The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.

ABSTRACT. Introduction. Chronic constipation is a common symptom in pediatrics, and physicians often use mineral oil to treat chronic constipation in children. Mineral oil, a hydrocarbon, may not elicit a normal protective cough reflex and may impair mucociliary transport. These effects can increase the likelihood of its aspiration and subsequent impaired clearance from the respiratory tract. We report a case of a child with neurodevelopmental delay with chronic constipation and a history of chronic mineral oil ingestion presenting as asymptomatic exogenous lipoid pneumonia (ELP).Case History. A 6-year-old white boy with a history of developmental delay was found to have an infiltrate in his right upper lobe on a chest radiograph obtained during evaluation for thoracic scoliosis. The patient had a long history of constipation with daily use of mineral oil. He was fed by mouth and had occasional episodes of coughing and choking during feeding. He was asymptomatic at presentation and physical examination was unremarkable. The patient was advised to stop administration of the mineral oil and was treated empirically with antibiotics during a 3-month period. At follow-up examination the patient continued to be asymptomatic, with the radiologic persistence of the infiltrate. Diagnosis of lipoid pneumonia was made by diagnostic bronchoscopy with bronchoalveolar lavage (BAL). The exogenous origin of the lipid in the BAL fluid was confirmed by gas chromatography/mass spectrometry.Discussion. The clinical presentation of ELP is nonspecific and ranges from the totally asymptomatic patient with incidental radiologic finding, like our patient, to the patient with acute or chronic symptoms attributable to pneumonia, pulmonary fibrosis, or cor pulmonale. Bronchoscopy with BAL can be successful in establishing the diagnosis of ELP by demonstration of a high lipid-laden macrophage index. Treatment of ELP in children is generally supportive, with the symptoms and roentgenographic abnormalities resolving within months after stopping the use of mineral oil.Conclusion. Lipoid pneumonia as a result of mineral oil aspiration still occurs in the pediatric population. It can mimic other diseases because of its nonspecific clinical presentation and radiographic signs. In patients with swallowing dysfunction and pneumonia, a history of mineral oil use should be obtained and a diagnosis of ELP should be considered in the differential diagnoses if mineral oil use has occurred. Our case points to the need for increased awareness by the general pediatricians of the potential hazards of mineral oil use for chronic constipation. Pediatrics 1999;103(2). URL: http://www. pediatrics.org/cgi/content/full/103/2/e19; bronchoalveolar lavage, constipation, exogenous lipoid pneumonia, lipidladen macrophages, mineral oil.ABBREVIATIONS. RUL, right upper lobe; CXR, chest x-ray; BAL, bronchoalveolar lavage; ELP, exogenous lipoid pneumonia. C hronic constipation, a common symptom in pediatrics, accounts for 3% of referrals to teaching hospital clinics and 10...

Eleven polycyclic aromatic hydrocarbons (PAHs) and 14 acetylenic PAHs and biphenyls were used to analyze interactions with cytochrome P450 (P450) 1B1 in inhibiting catalytic activity, using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. Most of the chemicals examined were direct inhibitors of P450 1B1 except for 4-(1-propynyl)biphenyl, a mechanism-based inhibitor. In the case of direct inhibition of EROD activity {15 of 24 chemicals, e.g. benzo[a]pyrene, 1-(1-propynyl)pyrene, and 3-(1-propynyl)phenanthrene}, restoration of the EROD activity occurred with increasing incubation time, and kinetic analysis showed that EROD K m values were higher with these inhibitors at initial stages of incubation but became lower with increasing incubation time. With the other 9 chemicals, the K m values for P450 1B1-mediated EROD increased during the incubations. Acetylenic inhibitors, but not the 11 PAHs, induced reverse type I spectral changes with P450 1B1 and the low dissociation constants (K s ) suggested a role for such interaction in the inhibition of catalytic activity. Studies of quenching of P450 1B1-derived fluorescence with inhibitors demonstrated that acetylenic inhibitors and PAHs interacted rapidly with P450 1B1, with K d values <10 μM. However, studies of quenching of inhibitor-derived fluorescence with P450 1B1 showed these interactions to be different, i.e. B[a]P interacted with P450 1B1 more slowly. Molecular docking of P450 1B1, based on P450 1A2 crystal structure, suggested that there are clear differences in the interaction of PAH inhibitors with P450 1B1 and 1A2 and that these differences may explain why PAH inhibitors inhibit P450 1 enzymes by different mechanisms. The results suggest that P450 1B1 interacts with synthetic polycyclic aromatic acetylenes and PAHs in different ways, depending on the chemicals, and that these differences in interactions may explain how these chemicals inhibit P450 activities by different mechanisms.