The effect of inducing agents of cytochrome P-450 on the binding and metabolism of three local anaesthetic agents: lidocaine, mepivacaine and bupivacaine has been investigated. All three local anaesthetic agents bound to the type I binding site of cytochrome P-450, which is characteristic of substrate binding to cytochrome P-450, and stimulated the CO-inhibitable oxidation of NADPH. Lidocaine is shown to be metabolized by cytochrome P-450 to the products MEGX and acetaldehyde. The forms of cytochrome P-450 elevated with phenobarbital and/or pregnenolone-16α-carbonitrile were shown to play an important role in the binding of lidocaine to cytochrome P-450. Cytochrome P-448 did not appear to be involved in the binding of lidocaine to cytochrome P-450. These findings are supported by the ability of the inhibitors of cytochrome P-450 viz. metyrapone, SKF 525-A and CO:O₂ to inhibit binding of lidocaine to cytochrome P-450. No single form of cytochrome P-450 appears to preferentially metabolize lidocaine, but rather multiple forms of the enzyme appear to be involved in the metabolism of lidocaine. The phenobarbital inducible form of cytochrome P-450 appears to play a major role in the binding of mepivacaine to cytochrome P-450. Cytochrome P-450 in microsomes from rats pretreated with β-naphthoflavone and pregnenolone-16α-carbonitrile does not appear to have a significant role in the binding of mepivacaine to cytochrome P-450. All forms of cytochrome P-450 are involved in the metabolism of mepivacaine· to metabolic products as assessed by the oxidation of NADPH. However, the form of cytochrome P-450 induced by pretreatment of rats with phenobarbital may play a predominant role in the total metabolism of mepivacaine. Multiple forms of cytochrome P-450 appear to be involved in the binding and total metabolism of bupivacaine. As in the case of mepivacaine, the total metabolism of bupivacaine, as assessed by the oxidation of NADPH, may be predominantly catalyzed by the form of cytochrome P-450 found in microsomes from rats pretreated with phenobarbital. Partially purified cytochrome P-450 was found to bind lidocaine in a type I manner and, in the presence of the artificial electron donor H₂O₂, produce MEGX. This further supports the role of cytochrome P-450 in the in vitro metabolism of lidocaine. Hepatocytes were found to metabolize lidocaine to MEGX, indicating that lidocaine metabolism in vivo might well be mediated by cytochrome P-450.

A soluble form of cytochrome b_5, with a 413/280 nm ratio of nearly unity, has been isolated and purified from porcine kidney using fractional precipitation, ion exchange resins, and gel filtration procedures. The cytochrome b_5 shows heat stability up to 50° and is stable in the pH range 6.0 to 8.5. The oxidized protein exhibits an absorption maximum at 413 nm, while the reduced form shows three peaks: 423, 526, 556 nm. Molecular weight determinations have given variable results, suggesting the possibility of a polymer composed of four and eight units of a 13,500 molecular weight protomer. This study has used no lipases, proteases, or detergents in the solubilization of the cytochrome, and the possibility exists that the cytochrome found represents newly formed cytochrome b_5 which is as yet unattached to the microsomes, and which reflects the form found in intact cells.

Juvenile rainbow trout (Oncorhynchus mykiss) were fed either 5 or 20 μg 2,2′,4,4′,5,5′‐hexachlorobiphenyl (245‐HxCB)/g diet (wet wt.) for 4, 8, or 12 weeks. Hepatic xenobiotic‐metabolizing enzyme activities and dietary 245‐HxCB accumulation in liver, muscle, and remaining carcass were determined. Liver‐to‐body weight ratios were not altered by either of the two 245‐HxCB concentrations. Relative growth rate increased with time but was not altered by 245‐HxCB concentration. Bioaccumulation of 245‐HxCB was dose and time dependent in all tissues without reaching apparent steady state. Hepatic arylhydrocarbon hydroxylase (AHH) activities increased with 245‐HxCB dose and with time. Ethoxyresorufin‐O‐deethylase (EROD) activities also increased in fish fed 20 μg 245‐HxCB/g diet. No 245‐HxCB‐induced changes in uridine diphosphoglucuronosyl transferase (UDP‐GT) or NADPH‐cytochrome‐c reductase (NCCR) activities were determined. High‐resolution GC‐MS analysis of the 245‐HxCB standard revealed trace (0.4‐0.5%) contamination by two mono‐ortho pentachlorobiphenyls (PnCBs): 2,3,3′,4,4′‐PnCB and 2,3,4,4′,5‐PnCB. Total liver accumulation of these contaminants was inversely related with corresponding EROD and AHH activities and estimated to contribute minimally to their induction. Results from this study suggested that long‐term dietary 245‐HxCB exposures induced cytochrome P4501A activities in rainbow trout liver.

The objective of this study was to examine hepatic steroid inactivation and luteal function throughout bovine gestation. In pregnant beef cows, cytochrome P450 3A activity decreased from mid- to late-gestation, while progesterone concentrations tended to increase from mid- to late-gestation. Uridine diphosphate-glucuronosyltransferase activity per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Total corpus luteum (CL) blood perfusion tended to be increased in pregnant vs non-pregnant dairy cows. Hepatic portal blood flow per kg of body weight was increased in pregnant vs non-pregnant dairy cows. Hepatic steroid inactivating enzyme activity, CL blood perfusion, and portal blood flow did not differ between pregnant and non-pregnant beef cows. There was no difference in progesterone concentrations in pregnant vs non-pregnant dairy or beef cows. The current study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.

Interprotein electron transfer (ET) is crucial for natural energy conversion and a fundamental reaction in the pursuit of understanding the broader problem of proteinprotein interactions and reactivity. Simplifying the complicated nature of these natural systems has driven development of biomimetic approaches. Functionalized gold nanoparticles offer simplified, tunable surfaces that can serve as a proxy to study the reactivity and dynamics of proteins. Amino-acid functionalized gold nanoparticles (Au-TX) served as a complementary partner to cytochrome c (Cyt c) and catalyzed its ET reactivity without altering the native structure. Redox mediator and EPR experiments confirmed that the redox potential and coordination environment of the heme were unaltered. Varying the functionality of Au-TX under limiting redox reagent concentrations resulted in distinct ET reactivity. These conditions reflected the collision of a small redox reagent with the Cyt c/Au-TX adduct, introducing the possibility of Cyt c/Au-TX dynamics to modulate ET. Under high ionic strength conditions, the rate enhancement ranged from 0.0870 " 1011 for Cyt c/Au-TAsp to 1.95 " 1011 M-1 s-1 for Cyt c/Au-TPhe. Au-TAsp binds to a larger surface of the front face of Cyt c than Au-TPhe, likely reducing heme access and resulting in attenuated ET reactivity.Site-directed spin-labeling characterized the dynamic interactions and motion of Cyt c with Au-TX. Several mutants of Cyt c were utilized to extract information about the different dynamics of the Cyt c/Au-TPhe and Cyt c/Au-TAsp systems. Cyt c appeared to have a highly dynamic binding interaction with the surface of Au-TPhe while binding to Au-TAsp resulted in a more rigid interface, particularly at the heme crevice. The dynamic interaction of Cyt c/Au-TX at the heme crevice could promote a gated ET mechanism between Cyt c and its redox partner. Thus, the reduced reactivity of Cyt c/Au-TAsp is likely a result of both slower global dynamics and more rigid binding near the heme crevice.

Yes / The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation. / The authors would like to thank Yorkshire Cancer Research (Program grant B381PA) for supporting our work focused on exploring CYPs as targets for prodrug development. The human recombinant CYP1A1 was a gift from Prof Emily E. Scott, University of Michigan; the enzyme was produced via NIH funded grant (R37 GM076343).

TxtE is a protein related to cytochrome P450 enzymes, which catalyze a number of reactions that typically involve oxygen and not nitrogen. It has been discovered that TxtE can nitrate tryptophan through an unusual reaction in which it uses nitric oxide (NO) as a nitrogen donor to install the nitro group despite NO typically being considered toxic to bacteria. This project will determine if all cytochromes P450 can catalyze nitration as long as they are given NO. This will have an impact on understanding drug delivery and metabolism for which nitration is important.

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