Evaluation and optimization of the protocols for measuring cytochrome P450 activity in aphids

AbstractCytochrome P450 enzymes play major roles in insect detoxification of plant toxins and insecticides. However, measuring P450 activity in aphids has variable success, and a reliable method is not available yet. In this study, we evaluated and optimized the method for measuring P450 activity in aphids using the 7-ethoxycoumarin as the substrate. First, we found that nicotinamide adenine dinucleotide phosphate and protective agents are not needed in the aphid P450 activity assay, and homogenizing the green peach aphid,Myzus persicae, in the microplate resulted in significantly higher P450 activities than those in Eppendorf tube. Homogenizing aphids in Eppendorf tube could grind tissues thoroughly and released uncharacterized compounds that could inhibit aphid and pig liver P450 activities, whereas aphids in the microplate likely could not be thoroughly ground and thus released fewer such inhibitors. Then, the microplate homogenization method was optimized to follows: one or two aphids were put into one well of the microplate and ground in phosphate buffer using pipette tips for 20 cycles, followed by addition of 7-ethoxycoumarin, and then incubated for 1 h at room temperature, after which glycine buffer-ethanol mixture was added to stop the reaction. This method is also suitable for the pea aphid,Acyrthosiphon pisum, and the bird cherry □oat aphid,Rhopalosiphum padi.These results emphasize the importance of considering inhibitory effect of endogenous compounds in insects on their P450 activity and provide one possible method to reduce this inhibitory effect.

In vitro metabolism of helenalin and its inhibitory effect on human cytochrome P450 activity

Archives of Toxicology ◽

10.1007/s00204-021-03218-6 ◽

2022 ◽

Author(s):

Michaela Šadibolová ◽

Risto O. Juvonen ◽

Seppo Auriola ◽

Iva Boušová

Keyword(s):

Cytochrome P450 ◽

Inhibitory Effect ◽

In Vitro Metabolism ◽

Human Cytochrome ◽

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

Synthetic Biology ◽

10.1093/synbio/ysy009 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 7

Author(s):

Adokiye Berepiki ◽

John R Gittins ◽

C Mark Moore ◽

Thomas S Bibby

Keyword(s):

Cytochrome P450 ◽

Electron Flow ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Experimental System ◽

Type I ◽

Photosynthetic Electron Flow ◽

The Impact ◽

AbstractIn this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular adenosine triphosphate (ATP) and the ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis.

Cytochrome P450 enzymes inhibitor in the control of Digitaria insularis

Ciência e Agrotecnologia ◽

10.1590/1413-7054202145024520 ◽

2021 ◽

Vol 45 ◽

Author(s):

Gledson Soares de Carvalho ◽

Jessica Ferreira Lourenço Leal ◽

Amanda dos Santos Souza ◽

Francisco Freire de Oliveira Junior ◽

Ana Claudia Langaro ◽

...

Keyword(s):

Cytochrome P450 ◽

Block Design ◽

Antagonistic Effect ◽

Cytochrome P450 Enzymes ◽

Randomized Block Design ◽

Untreated Check ◽

Herbicide Mixtures ◽

ABSTRACT Herbicide interactions can be synergic, additive, or antagonist when mixed in the spray tank. A good example is an association between 2,4-D and graminicides. One hypothesis is that 2,4-D contributes to increasing the Cytochrome P450 activity, which may be one of the causes of antagonism. This study aimed to investigate the use of CYP450 enzymes inhibitor associated with the herbicide mixtures 2,4-D and ACCase inhibitors in vivo on the control of Digitaria insularis. The experiment was performed using a randomized block design in a factorial scheme of 6x2 with four replications. Factor A consisted of untreated check, 2,4-D (1005 g a.e ha-1), clethodim (192 g a.i ha-1) and haloxyfop (62.4 g a.i ha-1), 2,4-D + clethodim (1005 g a.e ha-1+192 g a.i ha-1), and 2,4-D + haloxyfop (1005 g a.e ha-1 +62.4 g a.i ha-1). Factor B represented the presence or absence of malathion (1000 g ha-1) applied two hours before applying the herbicides. A physicochemical test was performed to verify the compatibility of the herbicides in the tank. Malathion application performed two hours before applying the herbicide mixtures (2,4-D and clethodim/haloxyfop) did not provide adequate control of sourgrass, suggesting that CYP450 enzymes inhibited by malathion are not involved in the antagonistic effect between 2,4-D and both graminicides in the management of sourgrass. The 2,4-D + haloxyfop in tank mix demonstrated less efficacy in controlling sourgrass than 2,4-D + clethodim, but both mixtures were incompatible in the tank mix, which may be associated with reduced efficacy in sourgrass management.

Faculty Opinions recommendation of Perpetrators of pharmacokinetic drug-drug interactions arising from altered cytochrome P450 activity: a criteria-based assessment.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.10396957.11219055 ◽

2011 ◽

Author(s):

Geoffrey Tucker

Keyword(s):

Cytochrome P450 ◽

Drug Interactions ◽

P450 Activity ◽

Pharmacokinetic Drug

P.615 Effect of cocaine self-administration on hepatic cytochrome P450 activity in the rat model of drug addiction

European Neuropsychopharmacology ◽

10.1016/j.euroneuro.2020.09.453 ◽

2020 ◽

Vol 40 ◽

pp. S350-S351

Author(s):

J. Jastrzębska ◽

R. Pukło ◽

M. Frankowska ◽

M. Filip ◽

W.A. Daniel

Keyword(s):

Cytochrome P450 ◽

Drug Addiction ◽

Rat Model ◽

Self Administration ◽

P450 Activity ◽

Hepatic Cytochrome

In vitro study on the effect of cornin on the activity of cytochrome P450 enzymes

BMC Complementary Medicine and Therapies ◽

10.1186/s12906-021-03309-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Qun Zhang ◽

Zengqiang Qu ◽

Yanqing Zhou ◽

Jin Zhou ◽

Junwei Yang ◽

...

Keyword(s):

Cytochrome P450 ◽

Drug Interaction ◽

Liver Microsomes ◽

In Vitro Study ◽

Inhibitory Effect ◽

Dependent Manner ◽

Cytochrome P450 Enzymes ◽

Drug Drug Interaction ◽

Dose Dependent

Abstract Background Cornin is a commonly used herb in cardiology for its cardioprotective effect. The effect of herbs on the activity of cytochrome P450 enzymes (CYP450s) can induce adverse drug-drug interaction even treatment failure. Therefore, it is necessary to investigate the effect of cornin on the activity of CYP450s, which can provide more guidance for the clinical application of cornin. Methods Cornin (100 μM) was incubated with eight isoforms of CYP450s, including CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1, in pooled human liver microsomes. The inhibition model and corresponding parameters were also investigated. Results Cornin exerted significant inhibitory effect on the activity of CYP3A4, 2C9, and 2E1 in a dose-dependent manner with the IC50 values of 9.20, 22.91, and 14.28 μM, respectively (p < 0.05). Cornin inhibited the activity of CYP3A4 non-competitively with the Ki value of 4.69 μM, while the inhibition of CYP2C9 and 2E1 by cornin was competitive with the Ki value of 11.31 and 6.54 μM, respectively. Additionally, the inhibition of CYP3A4 by cornin was found to be time-dependent with the KI/Kinact value of 6.40/0.055 min− 1·μM− 1. Conclusions The inhibitory effect of cornin on the activity of CYP3A4, 2C9, and 2E1 indicated the potential drug-drug interaction between cornin and drugs metabolized by these CYP450s, which needs further investigation and validation.