Nicotine Metabolism

 

 

Cytochrome P450 2A6 --What is Cytochrome P450 2A6?

Cytochrome P450 2A6 is an enzyme belonging to the superfamily of cytochrome P450s. These enzymes catalyze the metabolism of a wide variety of substances including chemicals from the environment, bile acids, drugs, and steroid hormones. Much of the information gleaned from experimentation with P450s involves the study of drug effects. The P450s are also the major enzyme family required for oxidation of carcinogens, pesticides, steroids, fat-soluble vitamins, and eicosanoids. The level of microsomal P450s in the human liver remains fairly constant across a population, but the levels of individual members of the P450 family vary significantly. These variances are evident in the differing abilities of individuals to metabolize drugs. Genetic polymorphisms, lack of inducibility, production of altered forms of P450s, enzyme inhibition, and enzyme induction are all factors that contribute to the variability of P450s such as 2A6. Cytochrome P450 2A6 enzyme is induced specifically by ingestion/inhalation of tobacco products (Guengerich 1995).

Cytochrome P450 are largely involved in oxygen activation and transfer. The enzyme incorporates one of the atoms of an O2 molecule into a vast multitude of substrates, while reducing the other atom of oxygen to form water. This enzyme family can be found in many mammalian tissues (liver, kidney, intestine, and lung, to name a few) as well as in plants, yeasts, insects, and bacteria. One of the remarkable things about cytochrome P450 is the wide variety of reactions that it catalyzes. These include hydroxylations, epoxidations, N-, S-, and O- dealkylations, dehalogenations, sulfoxidations, N-oxidations, as well as many others (Groves & Han 1995).

The active site of the Cytochrome P450 enzyme comprises an iron protoporphyrin IX, with a cysteinate found at the fifth ligand position. A separate coordination site participates in the binding and activation of oxygen molecules. The basic scheme of oxygen incorporation is as follows: (1) the substrate binds to the enzyme (2) the ferric portion of P450 is reduced to the ferrous state (3) molecular oxygen binds to the ferrous P450 to yield a dioxygen complex (4) another reduction (one electron) to produce a peroxoiron(III) complex (5) cleavage of the O-O bond and concomitant incorporation of one oxygen atom into water, forming the reactive iron-oxo intermediate (6) transfer of the oxygen atom from the oxo complex to the substrate (7) dissociation of product (Groves & Han 1995).

Cytochrome P450 2A6 is a member of the P450 2A subfamily found in the liver. It functions in the metabolism of pharmaceutical compounds and metabolically activates various precarcinogens including NNK. CYP2A6 is the allele encoding for cytochrome P450 2A6, and is a highly polymorphic gene. The extreme variation between individual ability to metabolize drugs, including nicotine, is due largely to the variability of the CYP2A6 allele. Interestingly, extrahepatic expression of P450 2A subfamily enzymes have been found in animals, and it has been proposed that human nasal mucosa may also express P450s 2A6 and 2E1, both of which function to oxidize tobacco-specific nitrosamines (Guengerich 1995, Oscarson 1999).

The primary fate of nicotine is cotinine, a metabolite derived from the 5'-Hydroxylation pathway:

The 5'-Hydroxylation Pathway -- Most of the nicotine absorbed by the body from cigarettes is metabolized in two ways. Much of the previous research focuses on the 5'-hydroxylation pathway (See Figure 1):

1) Nicotine is hydroxylated at the 5' position, yielding an unstable intermediate 5'-hydroxynicotine in equilibrium with Delta1'(5')iminium ion. This first step is catalyzed by cytochrome P450 2A6, with cytochromes P450 2B6 and P450 2D6 also contributing to the hydroxylation.

2) 5'-Hydroxynicotine is then oxidized to cotinine, the major nicotine metabolite, by aldehyde oxidase.

3) Cotinine can then be converted either to cotinine-Gluc (via a detoxification mechanism) or trans-3'-hydroxycotinine. This compound can also be detoxified to trans-3'-hydroxycotinine-Gluc (see Figure 1). In humans, cotinine and its metabolites comprise 70-80% of the products of nicotine metabolism.

 

The secondary--and more dangerous fate--of nicotine is an aminoketone, pseudooxynicotine, derived from the 2'-hydroxylation pathway. Aminoketone is a direct precursor to potent lung carcinogens.

The 2' Hydroxylation Pathway -- The second metabolic pathway begins with hydroxylation of nicotine at the 2' position instead of the 5' position. An additional 10-15% of nicotine metabolites are derived from the 2' hydroxylation pathway. The major compounds of this pathway are 4-oxo-4(3-pyridyl)butanoic acid (keto acid), and 4-hydroxy-4(3-pyridyl)butanoic acid (hydroxy acid) (See Figure at right). Researchers Hecht et al. (2000) found that these metabolites are present in the urine of subjects that received doses of nicotine, but are not present in the urine of subjects dosed only with cotinine.

1) Nicotine is hydroxylated at the 2' position to yield 2'-hydroxynicotine in a reaction also catalyzed by P450 2A6.

2) 2'-hydroxynicotine spontaneously interconverts with Delta1'(2') iminium ion as well as 4-(methylamino)-1-(3-pyridyl)-1-butanone, an aminoketone known as pseudooxynicotine.

3) This aminoketone is the direct precursor to keto aldehyde, which subsequently becomes the same keto acid found in the 5'-Hydroxylation pathway.

4) Aminoketone can also be converted via simple nitrosation to the infamous NNK (4-(methylnitrosamino)-1-3(pyridyl)-1-butanone), the potent lung carcinogen.

The metabolism of NNK will be discussed later.

 

 

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