986) the total specific enzyme activity per nmol cytochrome P-450 of 7MBAC metabolism positively correlated with DBAJAC metabolism ( r = 0.66, P < 0.05).T h e correlation between BP and 7MBAC metabolism was weak (r=0,55,P
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes Sugiyanto, C. E. Scharping, M. E. McManus, D. J. Birkett, G. M. Holder & A. J. Ryan To cite this article: Sugiyanto, C. E. Scharping, M. E. McManus, D. J. Birkett, G. M. Holder & A. J. Ryan (1992) The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes, Xenobiotica, 22:11, 1299-1307 To link to this article: http://dx.doi.org/10.3109/00498259209053158
Published online: 23 Apr 2010.
Submit your article to this journal
Article views: 6
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of California, San Diego]
Date: 09 December 2015, At: 04:18
XENOBIOTICA,
1992, VOL. 22,
NO.
11, 1299-1 307
The formation of proximate carcinogens from three polycyclic aromatic compounds by human liver microsomes
Downloaded by [University of California, San Diego] at 04:18 09 December 2015
SUGIYANTOTS, C. E. SCHARPING?, M. E. McMANUS9, D. J. BIRKETTg, G. M. HOLDER*? and A. J. RYAN? Department of Pharmacy, University of Sydney, NSW 2006, Australia Clinical Pharmacology, Flinders Medical School, Bedford Park, South Australia 5042
5 Department of
Received 18 December 1991; accepted 30 April 1992
1. The metabolism of 3H-benzo[a]pyrene (BP), 3H-7-methylbenz[c]acridine (7MBAC) and 3H-dibenz[a,~lacridine(DBAJAC) have been studied in human liver microsomes from 13 subjects. 2. When the metabolism of these carcinogens to more polar ethyl acetate-soluble metabolites were compared, the activities towards the nitrogenous carcinogens were twice that determined for BP. 3. The specific rates of formation of the three proximate carcinogens, BP-7,Sdihydrodiol, 7MBAC-3,4-dihydrodioI and DBAJAC-3,4-dihydrodiol per nmol cytochrome P-450 for 12 subjects were positively correlated. 4. These dihydrodiols constituted 5.9 *0.7% (mean _+ SEM), 57.8 2.6%and 3.0 +0.4% of the total metabolites identified by cochromatography with standards, 7MBAC. DBAJAC and BP respectively.
Introduction Interest in the mechanism of initiation of cancer has extended to all aspects of investigations into the behaviour of carcinogens. This has included both the metabolic activation and detoxication of carcinogens, the mutagenic and carcinogenic potential of the parent carcinogen and its metabolites, and their reactivity with DNA (Jeffrey 1987, Yang and Silverman 1988). Among the different classes of chemical carcinogens the polycyclic aromatic hydrocarbons have probably received the greatest attention. T h e metabolic profiles of many of these compounds have been determined and the ultimate carcinogenic species identified (Yang and Silverman 1988, Thakker et al. 1985a). In addition, the stereochemistry associated with the formation of the carcinogenically active and inactive diolepoxides has been well characterized. T o date most of these studies have been carried out in vkvo in animals or in vitro using subcellular animal tissue fractions, cells and purified enzymes. Recently considerable attention has also been focused on the ability of human systems to metabolize benzo[a]pyrene (BP) (Ekstroem et al. 1982, Hall et al. 1989, Kremers et al. 1981, Prough et al. 1979, Souhali-El Amri et al. 1986). While polycyclic aromatic hydrocarbons such as BP have been extensively investigated, polycyclic aza-aromatic compounds which occur environmentally (Sawicki et al. 1965, van Duuren et al. 1960) have been essentially ignored. In the present study we report on *To whom correspondence should be addressed.
1Current address: Faculty of Pharmacy,
Gadjah Mada University, Yogyakarta 55281, Indonesia.
0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd
1300
Sugiyanto et al.
12
CH3
B
Downloaded by [University of California, San Diego] at 04:18 09 December 2015
7MBFlC
6
OBFlJRC
BP Figure 1. Structures and numbering of 7-methylbenz[c]acridine (7MBAC), dibenz[a, jlacridine (DBAJAC), and benzo[a]pyrene (BP).
the ability of human liver microsomes to metabolize 7-methylbenz[c]acridine (7MBAC) and dibenz[a, ilacridine (DBAJAC) (figure 1). For comparison we have also determined the ability of the same microsomal preparations to metabolize BP.
Methods Radiochemicals 3H-BP was purchased from Amersham Australia (17.4340 Ci/mmol), and 7-3H-7MBAC (2.6Cilrnmol) (Cheung et al. 1980) and 14-3H-DBAJAC (22.1 Ci/mmol) (Rosario et 01. 1987a) were available from their previous synthesis. All radioisotopically labelled chemicals were purified by extraction of impurities from a toluene solution with 0.1 5 M NaOH in 85% dimethyl sulphoxide (DMSO). Subsequent h.p.1.c. on the reverse-phase systems used to separate metabolites afforded materials of 97-99.5% radiochemical purity with no radioactive impurity peaks. Chemicals BP and NADPH were purchased from Sigma Chemical Co., St Louis, MO, USA, and BP metabolite standards were obtaimed from the NCI Chemical Carcinogen Repository at the Midwest Research Institute, Kansas City, MO, USA. These were trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene(BP4,5-DHD), trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-DHD), trans-9.10-dihydroxy9,10-dihydrobenzo[o]pyrene (BP-9,10-DHD), benzo[a]pyrene-1,6-quinone and its 6,12-, and 3,6isomers (BP-l,6-Q, BP-6,12-Q and BP-3, 6-Q respectively), and 3-hydroxybenzo[a]pyrene and the 9phenol (3-hydroxy-BP and 9-hydroxy-BP respectively). Four isomeric benzo[a]pyrene-7,8,9,10tetrahydrotetraols were also available these being the hydrolysis products of the two vicinal7,8-dioI9,10epoxides derived from BP-7,8-DHD. 7MBAC and standards, trans-3,4-dihydroxy-7-methy1-3,4-dihydrobenz[c]acridine(7MBAC-3,4DHD), trans-l,2-dihydroxy-7-methyl-l,2-dihydrobenz[c]acridine(7MBAC-1,2-DHD), trans-5,6dihydroxy-7-methyl-8,9-dihydrobenz[c]acridine (7MBAC-8,9-DHD), 7-hydroxymethylbenz[c]acridine (7-hydroxy-MBAQ 7MBAC-S,6-oxide, phenols 5-hydroxy-7-MBAC and 9-hydroxy-7MBAC and 7-hydroxymethylbenz[c]acridine-5,6-oxide(7HOMBAC-5,6-oxide) were available from previous syntheses (Boux et al. 1980,Duke et al. 1984, Lap et al. 1983). DBAJAC and its metabolites, trans-5,6-dihydroxy-5,6-dihydrodibenz[a, j ] acridine (DBAJAC-5,6DHD), trans-3,4-dihydroxy-3,4-dihydrodibenz[a,~~acridine (DBAJAC-3,4-DHD), the phenols 3hydroxy dibenz[a, jlacridine (3-hydroxy-DBAJAC) and 4-hydroxydibenz[a,~]acridine (4-hydroxyDBAJAC), dibenz[a,j]acridine-5,6-oxide (DBAJAC-5,6-oxide) and dibenz[a,jlacridine-N-oxide) had heen prepared previously (Rosario et al. 1987 b).
H.p.1.c. Separations of metabolites from each other and the parent compound were performed using methanol-water gradients on a twin pump Beckman 421 system with a Jasco Uvidec 100 V U.V.detector at 254nm for BP metabolites of 275nm for 7MBAC and DBAJAC metabolites. Separations of BP metabolites were performed at ambient temperature, while those of the nitrogenous compounds were performed at 40°C. T h e column was either a 10pm Merck Hibar RP-8 (25 x 0.46cm int. diam.) usingml/min and 1 min fractions for the radioactivity assay (system 1) o r a Brownlee 3 pm Velosep RP8
Human hepatic metabolism of two aza-arenes
1301
Table 1. Clinical data for liver donor
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Subject
Age Sex Smoking history
Alcohol consumption
H5
62
M Unknown
Unknown
H6 H7 H8 H9 H10
18 44 24 19 67
M F M M F
Social Social Social Social Social
H11
23
M None
Moderate
H12 H13 H14* H15
66 61 48 22
M None F None for 3 years F Unknown M Unknown
Minimal Minimal Unknown Unknown
sv 1
72
F None
Unknown
sv2
66
M Smoker
Unknown
Unknown None 20 cig/day None None
Drug treatment Phenytoin, dexamethasone, gentamicin, rinatidine, cotrimoxazole Penicillin, cotrirnoxazole Dexamethasone Dopamine Dopamine Dopamine, morphine, frusemide, rnidazolam Phenytoin, carbamazepine, fluphenazine Insulin Metoprolol, methyclothiazide Unknown Phenobarbitone, phenytoin, dexamethasone, methylprednisolone, morphine, dopamine, frusemide, atenolol, hydralazine, amoxil, gentamicin, vit. K, folic acid Metformin, propranolol, digoxin, hydrochlorthiazide, amiloride disopyramide Prazosin, pindolol
P-450 (nmol/mg) 0.77 0.30 0.32 0.2 1 0.33 0.22 0.26
0.53 0.3 3 0.22 033
0.73
0.22
*All liver donors were Caucasians except subject H14 who was of Asian origin.
(10x0.32cm int. diam.) using 0.7ml/min and 10 drop fractions (system 2). They were used interchangeably (Sugiyanto et al. 1990). T h e reverse-phase fractions of the 7MBAC metabolite which cochromatographed with the 3,4-dihydrodiol were combined, stripped of solvent, redissolved in about 2Opl CHCL, and further chromatographed on a Brownlee 10pm silica column (25 x 0.46cm int. diam.) using 2% ethanol in cyclohexane.
Human liver microsomes Thirteen samples of human liver microsomes were obtained principally from a liver bank stored at -80°C. Clinical details are shown in table 1. Most were obtained from organ donors with the exception of H12, H13 and SV2 which were taken at the time of liver resection for cancer, and SV1 which was taken from a patient with undiagnosed liver disease. T h e liver used in the last four samples was normal tissue. Microsomes were prepared and stored in 20% glycerol at -80°C until required (up to 4 years after collection), and no significant losses of cytochrome P-450 were seen between samples freshly assayed and at times of metabolic incubation in the current study. Cytochrorne P-450 was measured according to the method of Guengerich (1984). Metabolism Incubations (1.0ml) contained potassium phosphate buffer (100pmol) p H 7.4, NADP (0.5 pmol), glucose 6-phosphate (1.5 pmol), MgCI, (4.5 pmol), all added in buffer (100p1), and glucose 6- phosphate dehydrogenase (1 IU). Substrates, BP (60nmol), 7MBAC (40 nmol), or dibenz[a,jlacridine (60nmol) were added in acetone ( 5 0 ~ 1 )to mixtures containing 0.6rng, 0.4mg and 0.5mg microsomal protein respectively for the three substrates. In some cases 1,1,1-trichloropropene 2,3-oxide (TCPO) (1.5 mM) was added in ethanol (1Op1) to inhibit epoxide hydrase. Following a 2 min preincubation in the absence of cofactor, the reaction was started by the addition of NADP and proceeded at 37°C for 15 min, 20min and 20min for the three substrates respectively, Usually about 0.5 pCi of tritium was employed except with 7MBAC as a substrate when the assay of 7MBAC-3,4-DHD required rechromatography on a normalphase system. In this case about 5 pCi was used. Linear reaction conditions were determined with the assay previously described (Ireland et al. 1981) in which unreacted substrate was extracted into hexane
1302 Table 2.
Sugiyanto et al. Total mixed function oxidase activities of human liver microsomes towards PAH. Substrate Subject H5 H6 H7 H8
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H9 H10 H11 H12 H13 H14 H15 sv1 sv2 Meana kSEM
7MBAC 097' 1.32 1.88 238 078 2.50 098 019 030 036 035 020 1.20 1.03 +021d
DBAJAC
BP
0.7Sb 0.40 0.60 0.50 0.26 0.55 0.26 0.10 0.10 0.08 0.12 0.15 0.26
0.93" 1.38 0.66 1.46 0.85 1.50 1.73 0.32 0.36 0.77 0.62
0.72' 0.41 0.21 0.31 0.28 0.33 0.45 0.17 0.12 0.17 0.21 -
-E
-
0.82
0.18
0.36
0.08
0.33 +O-Obd
0.95 +0.13d
0.30 f0.05d
0.50 k0.07
0.15 +@05
-'
0.23" 040 060 1.08 0.53 0.55 051 0.15 0.38 073 042
0.18' 0.12 0.19 0.23 0.18 0.12 0.13 0.08 0.13 0.16 0.14
a Expressed as the mean of duplicate incubations as nmol substrate oxidized/nmol cytochrome P-450 per min. Expressed as nmol substrate oxidized/mg protein per min ' Insufficient liver was available to assay for M F O activity towards BP and DBAJAC Significantly different from BP metabolism ( P i0.05 by Student's t-test).
and metabolites were determined by counting aliquots of the aqueous alkaline DMSO phase. Using liver microsomes from subject H9 and substrate concentrations previously shown to be saturating for control and induced rat liver microsomes (Gill et al. 1987, Ireland et al. 1981) metabolism was shown to be proportional to time up to 30 min for 7MBAC and DBAJAC and 20 min for BP, and to be proportional to protein concentrations up to 0.4mg/ml for 7MBAC, 06mg/ml for DBAJAC and 0.8mg/ml for BP. Using the h.p.1.c. assay for H12, and H15, and the extraction assay for HIS, no inhibition of metabolism by acetone (1@5Oyl) used to introduce the substrate was seen for BP. Metabolite distributions were determined by extraction of the metabolites and unreacted substrate into ethyl acetate (3 x 1.5 ml) and evaporation of the organic phase. Metabolite markers were added, and the total dissolved in dimethyl formamide (20 pl) before chromatography on a reverse-phase system. Fractions were assayed for radioactivity. Total metabolic activities were determined from the fraction of radioactivity which chromatographed before the unchanged substrate. No more than 15% of substrate was consumed. T h e proportion of total metabolites cochromatographing with standards was greater than 88% for 7MBAC, 77% for DBAJAC and 76% for BP, and product distributions were determined as the percentage of total metabolites which cochromatographed with each standard. These were expressed also as specific enzyme activities. Because amounts of microsomal samples were limited, assays were performed only in duplicate. Results, expressed as total activities, agreed to within less than f 15%, and product profiles generally agreed within less than +20% of their means (Sugiyanto et al. 1990).
Results Comparison of aza-aromatic polycyclic compounds with benzo[alpyrene Total cytochrome P-4 50 levels determined ranged from 0.21-0.77 nmol/mg microsomal protein. Metabolism results are presented as total metabolism of the three substrates (table 2) and as mean of metabolite distributions for 7MBAC, DBAJAC and BP metabolites (tables 3, 4 and 5). BP was included in the study as a reference polycyclic aromatic hydrocarbon because it has been studied previously with human hepatic microsomes. Under conditions of limited substrate consumption, secondary more polar metabolites were minor (less than 3%) and the tables exclude tetraols and secondary metabolites of dihydrodiols. Activity towards the
Human hepatic metabolism of two aza-arenes Table 3.
Formation of individual metabolites" during incubation of 3H-7MBAC with human liver microsomes. Product
Specific activity
Relative amount
7-Hydroxy-MBAC-5,6-oxide
94 i37 285 f72 64+15 30k6
19.3 1.0 24.9 k 1.6 5.9 k 0.7 46k1.0
7-Hydroxy-MBAC & 7MBAC-10,ll-DHD 7MBAC-S,6-oxide Phenolsb
152230 90 f25 112+22
22.0 I 1.7 8.7k 1.1 11.0f07
7MBAC-5.6-DHD 7MBAC-8,9-DHD 7MBAC-3,4-DHD
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1303
*Expressed as the means fSEM (n = 13) as pmol metabolite/nmol cytochrome P-450 per rnin and as the percentage of total metabolites. The 9- and 5-hydroxy-7-methylbenz[c]acridinewere determined together because their separation was not complete. Table 4. Formation of individual metabolites" during incubation of 3H-DBAJAC with human fiver microsomes. Product DBAJAC-5,6-DHD DBAJAC-3,4-DHD Phenolic 5,6-oxide DBAJAC-5,6-oxide 3-Hydroxy-DBAJAC 4-Hydroxy-DBAJAC
Specific activity
Relative amount
68f3 532 k 74 41 +6 54f7 37k16 74 f 20
6.8f1.0 57.8 f2.6 4.5 _+ 0.5 6.0 f0.4 3.3f 1.1 7.4 & 1.o
'See table 3: n=12.
Table 5. Formation of individual BP metabolitesa during incubations of 3H-BP with human liver microsomes. Product BP-9,lO-DHD BP-4,S-DHD BP-7,S-DHD Phenols and quinonesb
Specific activity
Relative amount
21 k 3 77k 10 15f4 307 f46
4.6 f0.6 2 4 f 1.7 3.0 f0.4 60.9 f 1.7
See table 3; n = 12. bBecause separations of the 3-, and 9-phenol from each other and the quinones were not consistent over the study all are determined together. a
nitrogenous substrates was significantly greater than that expressed towards BP being about twice that of the latter. When the donors listed in table 1 are sorted according to age, two groups may be discerned, one aged between 18 and 48 years and the other aged between 61 and 77 years. There was a significant difference ( P < 0.05) between the younger ( n = 7) and older ( n = 5) group for BP metabolism (0.61 f0.24 (mean fSD) nmol/nmol cytochrome P-450/min; 0.33 0.15 nmol/nmol cytochrome P-450/min respectively). While the means of the group data for 7MBAC and DBAJAC metabolism were greater for younger donors (1.1 5 k0.76 and 1.07f0.45 respectively) than older donors (0.89 f0.90 and 0.79 f0.48 respectively), these differences were not as great as for BP metabolism and did not reach statistical significance (P>OO5).
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1304
Sugiyanto et al.
Oxidation of 7-methylbenz[c]acridine Human liver microsomes have the capacity to oxidize the nitrogenous carcinogen 7MBAC to a variety of products found as rat liver metabolites (table 3 ) (Boux et al. 1983, Gill et al. 1986a). T h e metabolite distributions indicated that the 8,9dihydrodiol and the K-region dihydrodiol (5,6-dihydrodiol) were the major metabolites. While the 7-hydroxy MBAC fraction and 5,6-oxide were both formed in substantial proportions, phenols and the 3,4-dihydrodiol were quantitatively minor. The extent of K- region oxidation was comparable with the fraction of 8,9-dihydrodiol found when the sum of the K-region dihydrodiol and oxide is considered. Coincubation of 7MBAC, cofactors, and the epoxide hydrase inhibitor T C P O (Oesch 1973) with microsomes from subjects H7, H 8 and H 9 decreased dihydrodiol formation expressed as a fraction of total metabolites by 35%, 40% and 92%, respectively. A concomitant increase in phenol and 5,6-oxide formation was seen. Oxidation of dibenz [a, jlacridine Human liver microsomal metabolite distributions for DBA JAC (table 4) showed considerable similarities to those of rat liver microsomes (Gill et al. 1986b; 1987). T h e 3,4-dihydrodiol with a vicinal double bond in the bay-region was the single dominant metabolite and the extent of its formation was considerably greater in human preparations at 57.8% compared to about 40% for rat preparations. T h e sum of the dihydrodiols reached about 65% of the total ethyl acetate-soluble metabolites. About 13% of oxidation occurred at the K-region. Incubation of H7, H8 and H 9 in the presence of T C P O resulted in 97%, 96% and 98% inhibition of dihydrodiol formation respectively, and fractions corresponding to phenols and the 5,6-oxide increased. Benzo[a]pyrene metabolism T h e amount of trans-BP-7,8-dihydrodiolformed was 3% of total extractable metabolites, while the relative amounts of the 9,lO- and K-region or 5,6dihydrodiols were greater. T h e total proportion of dihydrodiols formed in the absence of epoxide hydrase inhibition was about 20%. Incubations in the presence of T C P O reduced the relative amounts of the dihydrodiols formed by 83% and 72% for H 7 and H8 respectively. Phenols and BP-quinones were major metabolites even in the absence of epoxide hydrase inhibition.
Discussion Human hepatic cytochrome P-450 levels were similar to those found by several other groups using liver from renal transplant donors and other trauma victims (Beaune et al. 1986, Kremers et al. 1981, Souhali-El Amri et al. 1986, von Bahr et al. 1980), and measured rates of BP oxidation were either similar or less than literature values, namely, 0.5 (Prough et al. 1979), 0-11 (Kremers et al. 1981), 0 1 4 (Ekstroem et al. 1982), and 0.18 nmol product/min per mg protein (Beaune et al. 1986). T h e amount of BP-7,8-DHD formed, about 3% of total extractable metabolites in the present study, is less than reported data of 13% (Ekstroem et al. 1982), 8% (Hall et al. 1989), and 6% (Prough et al. 1979, Kremers et al. 1981). T h e nitrogenous polycyclic aromatic compounds, 7MBAC and DBAJAC, were preferred to BP, as substrates by human liver enzymes. Similar observations with BP and 7MBAC were made for rat liver microsomal metabolism (Ireland et al. 1981).
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However, activities of liver microsomal preparations obtained from untreated rats towards nitrogenous compounds were about 5-6 times greater than those of human preparations when expressed per mg protein. Human hepatic metabolite distributions followed patterns similar to those found for rodent liver preparations (Boux et al. 1983, Gill et al. 1986a) except for the greater total of dihydrodiols at more than 50% for the human liver. Using correlation analysis (Beaune et al. 1986) the total specific enzyme activity per nmol cytochrome P-450 of 7MBAC metabolism positively correlated with DBAJAC metabolism ( r = 0.66, P < 0.05).T h e correlation between BP and 7MBAC metabolism was weak (r=0,55,P
