The biochemistry and medical significance of the flavonoids.

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

Quantification of mRNA using real-time RT-PCR

Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology

Of about one dozen human P450 s that catalyze biotransformations of xenobiotics, CYP2D6 is one of the more important ones based on the number of its drug substrates. It shows a very high degree of interindividual variability, which is primarily due to the extensive genetic polymorphism that influences expression and function. This so-called debrisoquine/sparteine oxidation polymorphism has been extensively studied in many different populations and over 80 alleles and allele variants have been described. CYP2D6 protein and enzymatic activity is completely absent in less than 1% of Asian people and in up to 10% of Caucasians with two null alleles, which do not encode a functional P450 protein product. The resulting "poor metabolizer" (PM) phenotype is characterized by the inability to use CYP2D6-dependent metabolic pathways for drug elimination, which affect up to 20% of all clinically used drugs. The consequences are increased risk of adverse drug reactions or lack of therapeutic response. Today, genetic testing predicts the PM phenotype with over 99% certainty. At the other extreme, the "Ultrarapid Metabolizer" (UM) phenotype can be caused by alleles carrying multiple gene copies. "Intermediate Metabolizers" (IM) are severely deficient in their metabolism capacity compared to normal "Extensive Metabolizers" (EM), but in contrast to PMs they express a low amount of residual activity due to the presence of at least one partially deficient allele. Whereas the intricate genetics of the CYP2D6 polymorphism is becoming apparent at ever greater detail, applications in clinical practice are still rare. More clinical studies are needed to show where patients benefit from drug dose adjustment based on their genotype. Computational approaches are used to predict and rationalize substrate specificity and enzymatic properties of CYP2D6. Pharmacophore modeling of ligands and protein homology modeling are two complementary approaches that have been applied with some success. CYP2D6 is not only expressed in liver but also in the gut and in brain neurons, where endogenous substrates with high-turnover have been found. Whether and how brain functions may be influenced by polymorphic expression are interesting questions for the future.

Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry

Pharmacogenetics is the study of how interindividual variations in the DNA sequence of specific genes affect drug response. This article highlights current pharmacogenetic knowledge on important human drug-metabolizing cytochrome P450s (CYPs) to understand the large interindividual variability in drug clearance and responses in clinical practice. The human CYP superfamily contains 57 functional genes and 58 pseudogenes, with members of the 1, 2, and 3 families playing an important role in the metabolism of therapeutic drugs, other xenobiotics, and some endogenous compounds. Polymorphisms in the CYP family may have had the most impact on the fate of therapeutic drugs. CYP2D6, 2C19, and 2C9 polymorphisms account for the most frequent variations in phase I metabolism of drugs, since almost 80% of drugs in use today are metabolized by these enzymes. Approximately 5-14% of Caucasians, 0-5% Africans, and 0-1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant enzyme that demonstrates multiple genetic variants with a potentially functional impact on the efficacy and adverse effects of drugs that are mainly eliminated by this enzyme. Studies into the CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and *3 alleles. Extensive polymorphism also occurs in other CYP genes, such as CYP1A1, 2A6, 2A13, 2C8, 3A4, and 3A5. Since several of these CYPs (e.g., CYP1A1 and 1A2) play a role in the bioactivation of many procarcinogens, polymorphisms of these enzymes may contribute to the variable susceptibility to carcinogenesis. The distribution of the common variant alleles of CYP genes varies among different ethnic populations. Pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and currently available drugs. Further studies are warranted to explore the gene-dose, gene-concentration, and gene-response relationships for these important drug-metabolizing CYPs.

Polymorphism of human cytochrome P450 enzymes and its clinical impact

In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.

https://www.future-science.com/doi/pdf/10.2144/96206st02

Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.

Inhibition of estrone sulfatase in human liver microsomes by quercetin and other flavonoids

Alternative splicing of CYP2D mRNA in human breast tissue.

Allelic variants of the CYP2D6 gene, a member of the cytochrome P450 gene superfamily, have been implicated in susceptibility to lung carcinogenesis. Human breast CYP2D6 and CYP2D7P (from a pseudogene) mRNAs were previously reported to be expressed as a series of splice variants. In this study, the expression of full-length and splice variants of these mRNAs in human lung tissue and tumors are reported for the first time and are compared in order to probe the potential for differential CYP2D6 regulation in lung normal tissue and tumors. The splice variant profiles differed within the same individual, but no consistent differences were detected.

Zeqi Huang

Papers

Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.

Inhibition of estrone sulfatase in human liver microsomes by quercetin and other flavonoids

Alternative splicing of CYP2D mRNA in human breast tissue.

Comparisons of CYP2D Messenger RNA Splice Variant Profiles in Human Lung Tumors and Normal Tissues