Thin film voltammetry of metabolic enzymes in rat liver microsomes

Krishnan, Sadagopan; Rusling, James F.

doi:10.1016/j.elecom.2007.07.002

Cited by 21 publications

(30 citation statements)

References 15 publications

(28 reference statements)

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“…Voltammetric studies suggested that electrons enter the microsomal films via CYP reductases and are then transferred to CYPs to initiate the metabolic pathway [39, 40]. Here we evaluated the possibility that the reactive metabolites generated in this system can be trapped by DNA as adducts in the sensor films, and are sufficient to allow electrochemical detection.…”

Section: Resultsmentioning

confidence: 99%

Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

et al. 2009

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Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response.

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Section: Resultsmentioning

confidence: 99%

Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

et al. 2009

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“…CV peaks of CPR microsome, HLM or cyt P450 1B1 supersome films did not shift with CO in solution (Figure 1). This lack of midpoint potential shifts for CPR microsomes, HLM and cyt P450 1B1 films suggests that CV peaks originated not from cyt P450s but from CPR [8, 11, 13, 17]. CPR contains FAD and FMN cofactors, and CPR voltammetry has been interpreted previously [11, 13, 17].…”

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confidence: 81%

“…All these approaches substitute the electrode for NADPH as an electron donor. We have reported successful electrochemical activation of cyt P450 via CPR in films that combined CPR microsomes with excess cyt P450 1A2 and 2E1 [8, 10], as well as in films of rat liver microsomes [11, 12]. Such a pathway may also occur in genetically enriched cyt P450 1A2 and cyt P450 3A4 supersomes [13] in polyion films on electrodes.…”

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confidence: 99%

“…This lack of midpoint potential shifts for CPR microsomes, HLM and cyt P450 1B1 films suggests that CV peaks originated not from cyt P450s but from CPR [8, 11, 13, 17]. CPR contains FAD and FMN cofactors, and CPR voltammetry has been interpreted previously [11, 13, 17]. Further, midpoint potentials near −0.5 V vs. SCE are characteristic of CPR and not cyt P450s, which have CV midpoint potentials in similar films of about −0.30 to −0.35 V vs. SCE [6, 8].…”

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confidence: 99%

“…[11, 13, 15]. However, films of HLM or CPR microsomes did not show catalytic behavior in 2 mM H 2 O 2 (see Figure S1).…”

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Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

et al. 2012

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The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of −0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H2O2, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.

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Correlating the Electrochemical Kinetics of Myoglobin‐Films to pH Dependent Meat Color

Nerimetla¹,

Walgama²,

Ramanathan³

et al. 2014

Electroanalysis

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We report here for the first time application of electrochemical kinetics to understand the myoglobin redox‐properties at three relevant meat pH conditions. We investigated the kinetics of oxymyoglobin formation electrochemically using films of myoglobin coated on high‐purity graphite electrodes. Three pH conditions corresponding to the physiological state (pH 7.4), stressed animal muscle (pH 6.4), and post‐mortem meat (pH 5.6) were examined in this study. Our findings show that slower metmyoglobin reduction rate and diminished oxygen‐affinity cause the discoloration of red‐meat at low acidic pH‐conditions in comparison to the physiological pH 7.4.

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Thin film voltammetry of metabolic enzymes in rat liver microsomes

Cited by 21 publications

References 15 publications

Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

Correlating the Electrochemical Kinetics of Myoglobin‐Films to pH Dependent Meat Color

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