Mechanisms of hydroxylation by cytochrome P-450: metabolism of monohalobenzenes by phenobarbital-induced microsomes.

Abstract

The monohydroxylation of halobenzenes by phenobarbital-induced rat liver microsomes was studied. The.p-halophenol was found to be the major metabolite from all four halobenzenes; o-halophenol formation decreased as the halogen atom size increased. Vmasfor-total hydroxylation (ortho and para products) correlated well with the ar Hammett constant with a negative p value. This implies a positively charged intermediate in the rate-determining step. Vm.._ for either ortho orpara hydroxylation alone did not correlate with a Hammett constant, implying that the product-determining step occurs after the rate-determining step. Rate-determining formation of a radical cation intermediate is postulated to explain this data.Metabolism of monohalobenzenes, especially chlorobenzene and bromobenzene, has been the subject of literally scores of studies. These compounds are of interest not only as primary environmental contaminants but also because they serve as models for more structurally complex haloaromatics such as polychloro-and polybromobiphenyls and polyhalogenated diphenyldioxins and diphenylfurans (1). Our recent interest in the biological oxidation of heteroatoms (2-5) led us to consider the.possibility that oxidation of the halogen atom was the initial step in the cytochrome P-450-catalyzed metabolism of aromatic halides. Oxidation by removal of a hydrogen atom or an electron as an initial step in cytochrome P-450-catalyzed metabolism has been postulated for several substrates, including vinyl halides (6, 7), cyclopropyl amines (5, 8), sulfides (9), and norbornane (10). Evidence for a similar one-electron oxidation of aryl halides might be obtained from comparison of the kinetics of hydroxylation of the monohalobenzenes.Although the aryl halides are a thoroughly studied group of compounds, consultation of the literature revealed an almost complete lack of kinetic data concerning their in vitro metabolism. An excellent product study of chlorobenzene metabolism progressing in complexity from reconstituted soluble hemoprotein systems to perfused rat livers was reported by Selander et al. (11). A Lineweaver-Burk plot for the metabolism of bromobenzene by a supernatant obtained at 9,000 X g from rat liver can be found in a study by Zampaglione et al. (12). However, it became obvious that, to make a valid comparison of the hydroxylation kinetics for the monohalobenzenes, a set of data generated in one laboratory with microsomes from a single isolation and with a consistent method of acquiring and presenting the kinetic data would be required. MATERIALS AND METHODSAll chemicals were of the highest purity commercially available. Pentane was redistilled prior to use. Reduced pyridine nucleotide (NADPH) was obtained from Sigma; o-and m-iodoanisole were obtained by decomposing the corresponding diazonium salt with potassium iodide (13).Microsomes. Male Sprague-Dawley rats (80-100 g each) were treated with phenobarbital (0.1% in drinking water) for 5 days. Rats were then killed, and liver microsomes were prepared as descr...

Transformations of Aromatic Compounds by Nitrosomonas europaea

“…ortho and meta hydroxylations of phenol (producing 1,2-and 1,3-dihydroxybenzenes) by AMO in N. europaea were not observed previously (19). A cytochrome P-450 enzyme was shown to oxidize monohalobenzenes to ortho-and para-halophenols (with meta-phenols occurring as minor products), and substrate binding and orientation at the active site were suggested as factors which might influence the ratios of ortho-and para-halophenol products (4). The observations that halobenzenes were more inhibitory toward NH3 oxidation than benzene and alkylbenzenes ( Fig.…”

Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450cam)

“…Even in the case of 1,3‐DCB this mechanism could be operating in parallel with the NIH shift. Furthermore, although the NIH shift appeared to occur only with 1,3‐DCB, we cannot rule out the formation of intermediates, be they epoxides [32], σ‐bound cationic species [37] or radical cations [38–40], in the oxidation of the other benzene substrates which then underwent NIH shift rearrangement to give one product exclusively.…”

Quantum chemical simulation of cytochrome P450 catalyzed aromatic oxidation: Metabolism, toxicity, and biodegradation of benzene derivatives

“…In this article, we study these dependences on the basis of quantum chemical calculations using the so‐called oxenoid model 42. According to this model, the P450 enzyme breaks the dioxygen molecule and generates the active atomic oxygen species (oxens) 43. The oxens readily react with substrates.…”