Annelid cytochrome P-450

Both classes of Annelida--Polychaeta and Clitellata--have been shown to contain cytochrome P-450. The metabolism of a number of aromatic hydrocarbons, drugs and pesticides by annelids required oxygen and NADPH, and was inhibited by a variety of cytochrome P-450 inhibitors. A number of types I and II substrates bound to the cytochrome P-450 in polychaete microsomes to give typical types I and II binding spectra. These results suggest that xenobiotics in annelids are metabolized by a typical cytochrome P-450 mixed function oxygenase. In addition to xenobiotics, annelid cytochrome P-450 systems are likely to function in the biosynthesis and metabolism of sterols and hormones found in annelids, such as cholesterol, ecdysteroids and eicosanoids. The primary source of cytochrome P-450 isolated to date from annelids has been intestinal microsomes. Cytochrome P-450 concentrations in these microsomes varied from 8 to 580 pmol mg-1 of protein. The only cytochrome P-450s purified from annelids were the three isomers isolated from microsomes of the oligochaete, Lumbricus terrestris, whose molecular masses were 48,000, 51,000 and 53,000 Da. Work on the induction of cytochrome P-450 in polychaetes by exposure to polycyclic aromatic hydrocarbons or polychlorinated biphenyls has given conflicting results, since some groups found induction after such exposure, but others found no induction. One possible explanation may be exposure to natural soil and sediments inducers, e.g. plant alkaloids, during feeding. Since gene and protein sequences have yet to be carried out on the cytochrome P-450 of any annelid, the relationship of annelid cytochrome P-450s to the 74 families of P-450 so far found, remains to be carried out.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O-deethylation), CYP2C9 (tolbutamide 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation) and CYP3A4 (testosterone 6beta-hydroxylation) activities with IC50 = 40, 49, 213 and 32 microM respectively. Ki for propofol against all of these enzymes with the exception of CYP2D6, where propofol showed little inhibitory activity, was 30, 30 and 19 microM respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC50 = 0.8, 0.5, 0.2 and 0.1 microM; furafylline and sulphaphenazole yielded Ki = 0.6 and 0.7 microM respectively. 3. The therapeutic blood concentration of propofol (20 microM; 3-4 microg/ml) together with the in vitro Ki estimates for each of the major human P450 enzymes have been used to estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro-in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in > 190 million people since its launch in 1986, there are only single reports of possible drug interactions between propofol and either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.     

Influence of tryptophan on the level of hepatic microsomal cytochrome P-450 in well-fed normal, adrenalectomized, and phenobarbital-treated rats

In well-fed normal male rats, either force-feeding of tryptophan or a single injection of phenobarbital produced significant increments in hepatic microsomal cytochrome P-450 and the associated aniline hydroxylase activity. Administration of both tryptophan and phenobarbital together resulted in even greater stimulation than when the compounds were given alone. Adrenalectomy lowered instead the cytochrome P-450 concentration in comparison with that of normal rats, and administration of tryptophan and phenobarbital in this condition produced no significant increment in cytochrome P-450 concentration. In addition, phenobarbital administered either singly or in combination with tryptophan resulted in 80% mortality, which was reduced to zero by pretreatment with cortisol. While in cortisol-treated adrenalectomized rats administration of phenobarbital caused a 56% increment in cytochrome P-450 as compared to controls, tryptophan produced only a minor (9%) increase. In normal, as well as in adrenalectomized rats, tryptophan and phenobarbital administered either idividually or together increased microsomal protein concentration. In normal rats actinomycin-D treatment reduced both cytochrome P-450 and microsomal protein concentrations below that of the non-treated control levels. Further administration of either tryptophan or phenobarbital slightly increased the level of cytochrome P-450, and the two compounds together caused 40 and 21% increments of the same compared to actinomycin-treated and non-treated controls, respectively.     

Modulatory effect of hyperthermia on hepatic microsomal cytochrome P450 in mice

It is widely known that the clearance of drugs is often compromised during episodes of infectious disease via a down-regulation of cytochrome P450 (P450) at a pre-translational step in enzyme synthesis. Etiocholanolone (ETC), a potent inflammatory agent, induces fever in humans and causes a decrease in the clearance of certain drugs that are metabolized by P450. On this basis it is widely believed that the fever per se rather than the immune modulation that occurs during infections may have a major role in depression of microsomal P450 enzymes during viral infections in humans. In the present study, we demonstrated that although ETC did not induce hyperthermia in mice, it still evoked a depression of the levels of P450 in hepatic microsomes. Ethoxyresorufin O-deethylase (EROD) was also inhibited significantly when hepatic microsomes were incubated with various concentrations of ETC in vitro. P450 levels and EROD activities remained unchanged following hyperthermia that was induced by a non-inflammatory procedure using 2,4-dinitrophenol. Provided the response in rodents is similar to humans, these results indicate that the depression of drug biotransformation by ETC in humans is more likely to be caused by the direct effects of this agent or other mechanisms rather than by the fever it produces. This may suggest that the loss of drug metabolism in humans during infections is due to the activation of host defence responses rather than to the febrile nature of the illness.     

Amount and type of dietary lipid modulate rat hepatic cytochrome P-450 activity

Cardiovascular responses to sustained and rhythmic (5 s on, 2 s off) forearm isometric exercise to fatigue at 40% maximal voluntary contraction (MVC) and to a period of arterial occlusion were investigated in elite rock climbers (CLIMB) as a trained population compared to non-climbing sedentary subjects (SED). Blood pressure (BP), monitored continuously by Finapres, and forearm blood flow, by venous occlusion plethysmography, were measured and used to calculate vascular conductance. During sustained exercise, times to fatigue were not different between CLIMB and SED. However, peak increases in systolic (S) BP were significantly lower in CLIMB [25 (13) mmHg; (3.3 (1.7) kPa] than in SED [48 (17) mmHg; (6.4 (2.3) kPa] (P < 0.05), with a similar trend for increases in diastolic (D) BP. Immediately after sustained exercise, forearm conductance was higher in CLIMB than SED (P < 0.05) for up to 2 min. During rhythmic exercise, times to fatigue were two fold longer in CLIMB than SED [853 (76) vs 420 (69) s, P < 0.05]. Increases in SBP were not different between groups except during the last quarter of exercise when they fell in CLIMB. Conductance both during and after rhythmic exercise was higher in CLIMB than in SED. Following a 10-min arterial occlusion, peak vascular conductance was significantly greater in CLIMB than SED [0.597 (0.084) vs 0.431 (0.035) ml x min(-1) x 100 ml(-1) x mmHg(-1); P < 0.05]. The attenuated BP response to sustained isometric exercise could be due in part to enhanced forearm vasodilatory capacity, which also supports greater endurance during rhythmic exercise by permitting greater functional hyperaemia in between contraction phases. Such adaptations would all facilitate the ability of rock climbers to perform their task of making repetitive sustained contractions.     

Effects of cold preservation and reperfusion on microsomal cytochrome P-450-linked monooxygenase system of the rat liver

The routine application of capillary electrochromatography (CEC) is demonstrated by incorporating 75 microns I.D. capillaries packed with 3 microns octadecylsilica (ODS) particles into a commercial CZE instrument. A mixture of several neutral compounds is separated into its components with an average efficiency up to 181 000 plates/m in less than 8 min. Hundreds of consecutive runs are performed over a period of weeks from which it is concluded that the reproducibility of the capacity factors is better than 2% and that CEC separations can be achieved in a reliable and routine manner.     

An infrared study of the carbon monoxide complexes of cytochrome P-450 and cytochrome P-420

The infrared stretch vibrations (upsilonCO) of the CO-complexes of cytochrome P-450 and cytochrome P-420 have been determined from infrared difference spectra. The CO-complexes exhibit IR-bands at 1949 cm-1 and 1966 cm-1 with half widths of approximately 17 cm-1 and approximately 20 cm-1 respectively. These results are compared with the CO-stretch frequencies of other haemoproteins and discussed with respect to specific interactions of the CO-ligand with the protein moiety and to the ligand trans to CO of the cytochromes.     

On the formation of cytochrome P-450 product complexes during the metabolism of phenylalkylamines

Differences have been found, which are usually 1% or less, between the left ventricular ejection time measured from the external carotid pulse tracing, and from the rate of change of thoracic impedance (dZ/dt) waveform, using either the second heart sound or the X-point of the dZ/dt tracing as the end-point. The Heather Index obtained from the ECG and dZ/dt tracings has been correlated with other indices of cardiac performance. The changes observed in the physiological variables during head-up and head-down tilting were in the expected directions.     

Involvement of testosterone in the induction of hepatic microsomal cytochrome P-450 2B1/2 (P-450 2B1/2) by 1-benzylimidazole in male and female rats: sex-differentiated induction of P-450 2B1/2 species

Collagen-induced arthritis (CIA) is an animal model for the human autoimmune disease rheumatoid arthritis (RA). CIA can be induced in several species including primates by immunization with heterologous type-II collagen (CII). Polyclonal antibodies are formed upon immunization with CII that exhibit a broad range of epitope specificities (some that cross-react with hose CII); however, only antibodies directed against certain specific epitopes on CII are arthritogenic. Recently, the importance of cognate interactions between T-cells and B-cells to the induction of CIA was demonstrated by administration of monoclonal antibodies against a T-cell surface protein, gp39. Blocking the interaction of T-cell gp39, with its receptor/ligand on the surface of B-cells (CD40), completely blocked induction of CIA in mice. A concomitant reduction in the level of anti-CII IgG produced in anti-gp39-treated animals was observed, demonstrating the crucial importance of T-cell:B-cell interactions via gp39:CD-40 binding to the primary immune response to CII in vivo and therefore to the induction of CIA. Other features of CIA are important in elucidating the condition and this article will deal with some important issues.     

Inhibitor-induced conformational change in cytochrome P-450CAM

The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].     

Alkylation of the prosthetic heme in cytochrome P-450 during oxidative metabolism of the sedative-hypnotic ethchlorvynol

The clinically used sedative-hypnotic ethchlorvynol destroys hepatic microsomal cytochrome P-450 enzymes in a process catalyzed by the same hemoproteins. Abnormal porphyrins accumulate in the livers of phenobarbital-pretreated rats after administration of ethchlorvynol. The abnormal porphyrin fraction has been isolated and shown to consist of the four possible regioisomers of N-(5-chloro-3-ethyl-3-hydroxy-2-oxo-4-pentenyl)protoporphyrin IX. Cytochrome P-450 inactivation thus appears to result from alkylation of the prosthetic heme by the oxidatively activated acetylenic function in ethchlorvynol. The autocatalytic destruction of the hemoprotein is likely to alter the metabolism and elimination of ethchlorvynol and coadministered drugs and may be the cause of the porphyrinogenic properties of ethchlorvynol.     

Ligand interaction of sustituted pyridines with cytochrome P-450

A series of pyridyl ketones and alkyl pyridines was evaluated as type II ligands for cytochrome P-450. Activity as type II ligands was evaluated in terms of the lipid solubility and the pKa values of the compounds.     

Inducers of the cytochrome P-450 superfamily as promoters of carcinogenesis

This review summarizes data on the mechanisms of tumor-promoting activity of non-genotoxic compounds that are inducers of cytochrome P-450 isoforms. Their promoting activity is analyzed in term of synthesis of new cytochrome P-450 isoforms. Active oxygen species formed by cytochrome P-450 isoforms can simultaneously act as stimulators of proliferation and inhibitors of intercellular communications. Promoter effects can be associated with changes in the ratio of cellular signaling non-protein molecules induced by newly synthesized cytochrome P-450 isoforms. Data on induction of cytochrome P-450 isoforms of family 1 indicate that inducer interaction with its receptor causes cellular events resulting in the stimulation of cell proliferation.     

Update: clinically significant cytochrome P-450 drug interactions

Recent technologies have resulted in an explosion of information concerning the cytochrome P-450 isoenzymes and increased awareness of life-threatening interactions with such commonly prescribed drugs as cisapride and some antihistamines. Knowledge of the substrates, inhibitors, and inducers of these enzymes assists in predicting clinically significant drug interactions. In addition to inhibition and induction, microsomal drug metabolism is affected by genetic polymorphisms, age, nutrition, hepatic disease, and endogenous chemicals. Of the more than 30 human isoenzymes identified to date, the major ones responsible for drug metabolism include CYP3A4, CYP2D6, CYP1A2, and the CYP2C subfamily.     

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon beta in mice

AIMS/METHODS: Interferon beta is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon beta on cytochrome P-450 in mice. RESULTS: Interferon beta (2 x 10(5) units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon beta reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In beta-naphthoflavone-treated mice, interferon beta lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). CONCLUSIONS: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon beta, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon beta.