Development of an IgY Antibody-Based Immunoassay for the Screening of the CYP2E1 Inhibitor/Enhancer from Herbal Medicines

Abstract

Cytochrome P450 (CYP) 2E1 is an important enzyme involved in the metabolism of many endogenous and exogenous compounds. It is essential to evaluate the expression of CYP2E1 in the studies of drug–drug interactions and the screening of drugs, natural products, and foodstuffs. The present work is a feasibility study on the development of immunoassays using a specific and sensitive chicken-sourced anti-CYP2E1 IgY antibody. Cloning, expression, and purification of a recombinant CYP2E1 (mice origin) protein were carried out. Anti-CYP2E1 IgY antibodies were generated by immunizing white Leghorn chickens with purified recombinant CYP2E1 protein and were purified by immune affinity chromatography. The IgY titer attained a peak level (≥1:128,000) after the fifth booster injection. For evaluation of the expression of CYP2E1 in different herbal treatment samples, the mice were treated by oral gavage for 3 days with alcohol (50% 15 mL/kg), acetaminophen (APAP, 300 mg/kg), Cornus officinalis extract (100 mg/kg), Alhagi-honey extract (100 mg/kg), Apocynum venetum extract (100 mg/kg), hyperoside (50 mg/kg), isoquercetin (50 mg/kg), 4-hydroxyphenylacetic acid (50 mg/kg), 3-hydroxyphenylacetic acid (50 mg/kg), and 3,4-hydroxyphenylacetic acid (50 mg/kg). The expression of CYP2E1 was determined by Western blot analysis, immunohistochemistry, ELISA, and immunomagnetic beads (IMBs) using anti-CYP2E1 IgY in liver tissue. The results showed that C. officinalis extract, Alhagi-honey extract, A. venetum extract, hyperoside, isoquercetin, and their xenobiotics 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-hydroxyphenylacetic acid significantly decreased CYP2E1 levels. Alcohol and APAP treatments significantly increased CYP2E1 levels as analyzed with Western blot analysis, immunohistochemistry, and ELISA. The IMB method is suitable for large-scale screening, and it is a rapid screening (20 min) that uses a portable magnet and has no professional requirements for the operator, which makes it useful for on-the-spot analysis. Considering these results, the anti-CYP2E1 IgY could be applied as a novel research tool in screening for the CYP2E1 inhibitor/enhancer.

In vitro inhibitory effects of cepharanthine on human liver cytochrome P450 enzymes

“…As previous studies reported, CYP2E1 and CYP2C9 play a vital role in the metabolism of many drugs (He et al 2009;Lim et al 2014;Xu et al 2017;Bedada and Neerati 2018). There are also many drugs inhibiting the activity of CYP2E1 and CYP2C9 (Pan et al 2010;Jiang et al 2016;Liu et al 2017;Hao et al 2018), and we get similar results in the study of CEP. Different from CYP3A4, the activity of human liver microsomal CYP2E1 and CYP2C9 was inhibited by CEP competitively.…”

“…According to previous studies about the pharmacokinetic profiles of CEP in rats, the plasma C max values of CEP in orally administered with CEP (10 mg/kg) were approximately 0.05 lg/ mL (Jiang et al 2016), which is much lower than the Ki and IC 50 values of CEP. Consequently, the drug-drug interaction might not occur when CEP was co-administered with the substrates of the CYP3A4, CYP2E1 and CYP2C9.…”

Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review

“…CYP2E1 metabolizes a wide variety of chemicals with different structures, in particular small and hydrophobic compounds, including potential cytotoxic and carcinogenic agents. Anti-CYP2E1 IgY was specific, without reacting with other cytochromes, and was able to detect the reduction of hepatic CYPE2E1 expression due to the ingestion of natural products with hepatoprotective effects [120].…”

Unveiling the Impact of Antibiotics and Alternative Methods for Animal Husbandry: A Review

“…There is a wide range of benefits of IgY antibody. Immunoglobulin Y (IgY) and the functional counterpart of mammalian IgG are excellent candidates alternatively to antibiotics for preventive and therapeutic against bacteria and virus [ 109 , 110 ]. IgY is also effective in providing protection against a wide range of gastrointestinal pathogens in humans and animals.…”