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Effects of inhibitors of CYP1A and CYP2B on styrene metabolism in mouse liver and lung microsomes
Toxicology Letters 98 (1998) 131 – 137
Effects of inhibitors of CYP1A and CYP2B on styrene metabolism in mouse liver and lung microsomes Gary P. Carlson a,*, Dawn E. Hynes b, Nancy A. Mantick a b
a School of Health Sciences, Ci6il Engineering Building, Purdue Uni6ersity, West Lafayette, IN 47907 -1338, USA Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Uni6ersity, West Lafayette, IN 47907, USA
Received 2 April 1998; received in revised form 15 June 1998; accepted 16 June 1998
Abstract Much of the toxicity of styrene is associated with its bioactivation to styrene oxide. Both liver and lung have been shown to carry out this metabolic step, but there are differences reported as to which isomers of cytochrome P450 are responsible for this biotransformation in various species and tissues. CYP2E1, CYP2F, CYP2B, CYP1A1/2 and CYP2C11 have all been implicated. In the current study, a-naphthoflavone (aNF) and a-methylbenzylaminobenzotriazole (MBA), selective inhibitors of CYP1A and CYP2B, were used to ascertain the contributions of these isomers to styrene metabolism in mouse hepatic and pulmonary microsomes. aNF did not inhibit styrene metabolism with microsomal preparations from either tissue. This indicates that CYP1A is unimportant in the metabolism of styrene to styrene oxide. MBA at a very low concentration of 1 mM inhibited the hepatic metabolism of benzyloxyresorufin (a CYP2B substrate) by 87% but caused only a 16 to 19% inhibition of R- and S-styrene oxide formation. This demonstrates that CYP2B plays a minor role in styrene metabolism. At 10 mM, MBA caused an even greater inhibition of styrene metabolism but at that level it also inhibited p-nitrophenol hydroxylation, a CYP2E1-dependent reaction, suggesting a loss of selectivity for this inhibitor at higher concentrations. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Styrene; Liver; Lung; Mouse
1. Introduction Human exposure to styrene occurs in a number of occupational settings, particularly in the rein* Corresponding author. Tel.: + 1 765 4941412; fax: +1 765 4941414.
forced plastics industry (Miller et al., 1994). Styrene is both hepatotoxic and pneumotoxic in mice (Morgan et al. 1993a,b; Gadberry et al., 1996). Styrene oxide is generally considered to be the metabolite of styrene formed by cytochromes P450 which is responsible for many of its toxic activities (Bond, 1989). Styrene oxide itself has
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been shown to be toxic to mice (Gadberry et al., 1996). There is considerable interest, therefore, in this bioactivation process. Several cytochrome P450 isozymes have been implicated in the bioactivation of styrene to styrene oxide including CYP2E1, CYP2B1, CYP1A1/2 and CYP2C11 in rat lung and liver (Nakajima et al., 1994b) and CYP2E1, CYP2B6 and CYP2F1 in human lung (Nakajima et al., 1994a). A number of inducers of cytochromes P450 including pyridine, phenobarbital and bnaphthoflavone, inducers of CYP2E1, CYP2B and CYP1A, respectively, increase the toxicity of styrene to liver and/or lungs in mice (Gadberry et al., 1996). In comparing the effects of these inducers on the metabolism of styrene to styrene oxide, we found that pyridine increased S – SO but not R–SO formation in liver whereas phenobarbital increased the production of both enantiomers (Carlson, 1997b). b-Naphthoflavone had no effect. None of the inducers had any effect on styrene metabolism in mouse lung. In comparing the effects of inhibitors on styrene metabolism, the CYP2E1 inhibitor diethyldithiocarbamate decreased the formation of both enantiomers in both tissues whereas 5-phenyl-1-pentyne (an inhibitor of CYP2F2) inhibited metabolism primarily in lung. In both control and phenobarbitaltreated mice, SKF525A inhibited both R – SO and S – SO formation in liver but only S – SO production in lung. Thus there are some tissue-specific differences in rates of metabolism and susceptibility to induction and inhibition. In view of the apparent contribution of multiple cytochromes P450 to styrene metabolism, it was of interest in the present study to examine the effects of more specific inhibitors of CYP1A and CYP2B on styrene metabolism in both lung and liver. a-Naphthoflavone (aNF) was selected to inhibit CYP1A. For CYP2B, two inhibitors were initially compared for potency and selectivity. These were a-methylbenzylaminobenzotriazole (MBA) which has been shown to be an isozyme-selective and potent inhibitor (Mathews and Bend, 1986) and N-(2-p-nitrophenethyl) chlorofluoroacetamide (NCFA) which also has been found to be selective (Halpert et al., 1990).
Since the R-enantiomer of styrene oxide is more toxic than the S-enantiomer (Gadberry et al., 1996) and is preferentially produced in control mouse liver and lung (Carlson, 1997a,b) and in view of differences in the effects of previous studies on inhibition of styrene metabolism in regard to the ratio of the SO-enantiomers (Carlson, 1997b), it was important to determine if this ratio was altered in mice by the inhibitors used in the current study. The styrene concentration used in the metabolism assay was 2 mM which was not only the concentration used in our previous studies (Carlson 1997a,b), but was also the concentration used by Foureman et al. (1989) and close to the high concentration (1.85 mM) used by Nakajima et al. (1994b) where anti-CYP2B1/2 had a greater effect on styrene metabolism than at a lower substrate concentration (0.85 mM).
2. Materials and methods
2.1. Animals CD-1 (Crl:CD-1 (ICR) BR) mice were obtained from Charles River Laboratories (Wilmington, MA). They were housed in group cages in environmentally controlled rooms on a 12 h light:dark cycle. Rodent laboratory chow (No. 5001, Purina Mills, St. Louis, MO) and tap water were allowed ad libitum. All animals were allowed a minimum of 1 week to adapt to the animal facilities and diet before being used in any experiment.
2.2. Chemicals Styrene, styrene oxide and 3,3,3-trichloropropene oxide were obtained from Aldrich Chemical (Milwaukee, WI). NADPH, a-naphthoflavone and HEPES buffer were from Sigma Chemical (St. Louis, MO). The resorufins were obtained from Molecular Probes (Eugene, OR). a-Methylbenzoaminobenzotriazole and N-(2-pnitrophenethyl)chlorofluoroacetamide were obtained from James Mathews (Research Triangle Institute). Other chemicals were reagent grade or better.
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137
2.3. Metabolism assays Groups of 10 to 20 mice were killed and their livers and lungs were removed and pooled. The tissues were homogenized in 0.5 M Tris – HCl buffer (pH 7.4) containing 1.15% KCl. Microsomes were prepared by differential centrifugation with the first centrifugation at 9000 ×g for 20 min followed by centrifugation of this supernatant at 105000× g for 60 min. The pooled microsomal preparations were resuspended and used in each of the assays such that all treatments could be compared back to the same control. In all the assays, when the inhibitors were included, they were added in 10 ml of methanol and a similar volume of methanol was added to the control. A 10 min preincubation period was used. Preliminary experiments indicated that the methanol was not inhibitory. p-Nitrophenol hydroxylation was measured using a modification of the method of Reinke and Moyer (1985). The 2 ml incubation mixture contained 0.2 mM p-nitrophenol, 5 mM MgCl2, 1 mM NADPH and microsomes equivalent to approximately 3 mg protein in 0.05 M Tris buffer (pH 7.4). After 15 min at 37°C, the reaction was terminated with 0.5 ml 0.6 N perchloric acid. The samples were centrifuged in a microcentrifuge at 11500 rpm for 10 min and to 1.0 ml of the supernatant was added 0.2 ml of 10 N NaOH. The samples were centrifuged again and the absorbance was measured at 546 nm for comparison to a 4-nitrocatechol standard curve. 7-Ethoxyresorufin O-deethylation (EROD) and 7-benzyloxyresorufin O-debenzylation (BROD) were measured using the method of Burke et al. (1985). The reaction mixture contained Tris buffer (100 mM, pH 7.8) and 0.20 mM NADPH. Reactions were carried out in a F2000 fluorescence spectrophotometer (Hitachi, Japan) with substrate concentrations of 10 mM 7-ethoxyresorufin or 2.5 mM benzyloxyresorufin. Formation of resorufin was measured for 3 min using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. The metabolism of styrene to styrene oxide was determined as previously described (Carlson, 1997a). Microsomes (a minimum of 0.2 – 0.3 mg
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protein) from the livers or lungs of mice were incubated for 20 min in an incubation mixture containing 2 mM styrene, 5 mM MgCl2, 2 mM NADPH and 1 mM trichloropropene oxide to inhibit epoxide hydrolase in 0.1 M HEPES buffer (pH 7.4) with a total volume of 1.0 ml. Incubations were carried out at 37°C in 25 ml vials with caps with rubber/teflon septa (Pierce, Rockford, IL) in a Dubnoff metabolic shaker. The reaction was terminated by the addition of two ml of cold heptane. After vortexing to extract the styrene and styrene oxide, samples were frozen to remove the aqueous layer. Metabolites in the organic layer was then analyzed using a Chiralpak AS (Chiral Technologies, Exton, PA) guard column (4.6×50 mm) and analytical column (4.6× 250 mm) on a Shimadzu HPLC. The mobile phase was heptane/isopropanol (99:1) at a rate of 1 ml/min. UV detection was at 219 nm which gave a good response for the styrene oxide. Standards were prepared from styrene oxide in heptane. Proteins were determined by the bicinchoninic acid method (Redinbaugh and Turley, 1986) (Pierce).
2.4. Statistical analysis Each assay was carried out 2–4 times as indicated in the individual tables. Values are expressed as mean9S.E. In comparing the inhibitor value with control value, a paired Student t-test was utilized. The level of significance selected was PB 0.05. Where necessary because of the variability of the data, square root transformations were done prior to analysis.
3. Results In order to validate the specificity of the inhibitors for both the hepatic and pulmonary microsomes of these CD-1 mice, the rates of metabolism of ethoxyresorufin, which is primarily dependent upon CYP1A and benzyloxyresorufin, which is CYP2B mediated, were measured. As expected, aNF at a concentration of 1 or 10 mM was inhibitory of ethoxyresorufin dealkylation in hepatic microsomes but did not inhibit benzy-
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loxyresorufin metabolism (Table 1). On the other hand, MBA inhibited benzyloxyresorufin metabolism by 87 to 92% at 1 or 10 mM. The small difference in inhibition by the 10-fold increase in concentration of inhibitor suggests near maximum inhibition of CYP2B even at the lower concentration. There was some evidence of inhibition of ethoxyresorufin metabolism at the high concentration of MBA. Surprisingly, NCFA appeared to be less selective as an inhibitor and was only effective at the higher concentration even for the benzyloxyresorufin metabolism. A similar pattern for the influence of the inhibitors on alkoxyresorufin metabolism was demonstrated for the lung with the exception that the lung was resistant to the inhibitory effect of aNF (Table 2). Both MBA and NCFA had minimal inhibitory activity for ethoxyresorufin. However, both inhibited benzyloxyresorufin metabolism. The effect of MBA was particularly dramatic in causing over 95% inhibition at a concentration of 1 mM. To examine the influence of these inhibitors on a known CYP2E1 dependent pathway, p-nitrophenol hydroxylation was measured. aNF did not inhibit the activity of this enzyme in either liver or Table 1 Effects of inhibitors on alkoxyresorufin metabolism in livers of mice Treatment
Concentration Ethoxyre(mM) sorufina
Benzyloxyresorufina
Table 2 Effects of inhibitors on alkoxyresorufin metabolism in lungs of mice Treatment
NFb MBAc NCFAd
— 10 1
36.4 9 1.5
118.3 9 43.5
13.0 90.3e 24.692.3e
148.49 47.4 125.99 42.5e
10 1
22.794.1 37.399.6
9.0 9 2.7e 15.9 95.3e
250 50
26.09 5.6 29.494.5
60.4 923.2e 100.8 9 34.3e
11.2 91.9
86.9 933.7
10 1
8.99 0.9 10.7 91.8
84.1928.4 73.0 9 22.8
MBAc
10 1
9.5 91.1 9.4 91.7
0.290.1e 2.591.1e
250 50
9.6 90.1 9.3 9 1.7
36.8 9 10.9e 67.2 925.4
NCFAd
Values are mean9 S.E. for duplicate experiments for ethoxyresorufin and quadruplicate for benzyloxyresorufin and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
—
a Values are mean9S.E. for duplicate experiments for ethoxyresorufin and quadruplicate for benzyloxyresorufin and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
lung (Table 3). NCFA had no effect in the liver and the inhibitory activity in pulmonary microsomes was not significant. Interestingly, MBA had little or no effect on hepatic or pulmonary metabolism at the lower concentration of 1 mM. However, at the higher concentration of 10 mM, the inhibition was greater, particularly in lung. Table 3 Effects of inhibitors on p-nitrophenol hydroxylation in liver and lungs of mice
Control NF
b
MBAc NCFAd
a
Benzyloxyresorufina
NFb
Control
Treatment Control
Concentration Ethoxyre(mM) sorufina
Livera
Lunga
434 950
83.7 9 22.2
10 1
430 932 4399 51
70.9 9 23.7 82.0 922.0
10 1
301 95 424945
35.4 9 11.5e 56.6 9 17.1
250 50
438 953 422 947
56.0 917.1 69.4 9 23.4
Concentration (mM) —
a Values are mean 9S.E. for duplicate experiments and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137 Table 4 Effects of inhibitors on the metabolism of styrene to styrene oxide in livers of mice Treatment
Control NFb
Concentration R-styrene ox(mM) idea — 10
c
MBA
1 10
1.58 90.63
S-styrene oxidea 1.23 9 0.16
1.5490.63 d
1.3290.57 1.099 0.53d
1.19 9 0.17 1.009 0.15d 0.54 90.07d
a Values are mean9 S.E. for four replicates and units are nmols formed/mg protein per min. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d Significantly different from control (PB0.05) using paired Student’s t-test.
Because of the potent and profound effects of MBA on CYP2B, this inhibitor was selected for use along with aNF in studies on the inhibition of the metabolism of styrene to styrene oxide. Similar to the studies on p-nitrophenol hydroxylation, aNF was without effect on hepatic metabolism of styrene (Table 4). However, MBA at the low dose (1 mM) caused a 16 to 19% inhibition of the production of the R- and S-enantiomers of styrene oxide. Although there was variability among the experiments in which the higher concentration of MBA (10 mM) was used, there was a substantial effect with of 31 and 56% inhibition for the R- and S- enantiomers. Similar to what was observed in liver, in lung there was little or no inhibition of styrene metabolism by aNF (Table 5). One mM MBA inhibited styrene metabolism by 25 and 38% for the R- and S-enantiomers. Increasing the concentration of this inhibitor 10fold caused only a modest increase in inhibition to 29 and 41%.
cytochromes P450 indicate that CYP2B6, CYP2E1 and CYP2F1 are important (Nakajima et al., 1994a). Our studies demonstrating the ability of pyridine, phenobarbital and bNF to enhance the hepatotoxicity of styrene support the roles of CYP2E1, CYP2B and CYP1A (Gadberry et al., 1996). However, when styrene oxide formation itself was measured in mouse hepatic microsomes, pyridine and phenobarbital but not bNF were effective inducers (Carlson, 1997b). Diethyldithiocarbamate, which is a relatively specific inhibitor of CYP2E1 (Ono et al., 1996), inhibits the formation of both enantiomers of styrene in both liver and lung microsomal preparations (Carlson, 1997b). 5-Phenyl-1-pentyne inhibits styrene metabolism primarily in the lung (Carlson, 1997b) where CYP2F2 is located (Chang et al., 1996). In both cases there is no change in the ratio of the enantiomers formed. Inhibition by SKF525A occurs in both liver and lung, but the formation of R-styrene oxide is much less affected than that of the S-enantiomer, especially in the lung where there is no effect on R-styrene oxide formation and nearly complete suppression of S-styrene oxide production (Carlson, 1997b). The results with SKF525A are difficult to interpret since it is non-selective and inhibits several cytochromes P450 in the liver including 1A, 2A, 2B, 2C, 2C19, 2D and 2E to varying degrees (Ono et al., 1996) and the metabolism of a variety of substrates in lung (Litterst et al., 1977). Table 5 Effects of inhibitors on the metabolism of styrene to styrene oxide in lungs of mice Treatment
Control NFb
4. Discussion Studies by Nakajima et al. using anti-cytochrome P450 antibodies suggest that in rat lung and liver CYP2C11, CYP2B1, CYP1A1/2 and CYP2E1 are involved in the bioactivation of styrene to styrene oxide (Nakajima et al., 1994b) and studies using vaccinia virus-expressed human
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c
MBA
Concentration R-styrene ox(mM) idea —
1.50 90.23
S-styrene oxidea 0.63 9 0.06
10
1.43 90.22
0.56 9 0.04
1 10
1.12 90.22 1.07 9 0.26d d
0.39 9 0.03d 0.37 9 0.03d
a Values are mean 9 S.E. for four replicates and units are nmols formed/mg protein per min. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d Significantly different from control (PB0.05) using paired Student’s t-test.
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To understand the roles of CYP1A and CYP2B in styrene metabolism better than can be determined from the results obtained with SKF525A, more selective inhibitors were used in the current experiments. The selectivity of aNF for CYP1A was confirmed based on the alkoxyresorufin assays and it had no influence on the hydroxylation of p-nitrophenol. aNF did not inhibit the metabolism of styrene to styrene oxide. This is in agreement with our finding that treatment with bNF, an inducer of CYP1A, does not increase styrene oxide formation (Carlson, 1997b) although it is at odds with our finding that bNF increases the toxicity of styrene (Gadberry et al., 1996) and the suggestion by Nakajima et al. (1994b) that CYP1A is involved. The reason for this is not clear but could be related to other pathways of styrene metabolism. When the CYP2B inhibitors MBA and NCFA were compared, the former was found to be a more potent and better inhibitor while at the same time maintaining a selectivity for CYP2B compared with CYP1A based on the alkoxyresorufin assays and thus was chosen for the styrene metabolism studies. When p-nitrophenol hydroxylation was used as a measure of CYP2E1 activity in the liver, MBA at a concentration of 1 mM had no effect. When the concentration was increased to 10 mM there was a modest inhibition. When styrene metabolism was measured, the overall formation of styrene oxide was inhibited 17% by 1 mM MBA, a dose which caused an 87% inhibition of CYP2B-mediated benzyloxyresorufin metabolism and, as noted above, no inhibition of p-nitrophenol hydroxylase. These data indicate that CYP2B plays a minor role in styrene metabolism. MBA at the higher concentration of 10 mM caused a greater inhibition of styrene metabolism, but it also did so for p-nitrophenol hydroxylation suggesting that at higher concentrations it may not be as selective and may inhibit CYP2E1. This would be in agreement with some in vivo studies on MBA that demonstrate CYP2E1 inhibition (Mathews, unpublished observations). Similar results were observed with lung where 1 mM MBA caused a \95% inhibition of benzyloxyresorufin metabolism. There was slightly greater inhibition of styrene metabolism
by MBA in lung compared with liver, but this correlates with the limited data indicating that a greater inhibition of p-nitrophenol hydroxylation also occurs in lung. There was little differentiation with respect to inhibition of the formation of the two enantiomers. In summary the data using selected inhibitors indicate that CYP1A plays little or no role in the metabolism of styrene to styrene oxide in mouse hepatic or pulmonary microsomes and that CYP2B makes only a minor contribution in naive animals. This is in contrast to the greater involvement of CYP2E1 and CYP2F2 (especially in lung) as demonstrated by previous selective inhibitors of these cytochrome P450 isozymes (Carlson, 1997b). In those studies the use of diethyldithiocarbamate and 5-phenyl-1-pentyne indicated that 50–60% of the styrene metabolizing activity of the lung could be inhibited by either of these inhibitors.
Acknowledgements This study was supported in part by National Institutes of Health Grant ES04362 and a grant from the Styrene Information and Research Center. Our special thanks go to James M. Mathews for providing the inhibitors and reviewing the manuscript.
References Bond, J.A., 1989. Review of the toxicology of styrene. Crit. Rev. Toxicol. 19, 227 – 249. Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., Meyer, R.T., 1985. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P450. Biochem. Pharmacol. 34, 3337 – 3345. Carlson, G.P., 1997a. Comparison of mouse strains for susceptibility to styrene-induced hepatotoxicity and pneumotoxicity. J. Toxicol. Environ. Health 51, 177 – 187. Carlson, G.P., 1997b. Effects of inducers and inhibitors on the microsomal metabolism of styrene to styrene oxide in mice. J. Toxicol. Environ. Health 51, 477 – 488. Chang, A., Buckpitt, A., Plopper, C., Alworth, W., 1996. Suicide inhibition of CYP2F2, the enzyme responsible for naphthalene (NA) metabolism to a Clara cell toxicant. Toxicologist 30, 72.
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137 Foureman, G.L., Harris, C., Guengerich, F.P., Bend, J.R., 1989. Stereoselectivity of styrene oxidation in microsomes and in purified cytochrome P-450 enzymes from rat liver. J. Pharmacol. Exp. Ther. 248, 492–497. Gadberry, M.G., DeNicola, D.B., Carlson, G.P., 1996. Pneumotoxicity and hepatotoxicity of styrene and styrene oxide. J. Toxicol. Environ. Health 48, 273–294. Halpert, J., Jaw, J-Y., Balfour, C., Kaminsky, L.S., 1990. Selective inactivation by chlorofluoroacetamides of the major phenobarbital-inducible form(s) of rat liver cytochrome P-450. Drug Metab. Dispos. 18, 168–174. Litterst, C.L., Mimnaugh, E.G., Gram, T.E., 1977. Comparative alterations in extrahepatic drug metabolism by factors known to affect hepatic activity. Biochem. Pharmacol. 26, 749 – 755. Mathews, J.M., Bend, J.R., 1986. N-Alkylaminobenzotriazoles as isozyme-selective suicide inhibitors of rabbit pulmonary microsomal P-450. Mol. Pharmacol. 30, 25–32. Miller, R.R., Newhook, R., Poole, A., 1994. Styrene production, use and human exposure. Crit. Rev. Toxicol. 24 (S1), S1– S10. Morgan, D.L., Mahler, J.F., Dill, J.A., Price, H.C., O’Connor, R.W., Adkins, B., 1993a. Styrene inhalation toxicity studies in mice. III. Strain differences in susceptibility. Fundam. Appl. Toxicol. 21, 326–333. Morgan, D.L., Mahler, J.F., O’Connor, R.W., Price, H.C.,
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Adkins, B., 1993b. Styrene inhalation toxicity studies in mice. I. Hepatotoxicity in B6C3F1 mice. Fundam. Appl. Toxicol. 20, 325 – 335. Nakajima, T., Elovaara, E., Gonzalez, F.J., Gelboin, H.V., Raunio, H., Pelkonen, O., Vainio, H., Aoyama, T., 1994a. Styrene metabolism by cDNA-expressed human hepatic and pulmonary cytochromes P450. Chem. Res. Toxicol. 7, 891 – 896. Nakajima, T., Wang, R.S., Elovaara, E., Gonzalez, F.J., Gelboin, H.V., Vainio, H., Aoyama, T., 1994b. CYP2C11 and CYP2B1 are major cytochrome P450 forms involved in styrene oxidation in liver and lung microsomes from untreated rats, respectively. Biochem. Pharmacol. 48, 637 – 642. Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F.J., Tsutsui, M., 1996. Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26, 681 – 693. Redinbaugh, M.G., Turley, R.B., 1986. Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal. Biochem. 153, 267 – 271. Reinke, L.A., Moyer, M.J., 1985. p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos. 13, 548 – 552.
.
Effects of inhibitors of CYP1A and CYP2B on styrene metabolism in mouse liver and lung microsomes Gary P. Carlson a,*, Dawn E. Hynes b, Nancy A. Mantick a b
a School of Health Sciences, Ci6il Engineering Building, Purdue Uni6ersity, West Lafayette, IN 47907 -1338, USA Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Uni6ersity, West Lafayette, IN 47907, USA
Received 2 April 1998; received in revised form 15 June 1998; accepted 16 June 1998
Abstract Much of the toxicity of styrene is associated with its bioactivation to styrene oxide. Both liver and lung have been shown to carry out this metabolic step, but there are differences reported as to which isomers of cytochrome P450 are responsible for this biotransformation in various species and tissues. CYP2E1, CYP2F, CYP2B, CYP1A1/2 and CYP2C11 have all been implicated. In the current study, a-naphthoflavone (aNF) and a-methylbenzylaminobenzotriazole (MBA), selective inhibitors of CYP1A and CYP2B, were used to ascertain the contributions of these isomers to styrene metabolism in mouse hepatic and pulmonary microsomes. aNF did not inhibit styrene metabolism with microsomal preparations from either tissue. This indicates that CYP1A is unimportant in the metabolism of styrene to styrene oxide. MBA at a very low concentration of 1 mM inhibited the hepatic metabolism of benzyloxyresorufin (a CYP2B substrate) by 87% but caused only a 16 to 19% inhibition of R- and S-styrene oxide formation. This demonstrates that CYP2B plays a minor role in styrene metabolism. At 10 mM, MBA caused an even greater inhibition of styrene metabolism but at that level it also inhibited p-nitrophenol hydroxylation, a CYP2E1-dependent reaction, suggesting a loss of selectivity for this inhibitor at higher concentrations. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Styrene; Liver; Lung; Mouse
1. Introduction Human exposure to styrene occurs in a number of occupational settings, particularly in the rein* Corresponding author. Tel.: + 1 765 4941412; fax: +1 765 4941414.
forced plastics industry (Miller et al., 1994). Styrene is both hepatotoxic and pneumotoxic in mice (Morgan et al. 1993a,b; Gadberry et al., 1996). Styrene oxide is generally considered to be the metabolite of styrene formed by cytochromes P450 which is responsible for many of its toxic activities (Bond, 1989). Styrene oxide itself has
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been shown to be toxic to mice (Gadberry et al., 1996). There is considerable interest, therefore, in this bioactivation process. Several cytochrome P450 isozymes have been implicated in the bioactivation of styrene to styrene oxide including CYP2E1, CYP2B1, CYP1A1/2 and CYP2C11 in rat lung and liver (Nakajima et al., 1994b) and CYP2E1, CYP2B6 and CYP2F1 in human lung (Nakajima et al., 1994a). A number of inducers of cytochromes P450 including pyridine, phenobarbital and bnaphthoflavone, inducers of CYP2E1, CYP2B and CYP1A, respectively, increase the toxicity of styrene to liver and/or lungs in mice (Gadberry et al., 1996). In comparing the effects of these inducers on the metabolism of styrene to styrene oxide, we found that pyridine increased S – SO but not R–SO formation in liver whereas phenobarbital increased the production of both enantiomers (Carlson, 1997b). b-Naphthoflavone had no effect. None of the inducers had any effect on styrene metabolism in mouse lung. In comparing the effects of inhibitors on styrene metabolism, the CYP2E1 inhibitor diethyldithiocarbamate decreased the formation of both enantiomers in both tissues whereas 5-phenyl-1-pentyne (an inhibitor of CYP2F2) inhibited metabolism primarily in lung. In both control and phenobarbitaltreated mice, SKF525A inhibited both R – SO and S – SO formation in liver but only S – SO production in lung. Thus there are some tissue-specific differences in rates of metabolism and susceptibility to induction and inhibition. In view of the apparent contribution of multiple cytochromes P450 to styrene metabolism, it was of interest in the present study to examine the effects of more specific inhibitors of CYP1A and CYP2B on styrene metabolism in both lung and liver. a-Naphthoflavone (aNF) was selected to inhibit CYP1A. For CYP2B, two inhibitors were initially compared for potency and selectivity. These were a-methylbenzylaminobenzotriazole (MBA) which has been shown to be an isozyme-selective and potent inhibitor (Mathews and Bend, 1986) and N-(2-p-nitrophenethyl) chlorofluoroacetamide (NCFA) which also has been found to be selective (Halpert et al., 1990).
Since the R-enantiomer of styrene oxide is more toxic than the S-enantiomer (Gadberry et al., 1996) and is preferentially produced in control mouse liver and lung (Carlson, 1997a,b) and in view of differences in the effects of previous studies on inhibition of styrene metabolism in regard to the ratio of the SO-enantiomers (Carlson, 1997b), it was important to determine if this ratio was altered in mice by the inhibitors used in the current study. The styrene concentration used in the metabolism assay was 2 mM which was not only the concentration used in our previous studies (Carlson 1997a,b), but was also the concentration used by Foureman et al. (1989) and close to the high concentration (1.85 mM) used by Nakajima et al. (1994b) where anti-CYP2B1/2 had a greater effect on styrene metabolism than at a lower substrate concentration (0.85 mM).
2. Materials and methods
2.1. Animals CD-1 (Crl:CD-1 (ICR) BR) mice were obtained from Charles River Laboratories (Wilmington, MA). They were housed in group cages in environmentally controlled rooms on a 12 h light:dark cycle. Rodent laboratory chow (No. 5001, Purina Mills, St. Louis, MO) and tap water were allowed ad libitum. All animals were allowed a minimum of 1 week to adapt to the animal facilities and diet before being used in any experiment.
2.2. Chemicals Styrene, styrene oxide and 3,3,3-trichloropropene oxide were obtained from Aldrich Chemical (Milwaukee, WI). NADPH, a-naphthoflavone and HEPES buffer were from Sigma Chemical (St. Louis, MO). The resorufins were obtained from Molecular Probes (Eugene, OR). a-Methylbenzoaminobenzotriazole and N-(2-pnitrophenethyl)chlorofluoroacetamide were obtained from James Mathews (Research Triangle Institute). Other chemicals were reagent grade or better.
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137
2.3. Metabolism assays Groups of 10 to 20 mice were killed and their livers and lungs were removed and pooled. The tissues were homogenized in 0.5 M Tris – HCl buffer (pH 7.4) containing 1.15% KCl. Microsomes were prepared by differential centrifugation with the first centrifugation at 9000 ×g for 20 min followed by centrifugation of this supernatant at 105000× g for 60 min. The pooled microsomal preparations were resuspended and used in each of the assays such that all treatments could be compared back to the same control. In all the assays, when the inhibitors were included, they were added in 10 ml of methanol and a similar volume of methanol was added to the control. A 10 min preincubation period was used. Preliminary experiments indicated that the methanol was not inhibitory. p-Nitrophenol hydroxylation was measured using a modification of the method of Reinke and Moyer (1985). The 2 ml incubation mixture contained 0.2 mM p-nitrophenol, 5 mM MgCl2, 1 mM NADPH and microsomes equivalent to approximately 3 mg protein in 0.05 M Tris buffer (pH 7.4). After 15 min at 37°C, the reaction was terminated with 0.5 ml 0.6 N perchloric acid. The samples were centrifuged in a microcentrifuge at 11500 rpm for 10 min and to 1.0 ml of the supernatant was added 0.2 ml of 10 N NaOH. The samples were centrifuged again and the absorbance was measured at 546 nm for comparison to a 4-nitrocatechol standard curve. 7-Ethoxyresorufin O-deethylation (EROD) and 7-benzyloxyresorufin O-debenzylation (BROD) were measured using the method of Burke et al. (1985). The reaction mixture contained Tris buffer (100 mM, pH 7.8) and 0.20 mM NADPH. Reactions were carried out in a F2000 fluorescence spectrophotometer (Hitachi, Japan) with substrate concentrations of 10 mM 7-ethoxyresorufin or 2.5 mM benzyloxyresorufin. Formation of resorufin was measured for 3 min using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. The metabolism of styrene to styrene oxide was determined as previously described (Carlson, 1997a). Microsomes (a minimum of 0.2 – 0.3 mg
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protein) from the livers or lungs of mice were incubated for 20 min in an incubation mixture containing 2 mM styrene, 5 mM MgCl2, 2 mM NADPH and 1 mM trichloropropene oxide to inhibit epoxide hydrolase in 0.1 M HEPES buffer (pH 7.4) with a total volume of 1.0 ml. Incubations were carried out at 37°C in 25 ml vials with caps with rubber/teflon septa (Pierce, Rockford, IL) in a Dubnoff metabolic shaker. The reaction was terminated by the addition of two ml of cold heptane. After vortexing to extract the styrene and styrene oxide, samples were frozen to remove the aqueous layer. Metabolites in the organic layer was then analyzed using a Chiralpak AS (Chiral Technologies, Exton, PA) guard column (4.6×50 mm) and analytical column (4.6× 250 mm) on a Shimadzu HPLC. The mobile phase was heptane/isopropanol (99:1) at a rate of 1 ml/min. UV detection was at 219 nm which gave a good response for the styrene oxide. Standards were prepared from styrene oxide in heptane. Proteins were determined by the bicinchoninic acid method (Redinbaugh and Turley, 1986) (Pierce).
2.4. Statistical analysis Each assay was carried out 2–4 times as indicated in the individual tables. Values are expressed as mean9S.E. In comparing the inhibitor value with control value, a paired Student t-test was utilized. The level of significance selected was PB 0.05. Where necessary because of the variability of the data, square root transformations were done prior to analysis.
3. Results In order to validate the specificity of the inhibitors for both the hepatic and pulmonary microsomes of these CD-1 mice, the rates of metabolism of ethoxyresorufin, which is primarily dependent upon CYP1A and benzyloxyresorufin, which is CYP2B mediated, were measured. As expected, aNF at a concentration of 1 or 10 mM was inhibitory of ethoxyresorufin dealkylation in hepatic microsomes but did not inhibit benzy-
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137
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loxyresorufin metabolism (Table 1). On the other hand, MBA inhibited benzyloxyresorufin metabolism by 87 to 92% at 1 or 10 mM. The small difference in inhibition by the 10-fold increase in concentration of inhibitor suggests near maximum inhibition of CYP2B even at the lower concentration. There was some evidence of inhibition of ethoxyresorufin metabolism at the high concentration of MBA. Surprisingly, NCFA appeared to be less selective as an inhibitor and was only effective at the higher concentration even for the benzyloxyresorufin metabolism. A similar pattern for the influence of the inhibitors on alkoxyresorufin metabolism was demonstrated for the lung with the exception that the lung was resistant to the inhibitory effect of aNF (Table 2). Both MBA and NCFA had minimal inhibitory activity for ethoxyresorufin. However, both inhibited benzyloxyresorufin metabolism. The effect of MBA was particularly dramatic in causing over 95% inhibition at a concentration of 1 mM. To examine the influence of these inhibitors on a known CYP2E1 dependent pathway, p-nitrophenol hydroxylation was measured. aNF did not inhibit the activity of this enzyme in either liver or Table 1 Effects of inhibitors on alkoxyresorufin metabolism in livers of mice Treatment
Concentration Ethoxyre(mM) sorufina
Benzyloxyresorufina
Table 2 Effects of inhibitors on alkoxyresorufin metabolism in lungs of mice Treatment
NFb MBAc NCFAd
— 10 1
36.4 9 1.5
118.3 9 43.5
13.0 90.3e 24.692.3e
148.49 47.4 125.99 42.5e
10 1
22.794.1 37.399.6
9.0 9 2.7e 15.9 95.3e
250 50
26.09 5.6 29.494.5
60.4 923.2e 100.8 9 34.3e
11.2 91.9
86.9 933.7
10 1
8.99 0.9 10.7 91.8
84.1928.4 73.0 9 22.8
MBAc
10 1
9.5 91.1 9.4 91.7
0.290.1e 2.591.1e
250 50
9.6 90.1 9.3 9 1.7
36.8 9 10.9e 67.2 925.4
NCFAd
Values are mean9 S.E. for duplicate experiments for ethoxyresorufin and quadruplicate for benzyloxyresorufin and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
—
a Values are mean9S.E. for duplicate experiments for ethoxyresorufin and quadruplicate for benzyloxyresorufin and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
lung (Table 3). NCFA had no effect in the liver and the inhibitory activity in pulmonary microsomes was not significant. Interestingly, MBA had little or no effect on hepatic or pulmonary metabolism at the lower concentration of 1 mM. However, at the higher concentration of 10 mM, the inhibition was greater, particularly in lung. Table 3 Effects of inhibitors on p-nitrophenol hydroxylation in liver and lungs of mice
Control NF
b
MBAc NCFAd
a
Benzyloxyresorufina
NFb
Control
Treatment Control
Concentration Ethoxyre(mM) sorufina
Livera
Lunga
434 950
83.7 9 22.2
10 1
430 932 4399 51
70.9 9 23.7 82.0 922.0
10 1
301 95 424945
35.4 9 11.5e 56.6 9 17.1
250 50
438 953 422 947
56.0 917.1 69.4 9 23.4
Concentration (mM) —
a Values are mean 9S.E. for duplicate experiments and units are pmols/min per mg protein. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d N-(2-p-nitrophenethyl)chlorofluoroacetamide. e Significantly different from control (PB0.05) using paired Student’s t-test.
G.P. Carlson et al. / Toxicology Letters 98 (1998) 131–137 Table 4 Effects of inhibitors on the metabolism of styrene to styrene oxide in livers of mice Treatment
Control NFb
Concentration R-styrene ox(mM) idea — 10
c
MBA
1 10
1.58 90.63
S-styrene oxidea 1.23 9 0.16
1.5490.63 d
1.3290.57 1.099 0.53d
1.19 9 0.17 1.009 0.15d 0.54 90.07d
a Values are mean9 S.E. for four replicates and units are nmols formed/mg protein per min. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d Significantly different from control (PB0.05) using paired Student’s t-test.
Because of the potent and profound effects of MBA on CYP2B, this inhibitor was selected for use along with aNF in studies on the inhibition of the metabolism of styrene to styrene oxide. Similar to the studies on p-nitrophenol hydroxylation, aNF was without effect on hepatic metabolism of styrene (Table 4). However, MBA at the low dose (1 mM) caused a 16 to 19% inhibition of the production of the R- and S-enantiomers of styrene oxide. Although there was variability among the experiments in which the higher concentration of MBA (10 mM) was used, there was a substantial effect with of 31 and 56% inhibition for the R- and S- enantiomers. Similar to what was observed in liver, in lung there was little or no inhibition of styrene metabolism by aNF (Table 5). One mM MBA inhibited styrene metabolism by 25 and 38% for the R- and S-enantiomers. Increasing the concentration of this inhibitor 10fold caused only a modest increase in inhibition to 29 and 41%.
cytochromes P450 indicate that CYP2B6, CYP2E1 and CYP2F1 are important (Nakajima et al., 1994a). Our studies demonstrating the ability of pyridine, phenobarbital and bNF to enhance the hepatotoxicity of styrene support the roles of CYP2E1, CYP2B and CYP1A (Gadberry et al., 1996). However, when styrene oxide formation itself was measured in mouse hepatic microsomes, pyridine and phenobarbital but not bNF were effective inducers (Carlson, 1997b). Diethyldithiocarbamate, which is a relatively specific inhibitor of CYP2E1 (Ono et al., 1996), inhibits the formation of both enantiomers of styrene in both liver and lung microsomal preparations (Carlson, 1997b). 5-Phenyl-1-pentyne inhibits styrene metabolism primarily in the lung (Carlson, 1997b) where CYP2F2 is located (Chang et al., 1996). In both cases there is no change in the ratio of the enantiomers formed. Inhibition by SKF525A occurs in both liver and lung, but the formation of R-styrene oxide is much less affected than that of the S-enantiomer, especially in the lung where there is no effect on R-styrene oxide formation and nearly complete suppression of S-styrene oxide production (Carlson, 1997b). The results with SKF525A are difficult to interpret since it is non-selective and inhibits several cytochromes P450 in the liver including 1A, 2A, 2B, 2C, 2C19, 2D and 2E to varying degrees (Ono et al., 1996) and the metabolism of a variety of substrates in lung (Litterst et al., 1977). Table 5 Effects of inhibitors on the metabolism of styrene to styrene oxide in lungs of mice Treatment
Control NFb
4. Discussion Studies by Nakajima et al. using anti-cytochrome P450 antibodies suggest that in rat lung and liver CYP2C11, CYP2B1, CYP1A1/2 and CYP2E1 are involved in the bioactivation of styrene to styrene oxide (Nakajima et al., 1994b) and studies using vaccinia virus-expressed human
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c
MBA
Concentration R-styrene ox(mM) idea —
1.50 90.23
S-styrene oxidea 0.63 9 0.06
10
1.43 90.22
0.56 9 0.04
1 10
1.12 90.22 1.07 9 0.26d d
0.39 9 0.03d 0.37 9 0.03d
a Values are mean 9 S.E. for four replicates and units are nmols formed/mg protein per min. b Alpha-naphthoflavone. c a-Methylbenzylaminobenzotriazole. d Significantly different from control (PB0.05) using paired Student’s t-test.
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To understand the roles of CYP1A and CYP2B in styrene metabolism better than can be determined from the results obtained with SKF525A, more selective inhibitors were used in the current experiments. The selectivity of aNF for CYP1A was confirmed based on the alkoxyresorufin assays and it had no influence on the hydroxylation of p-nitrophenol. aNF did not inhibit the metabolism of styrene to styrene oxide. This is in agreement with our finding that treatment with bNF, an inducer of CYP1A, does not increase styrene oxide formation (Carlson, 1997b) although it is at odds with our finding that bNF increases the toxicity of styrene (Gadberry et al., 1996) and the suggestion by Nakajima et al. (1994b) that CYP1A is involved. The reason for this is not clear but could be related to other pathways of styrene metabolism. When the CYP2B inhibitors MBA and NCFA were compared, the former was found to be a more potent and better inhibitor while at the same time maintaining a selectivity for CYP2B compared with CYP1A based on the alkoxyresorufin assays and thus was chosen for the styrene metabolism studies. When p-nitrophenol hydroxylation was used as a measure of CYP2E1 activity in the liver, MBA at a concentration of 1 mM had no effect. When the concentration was increased to 10 mM there was a modest inhibition. When styrene metabolism was measured, the overall formation of styrene oxide was inhibited 17% by 1 mM MBA, a dose which caused an 87% inhibition of CYP2B-mediated benzyloxyresorufin metabolism and, as noted above, no inhibition of p-nitrophenol hydroxylase. These data indicate that CYP2B plays a minor role in styrene metabolism. MBA at the higher concentration of 10 mM caused a greater inhibition of styrene metabolism, but it also did so for p-nitrophenol hydroxylation suggesting that at higher concentrations it may not be as selective and may inhibit CYP2E1. This would be in agreement with some in vivo studies on MBA that demonstrate CYP2E1 inhibition (Mathews, unpublished observations). Similar results were observed with lung where 1 mM MBA caused a \95% inhibition of benzyloxyresorufin metabolism. There was slightly greater inhibition of styrene metabolism
by MBA in lung compared with liver, but this correlates with the limited data indicating that a greater inhibition of p-nitrophenol hydroxylation also occurs in lung. There was little differentiation with respect to inhibition of the formation of the two enantiomers. In summary the data using selected inhibitors indicate that CYP1A plays little or no role in the metabolism of styrene to styrene oxide in mouse hepatic or pulmonary microsomes and that CYP2B makes only a minor contribution in naive animals. This is in contrast to the greater involvement of CYP2E1 and CYP2F2 (especially in lung) as demonstrated by previous selective inhibitors of these cytochrome P450 isozymes (Carlson, 1997b). In those studies the use of diethyldithiocarbamate and 5-phenyl-1-pentyne indicated that 50–60% of the styrene metabolizing activity of the lung could be inhibited by either of these inhibitors.
Acknowledgements This study was supported in part by National Institutes of Health Grant ES04362 and a grant from the Styrene Information and Research Center. Our special thanks go to James M. Mathews for providing the inhibitors and reviewing the manuscript.
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