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Regulation and Mutations of Drug Metabolizing Enzymes

By Laura Hall
April 2010

Joyce Goldstein, Ph.D.
"Those individuals who have two null alleles in the CYP2C19 gene and take Plavix might as well be taking starch pills," said Joyce Goldstein (above). "The mutated CYP2C19 enzyme cannot metabolize the drug into its active form. Individuals with mutated genes are common, particularly among Asian populations." (Photo courtesy of Steve McCaw)

On February 25, Joyce Goldstein, Ph.D.(http://www.niehs.nih.gov/research/atniehs/labs/ltp/human/index.cfm), principal investigator of the NIEHS Human Metabolism Group discussed the factors that regulate the gene expression of a superfamily of drug metabolizing enzymes called the human cytochrome P450 enzymes (CYPs) and the effects of mutations or genetic polymorphisms in the CYP genes.

In her talk, Goldstein focused on the CYP2C subfamily - CYP2C8, CYP2C9, CYP2C18, and CYP2C19 - which metabolize approximately 25 percent of all clinically prescribed drugs. Understanding CYP2C gene regulation provides insight into drug-drug interactions and drug tolerance.

Goldstein's Laboratory of Toxicology and Pharmacology (LTP) (http://www.niehs.nih.gov/research/atniehs/labs/ltp/index.cfm) Seminar Series presentation, titled "The Human Cytochrome P450 2C(CYP2C) Xenobiotic Metabolizing Enzymes: Their Medicinal Importance, Genetic Polymorphisms, and Transciptional Regulation" was hosted by David Miller, Ph.D., chief of the LTP.

The Goldstein laboratory first cloned and identified two members of the human CYP2C subfamily enzymes in 1991 and expressed all four members of this subfamily in yeast - identifying drug substrates for all four human enzymes. Goldstein's studies first established the human genomic sequences for this subfamily and her collaborations with clinical researchers have helped to identify human genetic polymorphisms in the CYP2C genes and to test their clinical consequences.

CYP2C Gene Polymorphisms

CYP2C9 metabolizes warfarin, the anticoagulant drug prescribed to reduce the chance of blood clots that can cause heart attacks and strokes. Goldstein explained that two predominant mutant variants of the CYP2C9 gene affect the ability of the enzyme products to function properly. Individuals with one or two of any combination of these variants metabolize warfarin poorly, so that normal, "safe" doses can be toxic.

The lower the amount of warfarin metabolized, the greater the concentration of drug that can accumulate in the body - possibly resulting in overdosage - and polymorphisms have been shown to produce serious and life-threatening bleeding problems.

Research characterizing the function of and identifying the human mutations in the CYP2C9 gene has led to warnings on warfarin packaging inserts. The warning makes doctors aware that patients with these mutated genes require lower doses.

Mutations in the CYP2C19 gene discovered by the Goldstein laboratory affect the metabolism of the anti-clotting drug Plavix and have been shown to be associated with a 50 percent increase in death and a 5-fold increase in stent failures in patients with cardiovascular disease treated with Plavix, which now bears a boxed warning (http://fdanews.com/newsletter/article?issueId=13542&articleId=125542)Exit NIEHS about the drug's ineffectiveness in some patients with a genetic mutation.

Human CYP2C Substrates - CYP2C enzymes metabolize many drugs. Common mutations in the CYP2C19 gene are associated with increased adverse effects of Plavix in patients. Genetic tests for these mutations are covered by an NIEHS patent and are being considered for individualized medicine for CYP2C19 substrates, said Goldstein.
CYP2C enzymes metabolize many drugs. Common mutations in the CYP2C19 gene are associated with increased adverse effects of Plavix in patients. Genetic tests for these mutations are covered by an NIEHS patent and are being considered for individualized medicine for CYP2C19 substrates, said Goldstein. (Slide courtesy of Joyce Goldstein)


CYP2C Gene Regulation

Variability in CYP2C enzyme expression can also make a difference in how much drug can be metabolized. To understand how CYP2C gene expression can be altered, Goldstein is investigating the role of nuclear receptors - constitutive androstane receptor (CAR), pregnane X receptor (PXR), and the important liver enriched hepatocyte nuclear factor four alpha (HNF4α) - in regulating CYP2C gene transcription.

CAR and PXR are cellular proteins that sense exogenous chemicals and activate expression of the CYP2C and other detoxification enzymes to help eliminate these foreign chemicals from the body.

Goldstein's group has identified HNF4α binding sites and a CAR/PXR binding site in the promoter regions of the CYP2C genes. Promoter regions of a gene are not protein product template, but help initiate transciption or copying of DNA into the RNA that will be used to make protein.

In the liver, CYP2C genes are constantly expressed at a basal level, but they can be upregulated by exposure to chemicals. Goldstein's group has demonstrated that the HNF4α sites are involved in the basal expression, whereas both CAR/PXR and HNF4α sites are needed for optimal induction of CYP2C9 and CYP2C8 gene transcription.

The scientists determined that CAR and PXR were not directly bound to the HNF4α site. A coactivator, nuclear receptor coactivator 6 (NCOA6), appears to form a bridge between one HNF4α and the CAR/PXR site. A second coactivator, mediator complex subunit 25 (MED25), binds to HNF4α and helps recruit the transcription enzyme RNA polymerase II to a mediator complex needed to initiate gene transcription.

(Laura Hall is a biologist in the NIEHS Laboratory of Toxicology and Pharmacology currently on detail as a writer for the Environmental Factor.)



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