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The metabolism of 3-methylcholanthrene by liver and lung microsomes: Effect of enzyme inducing agents
Cancer Letters, 20 (1983) 323-331 Elsevier Scientific Publishers Ireland Ltd.
323
THE METABOLISM OF 3-METHYLCHO LANTHRENE BY LIVER AND LUNG MICROSOMES: EFFECT OF ENZYME INDUCING AGENTS
M.A. GANGAROSA
and T.A. STOMING
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 3091.2 (U.S.A.) (Received 8 June 1983) (Revised version received 11 July 1983) (Accepted 19 July 1983)
SUMMARY The metabolism of 3-methylcholanthrene (3-MC) by rat liver and lung microsomes was investigated. The animals were pretreated with various inducing agents to determine the effect which microsomal induction has on metabolic profile. Metabolites were separated by high pressure liquid chromatography. Major metabolism was associated with three peaks: 2hydroxy-8MC, 1-hydroxy-8MC and a peak that co-chromatographs with 3.MC 11,12-oxide and probably contains phenols. Pretreatment of animals with various inducing agents caused an increase in total metabolism as well as in specific metabolites. However, lung microsomes from phenobarbital treated animals were less efficient at metabolizing 3-MC than control microsomes. Liver and lung microsomes converted 3-MC to qualitatively similar products. No metabolites were formed by lung microsomes that were not also formed by those from liver.
INTRODUCTION
Polycyclic aromatic hydrocarbons @‘AH) are widespread environmental pollutants. Many of the PAHs in the environment are known or suspected carcinogens; of these, benzolalpyrene (BlalP) has been most extensively studied. This compound is known to undergo metabolic activation to elicit its carcinogenic response. Sims et al. have proposed that B[a]P is activated by conversion to a bay region diol-epoxide [8]. This metabolic activation required two microsomal enzyme systems, cytochrome P-450 and epoxide hydrase. Continued research has extended the dial-epoxide theory to include 03043835/83/$03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd. Published and F+inted in Ireland.
324
Fig. 1. 9,lO-Dihydro-9,lO-dihydroxy-3-MC-7,8-oxide.
A proposed ultimate
carcinogen
of 3-MC.
benzanthracene, 7-methylbenzanthracene [5], and 7,12dimethylbenzanthracene [1,5]. According to the bay region theory, the ultimate carcinogenic form of 3-MC would be 9,10-dihydro-9,10-dihydroxy-3-MC-7,8-oxide (Fig. 1). Recent metabolite studies using liver microsomes have not been able to demonstrate the formation of the 9,lOdihydrodiol of 3.MC in sufficient amounts to suggest that it is an important metabolite in the diol-epoxide scheme. The purpose of this study is to compare the metabolism of 3-MC by liver and lung microsomes and to determine the effect of enzyme induction on the metabolic profile. MATERIALS
AND METHODS
Animals Male Sprague-Dawley rats 80-100 g were obtained from Holtzman Co. (Madison, WI) and maintained ad libitum on Purina rat chow. The animals (8-9 per group) were pretreated for 3 consecutive days with either 3-MC in corn oil (40 mg/kg) or phenobarbital in saline (75 mg/kg) and then killed on the fourth day. In addition, a group *as treated once with Aroclor 1254 in corn oil (500 mg/kg), 5 days prior to killing. Corn oil and saline injections served as controls. Chemicals Reagents were purchased from reliable suppliers and were of high commercial purity. The radioactive [6-14C]3-MC was obtained from New England Nuclear Corporation (Boston, MA) and purified, and the specific activity was adjusted to 20 mCi/mmol as outlined previously 1111. IsoZation of microsomes Liver microsomes were isolated according to the procedure al. [7]. The microsomes were suspended in 0.25 M sucrose at tration of 30-40 mg/ml and frozen at -70°C overnight. Lung microsomes were isolated according to the procedure et al. 161. The microsomes were suspended in 0.25 M sucrose:
of Seifried et a final concenof Matsubara 0.05 M Tris
325
(Cl) (pH 7.4) at a concentration of 15-23 mg/ml. The microsomes were stored at -70°C overnight. Protein concentration was measured by the method of Lowry et al. using bovine serum albumin as a standard [4]. Metabolism To analyze for metabolite formation, the following incubation was used: 0.05 M Tris chloride buffer @H 7.4), 3 mM MgClz, 0.5 mM NADPH and 0.5 mg of liver microsomal protein or 2 mg of lung microsomal protein in a total volume of 1.0 ml. The mixture was incubated for 1 min at 37°C and V4C13-MC (100 nmol in 20 ~1 of acetone) was added. Samples containing liver microsomes were incubated for 7 min and those containing microsomes from lung were incubated 15 min. After incubation, 0.5 ml of acetone was added to each tube. Metabolites were extracted into ethyl acetate and work-up was conducted as previously described [9]. All samples were stored as a residue at -70°C until HPLC analysis. IB?LC analysis Analysis was conducted using a 35-95% acetonitrile/water gradient as previously described [9]; 0.5-min fractions were collected directly into Beckman Bio-vials. Scintillation cocktail (TT-21, Yorktown) was added and radioactivity was determined by scintillation spectroscopy. RESULTS AND DISCUSSION
The HPLC separation of the products resulting from the metabolism of 3-MC by liver microsomes from Aroclor-treated rats is shown in Figs. 2 and 3. Figure 2 represents the W metabolite profile including synthetically prepared standards added to the extraction residue prior to chromatography. Peaks A-T (Fig. 3) represent the radioactive profile of metabolites formed by liver microsomes. Table 1 shows the effect of inducing agents on metabolite formation by liver microsomes. There was little qualitative difference in the metabolism of 3-MC by the microsomes from different sources. However, as expected, Aroclor and 3-MC were the most potent inducing agents. Peaks M (2-OH-3MC), N (l-OH-3-MC) and P (co-migrating with 11,lBoxide and probably including phenols) represent the major metabolic products of 3-MC. The early eluting peaks (Fig. 2) A, B, C, D and F are again dihydrodiols and triols [9,3]. Peak E, although an early eluting peak, is most probably not a diol. This peak is not affected when the epoxide hydrase inhibitor TCPG is included in the incubation mixture (results not shown). Peaks P, P’, and Q are probably a mixture of 11,12-oxide and various phenols. Peak G COmigrates with trans-1,2-dihydroxy-3-MC. Peaks H, I and J are unidentified. Two of these have been reported to be the 4,5-dihydrodiol and 9,10-dihydrodiol of 3-MC [14]; however, in our hands none of these peaks were affected when TCPO was added to the incubation. Peaks K and L are at times resolved into two distinct peaks. This region
2
3
,-
II
I
0
!
I
5
lo
15
I
,
I
/
I
I
20
25
30
45
40
45
r50
1
55
TIME Fig. 2. HPLC separation of 3-MC metabolites. Ultraviolet profile (254 nm) obtained with reference compounds added to the extraction residue. Metabolism and separation were conducted as outlined in Materials and Methods. The peak assignments are: (1) tmns-ll,l&dihydro11,12-dihydroxy-3.MC; (2) tmns-1,2-dihydroxy-3-MC; (3) cis-1,2-dihydroxy-3.MC; (4) Zhydroxy3-MC; (5) 1-hydroxyd-MC; (6) arises from incubation; (7) 3-MC 11,12-oxide; (8) Zoxo-3-MC; (9) 1-oxo-&MC; (10) 3-methylcholanthrylene; (11) 3-MC.
co-chromatographs with cis-1,2-dihydroxy-3.MC. Peaks R, S and T are extremely variable and are unidentified. The metabolism of 3-MC by lung microsomes, Table 2, is qualitatively similar to that by liver microsomes. From a quantitative standpoint, however, lung microsomes were less efficient than liver microsomes at metabolizing 3-MC. When phenobarbital induced microsomes were used, total metabolism was less than that of control. None of the early eluting peaks (A-E) could be detected unless induced microsomes (Aroclor or 3-MC) were used. As in liver, major metabolism centered around 2.OH-3-MC (I@, l-OH-3-MC (N) and peak P. Tables 1 and 2 also show the percent contribution of each metabolite of total metabolism for liver and lung, respectively (numbers in parentheses). When induced liver microsomes are used, the contribution of both 2-OH-3-MC (M) and l-OH-SMC (N) to total metabolism is reduced. This might be due to a further metabolism of these compounds to triols, since it has been shown that some of the early assay peaks are indeed secondary metabolites. With lung microsomes the contribution of 2-OH-3.MC to total metabolism does not appear to be affected
III
3
Q x
B 0
0 I,
h
FRACTION Fig. 3. Chromatographic separation of radioactive metabolites. Metabolism and separation were conducted as outlined in Materials and Methods. Tentative assignments are: (A, B, C, D, F) diols and triols; (R) unknown; (G) tmns-1,2dihydroxy&MC; (H, I, J) unknown. others 1141 have reported two of these peaks to be 4,6dihydroxy&MC and S,lO-dihydroxy-3-MC, (K and L) sometime two distinct peaks 0 (co-migrates with cis-1,2-dihydroxy-3-MC; 0 2-hydroxy-3MC; (N) lhydroxy&MC; (0) unknown, small and variable; fP, P’, Q), probably phenols. (R, S, T) unknown, small and variable.
by induction, whereas the contribution of l-OH-BMC is reduced upon induction. There is a general increase in the contribution of the early eluting peaks (A-E) to total metabolism upon induction. The metabolite(s) associated with peak P contribute(s) 3 times as much to total metabolism when induced (Aroclor or 3.MC) microsomes are used as compared to control or PB treated. This effect is seen in both liver and lung. In light of the fact that l-OH-SMC has been shown to give rise to an ultimate carcinogen upon further metabolism 131,it is interesting to note that the contribution of l-OH-S-MC to total metabolism is higher in lung than in liver. In all cases, less than 2% of the radioactivity remained associated with the aqueous phase. The bay region theory of carcinogenesis [2] would suggest that 3.MC is first metabolized to a 9,10-dihydrodiol and then to a bay region diol-epoxide. While 3-MC does give rise to a number of primary and secondary metabolites, we have been unable to show the formation of the 9,lOdihydrodiol in sufficient quantity to suggest that it is an important intermediate in the
329
TABLE 2 3-MC METABOLISM BY LUNG MICROSOMES: EFFECT OF VARIOUS INDUCING AGENTS pmol/mg
proteidmin
Control
+ S.E. Phenobarbital
A
-cl
B
(-)
(-)
C
(-) ‘cl
(-)
D
(-)
(-)
E
(-)
(-)
F
(-)
(-)
G
(-)
(-)
H
(-)
(-) il
(-)
(-)
(2.5%)
(-)
I J K+L M N 0 P P’ & R S+T Total Procedure
is the same as in Table 1.
Aroclor
3.MC
,0.2:‘,.1,
330
formation of an ultimate carcinogen. We have previously shown that the major metabolism of 3-MC centers around the cyclopentano-ring system, namely l-OH-&MC and 2-OH-3-MC. Thakker et al. have shown that the early eluting metabolites arise in part from further metabolism of l-OH-3-MC [13], and that two of these early metabolites are the diastereomeric 9,lOdihydrodiols [12]. One of these diastereomers is a carcinogen in newborn mice [3], as are 1.OH-3-MC and 2.OH-3.MC. The exact chemical nature of the ultimate carcinogenic form of 3.MC remains as yet unknown. We have centered our attention around the cyclopentano-ring and are currently investigating the further metabolism of both l.OH.8MC and 2.OH.3.MC. ACKNOWLEDGEMENTS
This investigation was supported by grant 21431 from the National Cancer Institute. This is publjcation number 0773 of the Department of Cell and Molecular Biology. REFERENCES 1 Dipple, A. and Nebxydoski, J.A. (1978) Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbens[alanthracene to DNA in cells in culture. Chem.-Biol. Interact., 20, 17-26. 2 Jerina, D.M. and Daly, J.W. (1976) In: Drug Metabolism, p. 13. Editors: D.V. Parke and R.L. Smith. Taylor and Francis, London. 3 Levin, W., Buening, M.K., Wood, A.W., Chang, R.L., Thakker, D.R., Jerina, D.M. and Conney, A.H. (1979) Tumorigenic activity of 3-methylcholanthrene metabolites on mouse skin and in newborn mice. Cancer Res., 39, 3549-3553. 4 Lowry, O.H., Roeebrough, N.J., Farr, A.L. and Randall, N.J. (1951) Protein measurement with folin phenol reagent. J. Biol. Chem., 193, 265-275. 5 Marquardt, H., Grover, P.L. and Sims, P. (1976) In vitro malignant transformation of mouse fibroblasts by non-K-region dihydrodiols derived from 7,12dimethylbenz(a) anthracene and benzo(a)pyrene. Cancer Res., 36 2059-2064. 6 Mataubara, T., Prough, R.A., Burke, M.D. and Estabrook, R.W. (1974) The preparation of microsomal fractions of rodent respiratory tract and their characterization. Cancer Res., 34, 2196-2203. 7 Siefried, H.E., Brikett, D.J., Levin, W., Lu, A.Y.H., Conney, A.H. and Jerina, D.M. (1977) Metabolism of benzo(a)pyrene. Arch. B&hem. Biophys., 178256-263. 8 Sims, P., Grover, P.L., Swaisland, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzda)pyrene proceeds by a diol-epoxide. Nature, 252, 326-327. 9 Stoming, T.A., Bornstein, W. and Bresnick, E. (1977) The metabolism of 3-methylcholanthrene by rat liver microsomes -A reinvestigation. Biochem. Biophys. Res. Commun., 79, 461-468. 10 Swaisland, A.J., Hewer, A., Pal, K., Keysell, G.R., Booth, J., Grover, P.L. and Sims, P. (1974) Polycyclic hydrocarbon epoxides: The involvement of S$-dihydro-8,9-dihydroxybenz(a)anthracene-lO,ll-oxide in reactions with the DNA of benz(a)anthracene treated hamster embryo cells. FEBS Lett., 47, 34-38. 11 Stoming, T.A. and Gerardot, R.J. (1977) High pressure liquid chromatographic separation of the metabolites of 3-methylcholanthrene. Life Sci., 20, 113-116.
331 I2 Thakker, D.R., Levin, W., Wood, A.W., Conney, A.H., Stoming, T.A. and Jerina, D.M. (19761 Metabolic formation of 1,9,l0-trihydroxy-9,l0-dihydro-3-methylcholanthrene: A potential proximate carcinogen from 3.methylcholanthrene. J. Am. Chem. Sot., 100, 645-646. 13 Thakker, D.R., Levin, W., Stoming, T.A., Conney, A.H. and Jerina, D.M. (1978) Metabolism of 3-methylcholanthrene by rat liver microsomes and a highly purified monooxygenase system with and without epoxide hydrase. In: Carcinogenesis, Vol. 3, Polynuclear Aromatic Hydrocarbons, pp. 252-264. Editors: P.W. Jones and R.I. Freudenthal. Raven Press, New York. 14 Tierny, B., Bresnick, E., Sims, P. and Grover, P.L. (1979) Microsomal and nuclear metabolism of 3.methylcholanthrene. Bicchem. Pharmacol., 28, 2607-2610.
323
THE METABOLISM OF 3-METHYLCHO LANTHRENE BY LIVER AND LUNG MICROSOMES: EFFECT OF ENZYME INDUCING AGENTS
M.A. GANGAROSA
and T.A. STOMING
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 3091.2 (U.S.A.) (Received 8 June 1983) (Revised version received 11 July 1983) (Accepted 19 July 1983)
SUMMARY The metabolism of 3-methylcholanthrene (3-MC) by rat liver and lung microsomes was investigated. The animals were pretreated with various inducing agents to determine the effect which microsomal induction has on metabolic profile. Metabolites were separated by high pressure liquid chromatography. Major metabolism was associated with three peaks: 2hydroxy-8MC, 1-hydroxy-8MC and a peak that co-chromatographs with 3.MC 11,12-oxide and probably contains phenols. Pretreatment of animals with various inducing agents caused an increase in total metabolism as well as in specific metabolites. However, lung microsomes from phenobarbital treated animals were less efficient at metabolizing 3-MC than control microsomes. Liver and lung microsomes converted 3-MC to qualitatively similar products. No metabolites were formed by lung microsomes that were not also formed by those from liver.
INTRODUCTION
Polycyclic aromatic hydrocarbons @‘AH) are widespread environmental pollutants. Many of the PAHs in the environment are known or suspected carcinogens; of these, benzolalpyrene (BlalP) has been most extensively studied. This compound is known to undergo metabolic activation to elicit its carcinogenic response. Sims et al. have proposed that B[a]P is activated by conversion to a bay region diol-epoxide [8]. This metabolic activation required two microsomal enzyme systems, cytochrome P-450 and epoxide hydrase. Continued research has extended the dial-epoxide theory to include 03043835/83/$03.00 @ 1983 Elsevier Scientific Publishers Ireland Ltd. Published and F+inted in Ireland.
324
Fig. 1. 9,lO-Dihydro-9,lO-dihydroxy-3-MC-7,8-oxide.
A proposed ultimate
carcinogen
of 3-MC.
benzanthracene, 7-methylbenzanthracene [5], and 7,12dimethylbenzanthracene [1,5]. According to the bay region theory, the ultimate carcinogenic form of 3-MC would be 9,10-dihydro-9,10-dihydroxy-3-MC-7,8-oxide (Fig. 1). Recent metabolite studies using liver microsomes have not been able to demonstrate the formation of the 9,lOdihydrodiol of 3.MC in sufficient amounts to suggest that it is an important metabolite in the diol-epoxide scheme. The purpose of this study is to compare the metabolism of 3-MC by liver and lung microsomes and to determine the effect of enzyme induction on the metabolic profile. MATERIALS
AND METHODS
Animals Male Sprague-Dawley rats 80-100 g were obtained from Holtzman Co. (Madison, WI) and maintained ad libitum on Purina rat chow. The animals (8-9 per group) were pretreated for 3 consecutive days with either 3-MC in corn oil (40 mg/kg) or phenobarbital in saline (75 mg/kg) and then killed on the fourth day. In addition, a group *as treated once with Aroclor 1254 in corn oil (500 mg/kg), 5 days prior to killing. Corn oil and saline injections served as controls. Chemicals Reagents were purchased from reliable suppliers and were of high commercial purity. The radioactive [6-14C]3-MC was obtained from New England Nuclear Corporation (Boston, MA) and purified, and the specific activity was adjusted to 20 mCi/mmol as outlined previously 1111. IsoZation of microsomes Liver microsomes were isolated according to the procedure al. [7]. The microsomes were suspended in 0.25 M sucrose at tration of 30-40 mg/ml and frozen at -70°C overnight. Lung microsomes were isolated according to the procedure et al. 161. The microsomes were suspended in 0.25 M sucrose:
of Seifried et a final concenof Matsubara 0.05 M Tris
325
(Cl) (pH 7.4) at a concentration of 15-23 mg/ml. The microsomes were stored at -70°C overnight. Protein concentration was measured by the method of Lowry et al. using bovine serum albumin as a standard [4]. Metabolism To analyze for metabolite formation, the following incubation was used: 0.05 M Tris chloride buffer @H 7.4), 3 mM MgClz, 0.5 mM NADPH and 0.5 mg of liver microsomal protein or 2 mg of lung microsomal protein in a total volume of 1.0 ml. The mixture was incubated for 1 min at 37°C and V4C13-MC (100 nmol in 20 ~1 of acetone) was added. Samples containing liver microsomes were incubated for 7 min and those containing microsomes from lung were incubated 15 min. After incubation, 0.5 ml of acetone was added to each tube. Metabolites were extracted into ethyl acetate and work-up was conducted as previously described [9]. All samples were stored as a residue at -70°C until HPLC analysis. IB?LC analysis Analysis was conducted using a 35-95% acetonitrile/water gradient as previously described [9]; 0.5-min fractions were collected directly into Beckman Bio-vials. Scintillation cocktail (TT-21, Yorktown) was added and radioactivity was determined by scintillation spectroscopy. RESULTS AND DISCUSSION
The HPLC separation of the products resulting from the metabolism of 3-MC by liver microsomes from Aroclor-treated rats is shown in Figs. 2 and 3. Figure 2 represents the W metabolite profile including synthetically prepared standards added to the extraction residue prior to chromatography. Peaks A-T (Fig. 3) represent the radioactive profile of metabolites formed by liver microsomes. Table 1 shows the effect of inducing agents on metabolite formation by liver microsomes. There was little qualitative difference in the metabolism of 3-MC by the microsomes from different sources. However, as expected, Aroclor and 3-MC were the most potent inducing agents. Peaks M (2-OH-3MC), N (l-OH-3-MC) and P (co-migrating with 11,lBoxide and probably including phenols) represent the major metabolic products of 3-MC. The early eluting peaks (Fig. 2) A, B, C, D and F are again dihydrodiols and triols [9,3]. Peak E, although an early eluting peak, is most probably not a diol. This peak is not affected when the epoxide hydrase inhibitor TCPG is included in the incubation mixture (results not shown). Peaks P, P’, and Q are probably a mixture of 11,12-oxide and various phenols. Peak G COmigrates with trans-1,2-dihydroxy-3-MC. Peaks H, I and J are unidentified. Two of these have been reported to be the 4,5-dihydrodiol and 9,10-dihydrodiol of 3-MC [14]; however, in our hands none of these peaks were affected when TCPO was added to the incubation. Peaks K and L are at times resolved into two distinct peaks. This region
2
3
,-
II
I
0
!
I
5
lo
15
I
,
I
/
I
I
20
25
30
45
40
45
r50
1
55
TIME Fig. 2. HPLC separation of 3-MC metabolites. Ultraviolet profile (254 nm) obtained with reference compounds added to the extraction residue. Metabolism and separation were conducted as outlined in Materials and Methods. The peak assignments are: (1) tmns-ll,l&dihydro11,12-dihydroxy-3.MC; (2) tmns-1,2-dihydroxy-3-MC; (3) cis-1,2-dihydroxy-3.MC; (4) Zhydroxy3-MC; (5) 1-hydroxyd-MC; (6) arises from incubation; (7) 3-MC 11,12-oxide; (8) Zoxo-3-MC; (9) 1-oxo-&MC; (10) 3-methylcholanthrylene; (11) 3-MC.
co-chromatographs with cis-1,2-dihydroxy-3.MC. Peaks R, S and T are extremely variable and are unidentified. The metabolism of 3-MC by lung microsomes, Table 2, is qualitatively similar to that by liver microsomes. From a quantitative standpoint, however, lung microsomes were less efficient than liver microsomes at metabolizing 3-MC. When phenobarbital induced microsomes were used, total metabolism was less than that of control. None of the early eluting peaks (A-E) could be detected unless induced microsomes (Aroclor or 3-MC) were used. As in liver, major metabolism centered around 2.OH-3-MC (I@, l-OH-3-MC (N) and peak P. Tables 1 and 2 also show the percent contribution of each metabolite of total metabolism for liver and lung, respectively (numbers in parentheses). When induced liver microsomes are used, the contribution of both 2-OH-3-MC (M) and l-OH-SMC (N) to total metabolism is reduced. This might be due to a further metabolism of these compounds to triols, since it has been shown that some of the early assay peaks are indeed secondary metabolites. With lung microsomes the contribution of 2-OH-3.MC to total metabolism does not appear to be affected
III
3
Q x
B 0
0 I,
h
FRACTION Fig. 3. Chromatographic separation of radioactive metabolites. Metabolism and separation were conducted as outlined in Materials and Methods. Tentative assignments are: (A, B, C, D, F) diols and triols; (R) unknown; (G) tmns-1,2dihydroxy&MC; (H, I, J) unknown. others 1141 have reported two of these peaks to be 4,6dihydroxy&MC and S,lO-dihydroxy-3-MC, (K and L) sometime two distinct peaks 0 (co-migrates with cis-1,2-dihydroxy-3-MC; 0 2-hydroxy-3MC; (N) lhydroxy&MC; (0) unknown, small and variable; fP, P’, Q), probably phenols. (R, S, T) unknown, small and variable.
by induction, whereas the contribution of l-OH-BMC is reduced upon induction. There is a general increase in the contribution of the early eluting peaks (A-E) to total metabolism upon induction. The metabolite(s) associated with peak P contribute(s) 3 times as much to total metabolism when induced (Aroclor or 3.MC) microsomes are used as compared to control or PB treated. This effect is seen in both liver and lung. In light of the fact that l-OH-SMC has been shown to give rise to an ultimate carcinogen upon further metabolism 131,it is interesting to note that the contribution of l-OH-S-MC to total metabolism is higher in lung than in liver. In all cases, less than 2% of the radioactivity remained associated with the aqueous phase. The bay region theory of carcinogenesis [2] would suggest that 3.MC is first metabolized to a 9,10-dihydrodiol and then to a bay region diol-epoxide. While 3-MC does give rise to a number of primary and secondary metabolites, we have been unable to show the formation of the 9,lOdihydrodiol in sufficient quantity to suggest that it is an important intermediate in the
329
TABLE 2 3-MC METABOLISM BY LUNG MICROSOMES: EFFECT OF VARIOUS INDUCING AGENTS pmol/mg
proteidmin
Control
+ S.E. Phenobarbital
A
-cl
B
(-)
(-)
C
(-) ‘cl
(-)
D
(-)
(-)
E
(-)
(-)
F
(-)
(-)
G
(-)
(-)
H
(-)
(-) il
(-)
(-)
(2.5%)
(-)
I J K+L M N 0 P P’ & R S+T Total Procedure
is the same as in Table 1.
Aroclor
3.MC
,0.2:‘,.1,
330
formation of an ultimate carcinogen. We have previously shown that the major metabolism of 3-MC centers around the cyclopentano-ring system, namely l-OH-&MC and 2-OH-3-MC. Thakker et al. have shown that the early eluting metabolites arise in part from further metabolism of l-OH-3-MC [13], and that two of these early metabolites are the diastereomeric 9,lOdihydrodiols [12]. One of these diastereomers is a carcinogen in newborn mice [3], as are 1.OH-3-MC and 2.OH-3.MC. The exact chemical nature of the ultimate carcinogenic form of 3.MC remains as yet unknown. We have centered our attention around the cyclopentano-ring and are currently investigating the further metabolism of both l.OH.8MC and 2.OH.3.MC. ACKNOWLEDGEMENTS
This investigation was supported by grant 21431 from the National Cancer Institute. This is publjcation number 0773 of the Department of Cell and Molecular Biology. REFERENCES 1 Dipple, A. and Nebxydoski, J.A. (1978) Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbens[alanthracene to DNA in cells in culture. Chem.-Biol. Interact., 20, 17-26. 2 Jerina, D.M. and Daly, J.W. (1976) In: Drug Metabolism, p. 13. Editors: D.V. Parke and R.L. Smith. Taylor and Francis, London. 3 Levin, W., Buening, M.K., Wood, A.W., Chang, R.L., Thakker, D.R., Jerina, D.M. and Conney, A.H. (1979) Tumorigenic activity of 3-methylcholanthrene metabolites on mouse skin and in newborn mice. Cancer Res., 39, 3549-3553. 4 Lowry, O.H., Roeebrough, N.J., Farr, A.L. and Randall, N.J. (1951) Protein measurement with folin phenol reagent. J. Biol. Chem., 193, 265-275. 5 Marquardt, H., Grover, P.L. and Sims, P. (1976) In vitro malignant transformation of mouse fibroblasts by non-K-region dihydrodiols derived from 7,12dimethylbenz(a) anthracene and benzo(a)pyrene. Cancer Res., 36 2059-2064. 6 Mataubara, T., Prough, R.A., Burke, M.D. and Estabrook, R.W. (1974) The preparation of microsomal fractions of rodent respiratory tract and their characterization. Cancer Res., 34, 2196-2203. 7 Siefried, H.E., Brikett, D.J., Levin, W., Lu, A.Y.H., Conney, A.H. and Jerina, D.M. (1977) Metabolism of benzo(a)pyrene. Arch. B&hem. Biophys., 178256-263. 8 Sims, P., Grover, P.L., Swaisland, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzda)pyrene proceeds by a diol-epoxide. Nature, 252, 326-327. 9 Stoming, T.A., Bornstein, W. and Bresnick, E. (1977) The metabolism of 3-methylcholanthrene by rat liver microsomes -A reinvestigation. Biochem. Biophys. Res. Commun., 79, 461-468. 10 Swaisland, A.J., Hewer, A., Pal, K., Keysell, G.R., Booth, J., Grover, P.L. and Sims, P. (1974) Polycyclic hydrocarbon epoxides: The involvement of S$-dihydro-8,9-dihydroxybenz(a)anthracene-lO,ll-oxide in reactions with the DNA of benz(a)anthracene treated hamster embryo cells. FEBS Lett., 47, 34-38. 11 Stoming, T.A. and Gerardot, R.J. (1977) High pressure liquid chromatographic separation of the metabolites of 3-methylcholanthrene. Life Sci., 20, 113-116.
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