Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6680-6684, November 1983 Medical Sciences Mechanisms of hydroxylation by cytochrome P-450: Metabolism of monohalobenzenes -by phenobarbital-induced microsomes (Idnetics/monohalophenols/Hammett constants/radical cation intermediate) LEO T. BURKA*, TERESAM. PLUCINSKIt, AND TIMOTHY L. MACDONALDt Department of Chemistry and Center in Environmental Toxicology, Vanderbilt University, Nashville, TN 37235 Communicated by Ronald Breslow, July 28, 1983 ABSTRACT The monohydroxylation of halobenzenes by phe- nucleotide (NADPH) was obtained from Sigma; o- and m-io- nobarbital-induced rat liver microsomes was studied. The.p-halo- doanisole were obtained by decomposing the corresponding phenol was found to be the major metabolite from all four halo- diazonium salt with potassium iodide (13). benzenes; o-halophenol formation decreased as the halogen atom Microsomes. Male Sprague-Dawley rats (80-100 g each) were size increased. Vmasfor-total hydroxylation (ortho and para prod- treated with phenobarbital (0.1% in drinking water) for 5 days. ucts) correlated well with the ar Hammett constant with a neg- Rats were then killed, and liver microsomes were prepared as ative pvalue. This implies a positivelycharged intermediate in the described (7). Microsomes were stored at -700C in 10mM Tris rate-determining step. Vm.._ for either ortho orpara hydroxylation acetate buffer, pH 7.4/1 mM EDTA/20% (vol/vol) glycerol. alone did not correlate with a Hammett constant, implying that Protein concentration was determined by the Lowry method the product-determining step occurs after the rate-determining (14) with bovine serum albumin as standard. step. Rate-determining formation of a radical cation intermediate Incubations. All incubations were carried out at 370C in a is postulated to explain this data. final volume of 2.0 ml per tube. Each tube contained potassium phosphate buffer (pH 7.4; 0.05 M), NADPH (2 mM for sub- Metabolism of monohalobenzenes, especially chlorobenzene strate concentrations <8.0 mM and 3 mM for substrate con- and bromobenzene, has been the subject of literally scores of centrations .8.0 mM),- microsomes (4.68 mg of microsomal studies. These compounds are of interest not only as primary protein), and various concentrations of benzene, bromoben- environmental contaminants but also because they serve as zene, chlorobenzene, fluorobenzene, or iodobenzene. Con- models for more structurally complex haloaromatics such as centrated solutions of benzene or halobenzenes were prepared polychloro- and polybromobiphenyls and polyhalogenated di- in acetonitrile. The maximum volume of acetonitrile added per phenyldioxins and diphenylfurans (1). Our recent interest in tube was 2% or less of the total incubation volume. All incu- the biological oxidation of heteroatoms (2-5) led us to consider bations were started by the addition of NADPH. the.possibility that oxidation of the halogen atom was the initial After 2 min, reactions were stopped by the addition of 200 step in the cytochrome P-450-catalyzed metabolism of aromatic ,ul of 10 M NaOH, and the tubes were placed on ice. A 50-1.l halides. Oxidation by removal of a hydrogen atom or an elec- volume of a standard solution of either p-chlorophenol (ben- tron as an initial step in cytochrome P-450-catalyzed metabo- zene and bromobenzene incubations) or p-bromophenol (chlo- lism has been postulated for several substrates, including vinyl robenzene, fluorobenzene, and iodobenzene incubations) in halides (6, 7), cyclopropyl amines (5, 8), sulfides (9), and nor- acetonitrile was added to each tube as internal standard. The bornane (10). Evidence for a similar one-electron oxidation of contents of the tubes were extracted once with 2 ml of pentane, aryl halides might be obtained from comparison of the kinetics and the supernatant was discarded. Dimethyl sulfate (200 1.d) of hydroxylation of the monohalobenzenes. was added to each tube to convert phenols to anisoles. The tubes Although the aryl halides are a thoroughly studied group of were heated at 400C for 30 min with frequent vigorous shaking. compounds, consultation of the literature revealed an almost The contents of the tubes were extracted twice with 2 ml of complete lack of kinetic data concerning their in vitro metab- pentane. The upper phases were combined and transferred to olism. An excellent product study of chlorobenzene metabo- 5-ml conical vials. The pentane solution was carefully concen- lism progressing in complexity from reconstituted soluble he- trated by distillation at 380C to a volume of about 50 1l. Control moprotein systems to perfused rat livers was reported by experiments established that selective methylation of one of Selander et al. (11). A Lineweaver-Burk plot for the metabo- the halophenols or evaporation of one of the derived anisoles lism of bromobenzene by a supernatant obtained at 9,000 X g under these conditions was not significant. Carbon tetrachlo- from rat liver can be found in a study by Zampaglione et al. (12). ride (50 Al) was added to each vial. The vials were tightly capped However, it became obvious that, to make a valid comparison and stored at -200C until the contents were assayed. of the hydroxylation kinetics for the monohalobenzenes, a set Measurement of Phenol Production. Phenols were mea- of data generated in one laboratory with microsomes from a sured as methyl ethers (anisoles) by using a Shimadzu Scientific single isolation and with a consistent method of acquiring and (Columbia, MD) model GC-Mini 2 gas chromatograph equipped presenting the kinetic data would be required. with a flame ionization detector. Data were recorded, and peak MATERIALS AND METHODS areas were calculated with a Shimadzu C-RIB integrating re- corder. A Supelco OV-1 glass capillary (30 m) was used to mea- All chemicals were of the highest purity commercially avail- able. Pentane was redistilled prior to use. Reduced pyridine * To whom reprint requests should be addressed at: Toxicology, Re- search and Testing Program, National Institute of Environmental Health The publication costs of this article were defrayed in part by page charge Sciences, Research Triangle Park, NC 27709. payment. This article must therefore be hereby marked "advertise- t Present address: Department of Chemistry, University of Virginia, ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Charlottesville, VA 22901. 6680 Downloaded by guest on October 1, 2021 Medical Sciences: Burka et al. Proc. Natl. Acad. Sci. USA 80 (1983) 6681 Table 1. Michaelis-Menten parameters for hydroxylation of benzene and halobenzenes by phenobarbital-induced microsomes VIMax K6, Km, nmol min-' Substituent mM mM (mg of protein)-' VIm/Km I H 2.9 3.0 3.3 1.1 0.0 F 0.56 6.4 11 1.7 -0.07 0.14 Cl 1.8 1.9 6.7 3.5 0.11 0.17 Br 1.4 0.41 6.4 16 0.15 0.86 I 0.69 0.30 6.3 21 0.13 1.12 K., Km, and V.. were determined as described. Values for o+ are taken from ref. 16. Values for ir are taken from ref. 17. sure phenols produced from benzene, chlorobenzene, fluo- portion with no generating system), the rate of formation of robenzene, and iodobenzene metabolism. A Supelco SE 54 glass chlorophenols was biphasic. A steep initial linear phase was ob- capillary column (15 m) was used to measure phenols from bro- served for the first 2-3 min followed by another, more shallow, mobenzene metabolism. Column and detector temperatures linear phase fbr up to 1 hr, the longest incubation time used. were 1500C and 2000C, respectively, for chloroanisole, anisole, The linearity of the rate of hydroxylation with time was not in- and iodoanisole determinations; 105'C and 1500C, respectively, vestigated as thoroughly for the other substrates, but the rates for measuring fluoroanisoles; and 1100C and 2000C, respec- were determined to be linear for at least 2 min. tively, for measuring bromoanisoles. Standard solutions of o-, Initial rates of formation of chlorophenols were determined p-, and m-anisoles for each compound were used to determine for six concentrations of chlorobenzene ranging from 0.3 to 8.0 retention times, the detector-response ratios for the com- mM. The Michaelis-Menten parameters, 4m and Vm., for or- pounds, and the internal standard. The detector-response ra- tho, para, and total (ortho plus para) hydroxylation were de- tios were determined to be linear over the range used in the termined from Lineweaver-Burk plots of the data with a least- analysis. Each incubation was done in duplicate; each incu- squares fit. The correlation coefficient (r) was greater than 0.99 bation mixture was analyzed at least twice. The minimum mi- in each case. crosomal enzyme activity detectable by this procedure is about Likewise, 4m and V., were determined from initial rate data 0.1 nmol/min-mg of protein. from three concentrations of fluorobenzene, 1.0-4.0 mM; four Determination of K1. The interaction of halobenzenes and concentrations of bromobenzene, 0.3-4 mM; five concentra- benzene with microsomal cytochrome P450 was examined by tions of iodobenzene, 0.15-1.5 mM; and four concentrations of determining Ks values for these compounds. The procedure used benzene, 1.0-8.0 mM. The correlation coefficient for the least- was based on that of Schenkman et al. (15). A Cary model 219 squares fit was 0.97 or greater for all determinations. Apparent (Varian) spectrophotometer was used. Microsomes from the same inhibition was observed with concentrations of fluorobenzene batch used for K4 and Vm. determinations were suspended in and bromobenzene above 4.0 mM. sample and reference cuvettes containing 0.1 M phosphate buffer m-Halophenols were always minor products, detectable with (pH 7.4) to a final protein concentration of 4.68 mg per each certainty only at the highest concentrations of chloro- and fluo- 3-ml volume.