CYP450 Upregulation

Cytochrome P450 (CYP450) enzymes are a class of membrane-bound hemo-protiens that work to detoxify various small exogenous xenobiotics and endogenous substances through oxidation, reduction, or hydrolysis, also referred to as Phase 1 metabolism (Zhao et al., 2021, Gilani & Cassagnol, 2023). CYP450 enzymes have various locations within organs such as the liver, kidney, and the intestine but these enzymes can most prominently be found in the liver. On a cellular level, CYP450 is expressed within eukaryotes cells and some prokaryotes, mainly in the mitochondria or the endoplasmic reticulum (Gilani & Cassagnol, 2023).  Typically but not always, the endoplasmic reticulum CYP450 mostly metabolizes exogenous substances such as drugs, whereas the mitochondrial enzymes interact with endogenous process such as steroid hormone metabolism and fatty acid regulation. There are 18 mammalian CYP450 families which encode for 57 genes found in the human genome (Nebert et al., 2013). As such many isoforms in the CYP450 family exist and exhibit different metabolic activities, substrate affinities, conformation, and expression. In addition, most CYP enzyme expression depends on a transcription inducing substrate for upregulation or downregulation. However, in an attempt to metabolize compounds, occasionally CYP450 enzymes can bioactivate a substrate into a more toxic metabolite, such as turning vinyls into vinyl epoxides (Guengerich, 2003).

**Please note that the Cell term and Organ term only allow for one selection in the traning AOP wiki

• P450 Spectral assay:  Total CYP450 enzyme detection and activity can be measured through its spectral properties and has been outlined by Guengerich et al., (2009). In this paper, the spectral determination of total CYP450 is completed by monitoring the reaction of CYP450 hemeprotein with carbon monoxide via spectrophotometry. The carbon monoxide CYP450 complex creates spectrum with at a maximum wavelength of ~450 nm and an extinction coefficient of 91, 000 M-1 cm -1 which corresponds to the cysteine thiolate axial ligand connection to the heme iron present in the enzyme (Johnston et al., 2008, Guengerich et al. 2009., Omura and Sato 1964).

• Immunochemical assays: The detection of many CYP450 enzymes has been discovered by obtaining microsomal fractions from various animals including primates and humans (Uehara et al., 2014, Nakanishi et al., 2011, Uehara et al., 2010, Uehara et al., 2011). A notable study outlined the use of cynomolgus monkey small intestine microsomal fractions to identify different types of CYP450's (Uehara et al., 2014). The enzymes once separated via SDS PAGE were reacted with primary anti-human antibody corresponding to multiple CYP's of interest followed by secondary antibody and visualized via chemiluminescence. This technique is known as Western blotting which allows for the visualization of CYP450 upregulation or downregulation when compared to a control by comparison of chemiluminescent protein band intensity (Mahmood and Yang., 2012). 

• Mass spectroscopy : Although in most studies CYP450 is not detected via analytical instruments such as the LC-MS/MS or GC-MS/MS, metabolites and substrates of these enzymes have been detected using mass spectroscopy (Nguyen et al., 2020, Behera et al., 2013, Jiang et al., 2022, Oh et al., 2015). After incubation with a stressor/ substrate, the metabolites can be extracted and separated via liquid or gas chromatography then the chemical composition extrapolated from the resulting mass to charge ratio obtained by the tandem mass spectroscopy.

There are also other methods such as knockout experiments and mRNA assays in animals but those typically target specific CYP's! 

• D: Taxonomic applicability In all studies gathered, the taxonomic applicability is quite wide spanning most eukaryotes including humans, plants, and rats (Uehara et al., 2014, Nakanishi et al., 2011, Uehara et al., 2010, Uehara et al., 2011, Zhao et al., 2021, Gilani & Cassagnol, 2023).
• E: Life stages: The domain of applicability for life stages is all life stages. 
• F: Sex applicability: The domain of applicability for sex is both males and females.

Behera D, Pattem R, Kumar MS, Gudi GS. Utility of a column-switching LC/MS/MS method in cytochrome P450 inhibition assays using human liver microsomes. Drug Metabol Drug Interact.

Gilani, B., Cassagnol, M. (2023) Biochemistry, Cytochrome P450. 

Guengerich, P., (2003). Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions, Archives of Biochemistry and Biophysics, 409(1),  59-71

Jiang F, Zhang C, Lu Z, Liu J, Liu P, Huang M, Zhong G., (2022) Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method. Front Pharmacol. 

Johnston WA, Huang W, De Voss JJ, Hayes MA, Gillam EM. (2008)Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application. J Biomol Screen. 13(2):135-41. 

Mahmood T, Yang PC. (2012)Western blot: technique, theory, and trouble shooting.  N Am J Med Sci. 2014(9):429-34. 

Nebert DW, Wikvall K, Miller WL. (2013)  Human cytochromes P450 in health and disease. Philos Trans R Soc Lond B Biol Sci

Nguyen V, Espiritu M, Elbarbry F.  (2020) Development and validation of a sensitive and specific LC–MS/MS cocktail assay for CYP450 enzymes: Application to study the effect of catechin on rat hepatic CYP activity. Biomedical Chromatography. 

Oh HA, Lee H, Kim D, Jung BH. (2017) Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction. Anal Biochm. 15;519:71-83. 

Tsuneo Omura, Ryo Sato, (1964) The Carbon Monoxide-binding Pigment of Liver Microsomes: I. EVIDENCE FOR ITS HEMOPROTEIN NATURE, Journal of Biological Chemistry, 239 (7) 2370-2378

Uehara S, Murayama N, Yamazaki H, Uno Y. (2010) A novel CYP2A26 identified in cynomolgus monkey liver metabolizes coumarin. Xenobiotica 40:621–9.

Uehara S, Murayama N, Nakanishi Y, Zeldin DC, Yamazaki H, Uno Y. (2011) Immunochemical detection of cytochrome P450 enzymes in liver microsomes of 27 cynomolgus monkeys. J Pharmacol Exp Ther. 2011;339:654–61

Uehara S, Murayama N, Nakanishi Y, Nakamura C, Hashizume T, Zeldin DC, Yamazaki H, Uno Y. (2014) Immunochemical detection of cytochrome P450 enzymes in small intestine microsomes of male and female untreated juvenile cynomolgus monkeys. Xenobiotica. 

Zhao, M., Ma, J., Li, M., Zhang, Y., Jiang, B., Zhao, X., Huai, C., Shen, L., Zhang, N., He, L., & Qin, S. (2021). Cytochrome P450 Enzymes and Drug Metabolism in Humans. International journal of molecular sciences, 22(23)