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PubMed1998

Effects of volatile compounds on consumption of alfalfa pellets by sheep.

We examined the effects of six volatile compounds on alfalfa pellet consumption by lambs. In each experiment, 45 lambs were individually fed alfalfa pellets sprayed with a selected compound (camphor, limonene, cis-jasmone, beta-caryophyllene, borneol, or alpha-pinene) at one of five concentrations. Treatment concentrations were multiples (0, .5, 1, 2, and 10) of the concentration of a specific compound (X) that was related to differential herbivory of tarbush by livestock in previous studies. Treatments were applied to alfalfa pellets (.64 kg x lamb(-1) x d(-1), DM basis), and consumption was measured during a 20-min interval each morning for 5 d. Lambs were adapted to handling procedures and the pelleted diet (without treatments) for 10 d. Lambs were maintained and fed (approximately 4.5 to 5% of BW) as one group except during 20-min tests. A negative linear effect of treatment concentration on intake was observed for camphor (P < .02) and alpha-pinene (P < .01), and a quadratic response was detected for borneol (P < .02). The other three compounds had no discernible effect on consumption. Although volatile compounds generally had only minor influences on consumption, the negative influences of alpha-pinene and camphor concentrations on pellet consumption suggest that these monoterpenes may partially explain differential herbivory of individual tarbush plants by livestock.

Administration, Oral↗
PubMed2001

[Effects of different disinfectants on levels of endotoxin in canals of chronic periapical periodontitis].

OBJECTIVE: The objective of this study is to compare effects of formocresol, camphorated phenol and Ya Kan (metronedazole) on endotoxin levels in canals of chronic periapical periodontitis. METHODS: A total of 30 patients with chronic periapical periodontitis were selected from outpatients, and 10 cases each were randomly picked out to treat by sealing with formocresol, camphorated phenol and Ya Kan respectively. All patients were both sampled before and after sealing of these disinfectants, and chromogenic substrate method of limulus amebocyte lysate (LAL) test was used to measure levels of endotoxin. RESULTS: The effects of Ya Kan, formocresol and camphorated phenol on levels of endotoxin did not showed significant difference. CONCLUSION: Some anti-anaerobic bacterial drugs can be used to treat chronic periapical periodontitis.

Anti-Infective Agents, Local↗
PubMedDate not supplied

[Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme].

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.

Binding Sites↗
PubMed1985

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam.

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.

Camphor↗
PubMed1993

Active sites of the cytochrome p450cam (CYP101) F87W and F87A mutants. Evidence for significant structural reorganization without alteration of catalytic regiospecificity.

Ferricyanide oxidation of the aryl-iron complexes formed by the reaction of cytochrome P450 enzymes with arylhydrazines causes in situ migration of the aryl group from the iron to the porphyrin nitrogen atoms. The regiochemistry of this migration, defined by the ratio of the four possible N-arylprotoporphyrin IX isomers, provides a method for mapping the topologies of cytochrome P450 active sites. The method has been validated by using it to examine the active site of cytochrome P450cam (CYP101), for which a crystal structure is available. In agreement with the crystal structure, reaction with phenylhydrazine gives a 5:25:70 ratio of the NA:NC:ND (subscript indicates pyrrole ring) N-phenylprotoporphyrin IX isomers. Naphthylhydrazine, however, yields exclusively the NC regioisomer and 4-(phenyl)phenylhydrazine the NA:NC:ND isomers in a 14:40:46 ratio. These isomer ratio differences are readily explained by topological differences between the upper and lower reaches of the active site. Having validated the aryl-iron shift as a topological probe, we used it to investigate the structural changes caused by mutation of Phe-87, a residue that provides the ceiling over pyrrole ring D in the crystal structure of cytochrome P450cam. Mutation of Phe-87 to a tryptophan causes no detectable change in the regiochemistry of camphor hydroxylation and only minor changes in the N-aryl isomer ratios. However, mutation of Phe-87 to an alanine, which was expected to open up the region above pyrrole ring D, severely decreased the proportion of the ND in favor of the NA isomer. Less rather than more space is therefore available over pyrrole ring D in the F87A mutant despite the fact that the regiochemistry of camphor hydroxylation remains unchanged. These results provide evidence for significant structural reorganization in the upper regions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant.

Alanine↗
PubMed2004

Antispasmodic activity of extracts and compounds of Acalypha phleoides Cav.

The aerial parts of Acalypha phleoides are usually prescribed in the Mexican traditional medicine for a variety of gastrointestinal complaints. The MeOH-CHCl(3) (1:1) extract of the aerial part of A. phleoides showed an inhibitory effect on the gastrointestinal propulsion of a charcoal meal in mice. In isolated guinea-pig ileum, this extract produced a concentration dependent inhibition of the contractions induced by 5-hydroxytryptamine, but it was unable to inhibit the contractions elicited by acetylcholine, histamine, KCl and BaCl(2). This extract produced also a concentration dependent inhibition of the spontaneous pendular movement of the isolated rabbit jejunum. This inhibitory activity was partially blocked by propranolol. The essential oil, obtained from the aerial part of this plant, was more potent than the MeOH-CHCl(3) (1:1) extract in inhibiting the spontaneous pendular movement of the rabbit jejunum. Thymol, camphor and gamma-terpinene were identified from the essential oil by GC-MS. These monoterpenes showed antispasmodic activity in the rabbit jejunum preparation, thymol was the most active compound, followed by camphor and gamma-terpinene. Thymol and camphor in high concentrations also showed tracheal relaxant properties, but gamma-terpinene did not. These in vivo and in vitro results tend to support the traditional use of A. phleoides as an antispasmodic agent.

Animals↗
PubMed1981

Magnetic circular dichroism studies of cytochrome P-450cam. Characterization of axial ligands of ferric and ferrous low-spin complexes.

MCD was applied to ferric and ferrous low-spin complexes of cytochrome P-450 cam to elucidate the electronic states and the nature of the axial ligands of the heme in cytochrome P-450cam. (1) Low-spin complexes of ferric cytochrome P-450cam, produced either by ligation of external ligands such as pyridine and imidazole derivatives or by being freed of (-)-camphor, showed sinusoidal Soret and alpha-MCD bands. The magnitude ratio of the Soret vs. alpha-MCD bands was quite sensitive to the nature of axial ligands of the ferric low-spin complexes. The ratio (2.7) for the camphor-free form of cytochrome P-450cam, thus, was the smallest among those (2.7-9.0) for low-spin forms of cytochrome P-450cam and other corresponding low-spin hemoproteins (ratio 7.8-13.9). The ratio (4.2) for the alpha-picoline-bound form of cytochrome P-450cam, however, was the closest to that (2.7) for the camphor-free form of cytochrome P-450cam among those (4.2-9.0) for the external ligand-bound form of cytochrome P-450cam. The ratio for the 2-methylimidazole-bound form of cytochrome P-450cam was the smallest among those of cytochrome P-450cam bound with imidizole derivatives. Thus, among the nitrogen-bound low-spin forms, the low-spin form with a sterically hindered nitrogen ligand trans to the thiolate anion (-S-) most reproduced spectral characteristics of the native low-spin ferric form. Low-temperature absorption studies offered the same results. (2) It was found that MCD magnitudes of alpha-bands of ferrous low-spin complexes are intimately related to the electronic character of axial ligands. Thus, the CO, O2 and NO-bound forms of cytochrome P-450cam, which have two pi-type axial ligands, showed the smallest alpha-MCD bands ([theta]M = 5.2-7.5) among complexes, while ferrous cytochrome b5 and cytochrome c, which have two sigma-electron-donating axial ligands, showed the largest magnitude ([theta]M = 120-176). The data for the ferrous low-spin complexes of other hemoproteins so far available were well rationalized in consideration of the property of the axial ligands.

Animals↗
PubMed2003

Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandulaefolia essential oil.

In vitro anticholinesterase activities of eight commercially available terpenoid constituents of Salvia lavandulaefolia have been investigated. These included 1,8-cineole, camphor, alpha-pinene, beta-pinene, borneol, caryophyllene oxide, linalool and bornyl acetate. Dose-dependent inhibition of acetylcholinesterase (AChE) by these chemical constituents was determined using the method of Ellman [Biochem. Pharmacol. 7 (1961) 88]. The IC50 value of 1,8-cineole was 0.06+/-0.01 mg/ml similar to that of the essential oil (0.05+/-0.01 mg/ml). Analyses of the expected inhibitions based on the prediction of a zero interactive response of a combination at its naturally occurring ratios were carried out in comparison with observed inhibition. Minor synergy was apparent in 1,8-cineole/alpha-pinene and 1,8-cineole/caryophyllene oxide combinations, with interaction indexes not exceeding 0.5. In contrast, a combination of camphor and 1,8-cineole was antagonistic with an interaction index of 2. A combination of all eight compounds was zero interactive. A combination of six constituents, excluding 1,8-cineole and camphor, was used to compare the method of expected response of a combination with a method of summation. These findings reveal that the inhibitory activity of the oil results from a complex interaction between its constituents, which produce both synergistic and antagonistic responses between the component terpenes. Understanding such interactions is important in comparing species on the basis of chemical composition.

Cholinesterase Inhibitors↗
PubMed2005

Vapor phase toxicity of marjoram oil compounds and their related monoterpenoids to Blattella germanica (Orthoptera: Blattellidae).

The toxicity of marjoram, Origanum majorana L., oil, 41 monoterpenoids, and 2 sesquiterpenoids against adult females of the German cockroach, Blattella germanica L., was examined using direct contact and vapor phase toxicity bioassays and compared with those of deltamethrin, dichlorvos, permethrin, and propoxur, four commonly used insecticides. In a filter-paper contact toxicity bioassay, the adulticidal activities of pulegone (0.06 mg/cm2), (+/-)-camphor (0.07 mg/cm2), and verbenone (0.07 mg/cm2) were comparable to that of permethrin (0.05 mg/cm2) but more pronounced than that of propoxur (0.18 mg/cm2), as judged by the 24-h LC50 values. These compounds were less effective than either deltamethrin (0.013 mg/cm2) or dichlorvos (0.007 mg/cm2). The toxicity of marjoram oil, thymol, alpha-terpineol, (-)-alpha-thujone, linalool, 1,8-cineole, (-)-camphor, and (+)-carvone, ranging from 0.08 to 0.18 mg/cm2, was higher than that of propoxur. In vapor phase toxicity tests, verbenone (11.48 mg/L air) was the most toxic compound followed by (-)-alpha-thujone (18.43 mg/L of air), thymol (18.76 mg/L of air), alpha-terpineol (21.89 mg/L of air), (+/-)-camphor (24.59 mg/L of air), linalool (26.20 mg/L of air), and marjoram oil (38.28 mg/L of air) on the basis of the 24-h LC50 values. Dichlorvos (0.07 mg/L of air) was the most potent fumigant. Structure-activity relationships indicate that structural characteristics, such as degrees of saturation and types of functional groups rather than types of carbon skeleton, and hydrophobicity and vapor pressure parameters appear to play a role in determining the monoterpenoid toxicities to adult B. germanica. Marjoram oil and the monoterpenoids described merit further study as potential fumigants or leads for the control of B. germanica.

Animals↗
PubMed1990

Metabolism of Monoterpenes in Cell Cultures of Common Sage (Salvia officinalis) : Biochemical Rationale for the Lack of Monoterpene Accumulation.

Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from [U-(14)C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism.

Journal Article↗
PubMed2005

Functionalization of the cytochrome P450cam monooxygenase system in the cell-like aqueous compartments of water-in-oil emulsions.

The functionalization of the cytochrome P450cam monooxygenase system, which requires electron transfer among three different proteins, was investigated in the micro-scale aqueous compartments of stable water-in-oil (W/O) emulsions formed with the nonionic surfactant tetraethylene glycol dodecyl ether. Neither an organic-aqueous biphasic system nor a non-emulsified organic-aqueous solution containing the same amount of surfactant showed substantial hydroxylation of camphor, a natural substrate of P450cam, whereas substantial monooxygenation activity was detected when stable aqueous compartments were provided by the formation of W/O emulsions. Since the camphor hydroxylation in W/O emulsions was modest, we explored the integration of an enzymatic NADH regeneration system in order to effectively provide a reducing equivalent. Two different dehydrogenases, bacterial glycerol dehydrogenase (GLD) and yeast alcohol dehydrogenase (ADH), were selected, and each of these was coupled with the P450cam catalytic cycle in W/O emulsions. As a result, the camphor hydroxylation rate was successfully improved by approximately 5-fold when GLD was employed under optimized conditions. These results reveal the potential utility of the micro-scale cell-like aqueous compartments of W/O emulsions for multicomponent enzymatic reactions especially for substrates with low aqueous solubility.

Biomimetics↗
PubMed1981

Toxicity of some essential plant oils. Clinical and experimental study.

Commercial preparations of essences of sage, hyssop, thuja, and cedar have caused human intoxication in eight cases, from which tonico-clonic convulsions were the major symptom. The experimental study of the toxic properties of commercialized essential oils of sage and hyssop has revealed that their convulsant action was of central nervous system origin in unanesthetized rats, as proven by electrocortical records. The toxicity of the hyssop oil seems to be more powerful than that of sage, since the dose limit from which the cortical events are only subclinical is 0.08 g/kg for hyssop oil and 0.3 g/kg for sage oil. Above 0.13 g/kg for hyssop oil and 0.50 g/kg for sage oil, the convulsions appeared and became lethal above 1.25 g/kg with hyssop oil and 3.2 g/kg with sage oil. The daily repeated injection of subclinical doses revealed the cumulative toxic effect of hyssop oil, since the same low dose induced electrocortical clonic seizures. The toxicity of each oil appeared to be related to the presence of terpenic ketones, camphor in sage commercial oil, camphor and thujone in sage Dalmatian oil, thujone in thuja and cedar oils, and pinocamphone in hyssop oil. The convulsant properties of camphor are well known. The neurotoxicity of thujone and pinocamphone is demonstrated in rats for the first time.

Adolescent↗
PubMed2002

Assessment of olfactory status in allergic and non-allergic rhinitis patients.

A study undertaken to assess the olfactory acuity in allergic (group I) and non-allergic rhinitis (group II) patients in comparison with age and sex matched controls (group III). Patients presenting with atleast three of the five cardinal symptoms of rhinitis i.e. rhinorrhoea, sneezing, itching, headache and nasal obstruction were grouped as non-infective rhinitis and further divided into allergic rhinitis (group I, n = 20) and non-allergic rhinitis (group II, n = 20) based on nasal smear cytology positivity or negativity for eosinophils respectively. Detailed Ear, Nose and Throat examination was carried out in both the groups and peripheral blood samples were analysed for total, differential leukocyte and absolute eosinophil counts using standard techniques. In all the three groups the olfactory thresholds for 5 odorants i.e. musk (M), formalin (F), camphor (C), asafoetida (A, 10% aqueous solution) and oil of peppermint (P, 20%) were evaluated for testing musky, pungent, camphorous, putrid and minty odours respectively by the method described by Elsberg and Levy for quantitative olfactometry. The results indicated elevation of olfactory thresholds (delta %, calculated taking control values as 100%) for 4 or 5 odorants in group I and group II patients respectively as compared with controls (group I: delta % for P--89.6%; M--116.4%; A--55.8%; P < 0.001; C--73.1%; P < 0.02; F--26.6% N.S.; group II: P--96.9%; M--99.3%, P < 0.01 for both; A--66.8%; C--102.7%, P < 0.001; F--42%, P < 0.05). In the non allergic rhinitis group the magnitude of the olfactory loss was more severe except for the odorant musk. Further interpretations as per gender based specificities revealed more severe olfactory loss in males of both the groups for the odorants peppermint and musk and moderately severe olfactory loss for formalin and camphor as compared to females. However, for the odorant asafoetida females showed greater olfactory loss than males in both the groups (delta % 73.38% versus 52% in group I and 81.29% versus 69.7% in group II).

Adult↗
PubMed2003

Analysis of the essential oils of the leaves, stems, rhizomes and roots of the medicinal plant Alpinia galanga from southern India.

The essential oils of the leaves, stems, rhizomes and roots of the medicinal plant Alpinia galanga from southern India were investigated by GC-FID, GC-MS and olfactometry. In all four samples, mono- and sesquiterpenes as well as (E)-methyl cinnamate could be identified. They are responsible for the characteristic odor as well as for the reported use in (folk) medicine and in food products of A. galanga. The essential oil of A. galanga leaves is rich in 1,8-cineole (28.3%), camphor (15.6%), beta-pinene (5.0%), (E)-methyl cinnamate (4.6%), bornyl acetate (4.3%) and guaiol (3.5%). The stem essential oil contains 1,8-cineole (31.1%), camphor (11.0%), (E)-methyl cinnamate (7.4%), guaiol (4.9%), bornyl acetate (3.6%), beta-pinene (3.3%) and alpha-terpineol (3.3%). 1,8-cineole (28.4%), alpha-fenchyl acetate (18.4%), camphor (7.7%), (E)-methyl cinnamate (4.2%) and guaiol (3.3%) are the main constituents of the rhizome essential oil. The root essential oil contains alpha-fenchyl acetate (40.9%), 1,8-cineole (9.4%), borneol (6.3%), bornyl acetate (5.4%) and elemol (3.1%). In addition, biological and aroma effects of the main and minor compounds of the four essential oils of Alpinia galanga are discussed in terms of their possible use in medicine, cosmetics and foods.

Cosmetics↗
PubMed2000

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms.

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

Bacterial Proteins↗
PubMed2006

Increasing the catalytic performance of a whole cell biocatalyst harboring a cytochrome p450cam system by stabilization of an electron transfer component.

Catalytic activity of a recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system from Pseudomonas putida coupled with enzymatic co-factor regeneration was investigated. About 0.7 micromol camphor was hydroxylated per mg dry cells at 4 degrees C in 50 mM Tris/HCl buffer (pH 7.4) when utilizing a stable putidaredoxin (Pdx) mutant, C73S/C85S-Pdx (Cys73Ser, Cys85Ser double mutant), instead of wild-type Pdx, which was about two-fold improvement in the substrate conversion. Ten-micromole camphor was completely hydroxylated at 20 degrees C in 6 h by 15 mg dry cell weight of whole cell biocatalyst including C73S/C85S-Pdx. Thus, modulation of protein-protein interaction in multicomponent enzymatic catalysis in whole cells is important.

Camphor↗
PubMed1990

Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P450cam) by the chemopreventive agent 3-t-butyl-4-hydroxyanisole.

The efficacy of 2(3)-t-butyl-4-hydroxyanisole (BHA) and other chemicals as chemopreventive agents against chemically induced cancer or toxicity may involve direct modulation of cytochrome P450 activity. Direct interaction of BHA with cytochrome P450 was investigated using substrate-bound, oxyferrous cytochrome P450CIA1 either in a reconstituted system containing cytochrome P450CIA1, putidaredoxin, and putidaredoxin reductase with NADH as electron donor or in the absence of physiological electron donors. In the reconstituted system, BHA caused a concentration-dependent decrease in the production of 5-exo-hydroxycamphor and a substoichiometric increase in hydrogen peroxide production. However, BHA did not appreciably inhibit either NADH oxidation or oxygen utilization under conditions optimal for accumulation of oxyferrous cytochrome P450CIA1 during steady-state metabolism of camphor. In the absence of electron donor, BHA enhanced decomposition of the ternary oxyferrous substrate complex of cytochrome P450CIA1 without the formation of any apparent spectral intermediate(s). The rate of decomposition of the oxyferrous complex was pseudo-first order and was dependent upon the concentration of BHA present. Enhanced decomposition of the complex was not attributable to catalytic turnover of cytochrome P450CIA1 (i.e., acquisition of a second electron from an indeterminate source) since no appreciable metabolism of either camphor or BHA was observed. The enhanced decomposition was accompanied by a substoichiometric increase in hydrogen peroxide production, suggesting that BHA may facilitate four-electron reduction of molecular oxygen to water. These results indicate that BHA inhibits cytochrome P450 function, presumably by enhancing autoxidation of the substrate-bound oxyferrous complex.

Animals↗
PubMed1995

Substrate interactions in cytochrome P-450: correlation between carbon-13 nuclear magnetic resonance chemical shifts and C-O vibrational frequencies.

13CO NMR chemical shifts and 12CO infrared stretching frequencies have been measured for cytochrome P-450cam-CO in the presence of D-camphor and of various camphor analogues. A linear correlation between both parameters delta (13C) and v(CO) was found indicating that the steric and electrostatic interactions acting on the CO ligand are influenced by the substrate. It has been proved that P-450 complexes are on another line in this correlation than hemoglobins which is explained by the different proximal ligand.

Camphor↗