The main reaction involved in phase 1 metabolism is hydroxylation, catalyzed by a family of enzymes known as monooxygenases or cytochromes P450. There are at least 57 cytochrome P450 genes in the human genome.
Cytochrome P450 is a heme enzyme. It is so named because it was originally discovered when it was noted that preparations of microsomes (fragments of the endoplasmic reticulum) that had been chemically reduced and then exposed to carbon monoxide exhibited an absorption peak at 450 nm. Approximately 50% of the common drugs that humans ingest are metabolized by isoforms of cytochrome P450. They also act on steroid hormones, carcinogens, and pollutants. The major cytochromes P450 in drug metabolism are members of the CYP1, CYP2, and CYP3 families (see below). In addition to their role in metabolism of xenobiotics, cytochromes P450 are important in the metabolism of a number of physiological compounds—for example, the synthesis of steroid hormones (see Chapter 26) and the conversion of vitamin D to its active metabolite, calcitriol (see Chapter 44).
The overall reaction catalyzed by a cytochrome P450 is:
The role of NADPH is to reduce cytochrome P450; the reduced cytochrome then reduces oxygen to water and the hydroxyl group that is introduced into the substrate. The reaction mechanism is complex (see Figure 12–6). Using 18O2 it has been shown that one atom of oxygen forms the hydroxyl group of R–OH and the other forms water. This dual fate of the oxygen accounts for the former naming of monooxygenases as “mixed-function oxidases.”
Isoforms of Cytochrome P450 Make Up a Superfamily of Heme-Containing Enzymes
Because of the large number of isoforms of cytochrome P450 that have been discovered (about 150, in a wide range of organisms, including bacteria), it is important to have a systematic nomenclature for the enzymes and their genes. It is based on amino acid sequence homology of the enzymes. The abbreviated root symbol CYP denotes a cytochrome P450. This is followed by an Arabic numeral designating the family; cytochromes P450 are included in the same family if they exhibit 40% or more amino acid sequence identity. The Arabic number is followed by a capital letter indicating the subfamily; P450s are in the same subfamily if they exhibit greater than 55% sequence identity. The individual P450s are then assigned Arabic numerals in their subfamily. Thus, CYP1A1 denotes a cytochrome P450 that is a member of family 1 and subfamily A and is the first individual member of that subfamily. The nomenclature for the genes encoding cytochrome P450s is the same, except that italics are used; thus, the gene encoding CYP1A1 is CYP1A1. The families of cytochrome P450 in human tissues, and their principal functions, are shown in Table 47–1.
TABLE 47–1The Families of Cytochrome P450 in Human Tissues |Favorite Table|Download (.pdf) TABLE 47–1 The Families of Cytochrome P450 in Human Tissues
|Family ||Function ||Members |
|CYP1 ||Drug and steroid (especially estrogen) metabolism ||3 subfamilies |
|CYP2 ||Drug and steroid metabolism ||13 subfamilies |
|CYP3 ||Drug and steroid (including testosterone) metabolism ||1 subfamily |
|CYP4 ||Arachidonic acid and fatty acid metabolism ||6 subfamilies |
|CYP5 ||Thromboxane A2 synthase ||1 subfamily |
|CYP7 ||Bile acid biosynthesis and steroid 7α hydroxylase ||2 subfamilies |
|CYP8 ||Various, including prostacyclin synthase and bile acid synthesis ||2 subfamilies |
|CYP11 ||Steroid biosynthesis ||2 subfamilies |
|CYP17 ||Steroid biosynthesis, 17α hydroxylase ||1 subfamily |
|CYP19 ||Steroid biosynthesis, aromatase ||1 subfamily |
|CYP20 ||Unknown function ||1 subfamily |
|CYP21 ||Steroid biosynthesis ||2 subfamilies |
|CYP24 ||Vitamin D catabolism ||1 subfamily |
|CYP26 ||Retinoic acid hydroxylase ||3 subfamilies |
|CYP27 ||Various, including bile acid synthesis and calcidiol 1α hydroxylase. ||3 subfamilies |
|CYP39 ||7-Alpha hydroxylation of 24-hydroxycholesterol ||1 subfamily |
|CYP46 ||Cholesterol 24-hydroxylase ||1 subfamily |
|CYP51 ||Cholesterol biosynthesis ||1 subfamily |
In mammals, cytochromes P450 are present in highest amount in liver cells and enterocytes but are probably present in all tissues. In liver and most other tissues, they are present mainly in the membranes of the smooth endoplasmic reticulum, which constitute part of the microsomal fraction when tissue is subjected to subcellular fractionation. In hepatic microsomes, cytochromes P450 can comprise as much as 20% of the total protein. P450s are found in most tissues, though often in low amounts compared with liver. In the adrenal gland, they are found in mitochondria as well as in the endoplasmic reticulum; the various hydroxylases present in the gland are involved in cholesterol and steroid hormone biosynthesis. The mitochondrial cytochrome P450 system differs from the microsomal system in that it uses an NADPH-linked flavoprotein, adrenodoxin reductase, and a nonheme iron-sulfur protein, adrenodoxin. In addition, the P450 isoforms involved in steroid biosynthesis are generally much more restricted in their substrate specificity.
Not only is there a wide variety of cytochromes P450, but they also have overlapping substrate specificities, so that a very broad range of xenobiotics can be metabolized by one or other of the cytochromes P450.
NADPH, not NADH, is involved in the reaction mechanism of cytochrome P450, in a reaction catalyzed by NADPH-cytochrome P450 reductase. Electrons are transferred from NADPH to NADPH-cytochrome P450 reductase and then to cytochrome P450. This leads to the reductive activation of molecular oxygen, and one atom of oxygen is subsequently inserted into the substrate. Cytochrome b5, another hemoprotein found in the membranes of the smooth endoplasmic reticulum (see Chapter 12), may be involved as an electron donor in some cases.
Most isoforms of cytochrome P450 are inducible. For instance, the administration of phenobarbital or other drugs causes hypertrophy of the smooth endoplasmic reticulum and a three- to fourfold increase in the amount of cytochrome P450 within 4 to 5 days. In most cases this involves increased transcription of mRNA. However, in some cases, induction involves stabilization of mRNA or the enzyme protein itself, or an increase in the translation of mRNA.
Induction of cytochrome P450 underlies drug interactions, when the effects of one drug are altered by prior, concurrent, or later administration of another. For example, the anticoagulant warfarin is metabolized by CYP2C9, which is induced by phenobarbital. Induction of CYP2C9 by phenobarbital will increase the metabolism of warfarin, so reducing its efficacy, and the dose must be increased. Another example involves CYP2E1, which is induced by consumption of ethanol. This P450 metabolizes some widely used solvents and compounds found in tobacco smoke, many of which are established procarcinogens. If the activity of CYP2E1 induced by ethanol, this may increase the risk of carcinogenicity.
Naturally occurring compounds in foods may also affect cytochrome P450. Grapefruit contains a variety of furanocoumarins, which inhibit cytochrome P450 and so affect the metabolism of many drugs. Some drugs are activated by cytochrome P450, so that grapefruit will reduce their activity; others are inactivated by cytochrome P450, so that grapefruit increases their activity. Drugs that are affected include statins, omeprazole, antihistamines and benzodiazepine antidepressants.
Polymorphism of cytochromes P450 may explain much of the variations in drug responses noted among many patients – variants with low catalytic activity will lead to slower metabolism of the substrate, and hence prolonged drug action and accumulation of the drug in the body. One interesting polymorphism is that of CYP2A6, which is involved in the metabolism of nicotine to conitine. Three CYP2A6 alleles have been identified: a wild type and two null or inactive alleles. It has been reported that individuals with the null alleles, who have impaired metabolism of nicotine, are apparently protected against becoming tobacco-dependent smokers. These individuals smoke less, presumably because their blood and brain concentrations of nicotine remain elevated longer than those of individuals with the wild-type allele. It has been speculated that inhibiting CYP2A6 may provide a novel way to help smoking cessation.
Table 47–2 summarizes some principal features of cytochromes P450.
TABLE 47–2Some Properties of Human Cytochromes P450 |Favorite Table|Download (.pdf) TABLE 47–2 Some Properties of Human Cytochromes P450
Involved in phase I of the metabolism of a large number of xenobiotics, including perhaps 50% of the clinically used drugs; they may increase, decrease or not affect the activities of various drugs.
Involved in the metabolism of many endogenous compounds (eg, steroids).
All are hemoproteins.
Often exhibit broad substrate specificity, thus acting on many compounds; consequently, different P450s may catalyze formation of the same product.
Extremely versatile catalysts, perhaps catalyzing about 60 types of reactions. However, basically they catalyze reactions involving introduction of one atom of oxygen into the substrate and one into water.
Their hydroxylated products are more water-soluble than their generally lipophilic substrates, facilitating excretion.
Liver contains highest amounts, but found in most if not all tissues, including small intestine, brain, and lung.
Located in the smooth endoplasmic reticulum or in mitochondria (steroidogenic hormones).
In some cases, their products are mutagenic or carcinogenic.
Many have a molecular mass of about 55 kDa.
Many are inducible, resulting in one cause of drug interactions.
Many are inhibited by various drugs or their metabolic products, providing another cause of drug interactions.
Some exhibit genetic polymorphisms, which can result in atypical drug metabolism.
Their activities may be altered in diseased tissues (eg, cirrhosis), affecting drug metabolism.
Genotyping the P450 profile of patients (eg, to detect polymorphisms) may in the future permit individualization of drug therapy.