METABOLISM OF XENOBIOTICS

Teaching aims

• By the end of the lecture, students would be able to :– realize that humans are subjected

to exposure to various foreign chemicals (xenobiotics) and

– understand how xenobiotics are handled at the cellular level

Core topics

• Introduction• Metabolism of xenobiotics

– Phase 1– Phase 2

• Responses to xenobiotics

Introduction• A xenobiotic is a compound that is

stranger to the body (xenobiotics xenos = stranger)

• Humans are subjected to exposure to various (maybe thousands) foreign chemicals e.g. drugs, good additives, pollutants, etc.

• Those relevance to medicine are drugs, chemical carcinogens, polychlorinated biphenyls (PCB) & certain insecticides

Introduction (cont.)

• They must be metabolized (chemical alteration) before being excreted

• Knowledge of the metabolism of xenobiotics is basic to a rational understanding of pharmacology & therapeutics, pharmacy, toxicology, cancer research, drug addiction etc.

Metabolism of xenobiotics

• Liver is the main organ involved in xenobiotics metabolism

• Occasionally, a xenobiotic may be excreted unchanged

• At least 30 different enzymes catalyze reactions involved in xenobiotics metabolism

• It is convenient to consider the metabolism of xenobiotics in two phases:

• Phase 1– The major reaction involved is

hydroxylation, catalyzed by members of a class of enzymes called monooxygenases or cytochrome P450s. In addition to hydroxylation, these enzymes catalyze wide range of reactions, including those involving deamination, dehalogenation, desulfuration,epoxidation, peroxygenation, & reduction

– Reactions involving hydrolysis (eg. Catalyzed by esterases) & certain other non-P450-ctalyzed reactions

• Phase 2– Hydroxylated or other compounds

produced in phase 1 are converted by specific enzymes to various polar metabolites by conjugation with glucuronic acid, sulfate, acetate, glutathione, or certain amino acids, or by methylation.

• The overall purpose of the 2 phases is to increase their water solubility (polarity) and thus facilitate their excretion from the body

• Very hydrophobic xenobiotics would persist in adipose tissue almost indefinitely unless they are converted to more polar forms

• In certain cases, phase 1 metabolic reactions convert xenobiotics from inactive to biologically active compounds in these instances, the original xenobiotics are referred to as “prodrugs” or “procarcinogens”

• In other cases, additional phase 1 reaction (eg. further hydroxylation reactions) convert the active compounds to less active or inactive forms prior to conjugation.

• In other cases, it is the conjugation reactions themselves that convert the active products of phase 1 reactions to less active or inactive products, which are subsequently excreted in the urine or bile. In a very few cases, conjugation may actually increase the biologic activity of a xenobiotic

• The term “detoxification” is sometimes used for many of the reactions involved in xenobiotics metabolism, but the term is not always appropriate

Hydroxylation Reaction in phase 1:Role of Cytochrome P450

• The chief reaction involved in phase 1 is hydroxylation & the responsible enzymes are called monooxygenases or cytochrome P450s.

• Human genome encodes at least 14 families of these enzymes. The number of distinct cyt. P450s in human tissues range from approx. 35 to 60

RH + O2 + NADPH + H+ R-OH + H2O + NADPorReduced cyt. P-450 Oxidized cyt P-450

RH + O2 R – OH + H2O • RH can represent a variety of xenobiotics,

including drugs, carcinogens, pesticides, petroleum products & pollutants (such as a mixture of PCBs)

Cyt.P-450

• Endogenous compounds, such as certain steroids, eicosanoids, fatty acids & retinoids are also substrate (generally lipophilic)

• Cyt. P-450 is major monooxygenases in endoplasmic reticulum. It is important because approx. 50% of the drugs that humans ingest are metabolized by isoforms of cyt. P-450, and these enzymes also act on various carcinogens & pollutants

• Cyt. 450 is considered the most versatile biocatalyst known

• Isoforms of cyt. P-450 make up a superfamily of heme-containing enzymes (they exhibit 40% or more sequence identity):– Needs a systematic nomenclature

(Arabic number followed by a capital letter indicating the subfamily & individual number) CYP1A1 is cyt. P-450,a member of family 1 & subfamily A & the first individual member. The gene encoding this enzyme is CYP1A1

– They are hemoproteins– They are widely distributed across

species– They are present in highest amount in

liver & small intestine present mainly in membranes of the smooth endoplasmic reticulum. In the adrenal they are found in mitochondria as well as in the endoplasmic retic.

– Mitochondrial cyt. P-450 system differs from the macrosomal system in that it uses an NADPH-linked flavoprotein

– There are at least six isoforms of cyt. P-450 present in the endopl. retic. of human liver, each with wide & somewhat overlapping substrate specificities & acting on both xenobiotics and endogenous compounds

– NADPH, not NADH is involved in the reaction mechanism of cyt. P-450

– Lipids, particularly phosphatidyl-choline are also components of cyt. P-450 system

– Most isoforms of cyt. P-450 are inducible (has important clinical implication due to biochemical mechanism of drug interaction.

– Certain isoforms of cyt. P-450 (eg. CYP2E1) are particularly involved in the metabolism of Polycyclic Aromatic Hydrocarbon (PAHs) & related molecules

– Certain cyt. P-450s exist in polymorphic forms (genetic isoforms), some of which exhibit low catalytic activity

Conjugation reaction in phase 2

• Hydroxylated derivatives of xenobiotics from phase 1 are conjugated with molecules such as glucuronic acid, sulfate or glutathione:a. Glucuronidation (UDP-glucuronic acid as

glucuronyl donor. Glucuronosyltransferases in endoplasmic retic. & cytosol are the catalysts) the most frequent conjugation reaction. Substrates: 2-acetylaminofluorene, aniline, benzoic acid, meprobamate, phenol & many steroids

b. Sulfation (Adenosine 3’-phosphate-5’-phosphosulfate=PAPS as sulfate donor). Substrates: steroids, glycosaminoglycans, glycolipids & glycoproteins

c. Conjugation with glutathione (tripeptide -glutamylcysteinylglycine = GSH). Substrates: electrophilic xenobiotics R + GSH R—S—G R = an electrophilic xenobiotic and the enzymes catalysing the reaction are : glutathione S-transferase.

Glutathione conjugates are subjected to further metabolism before excretion (eg. Mercapturic acid excreted in the urine)

d. Other reactions:1. acetylationX + Acetyl-CoA acetyl-X + CoA(using acetyltransferases in the cytosol of various tissues, particularly liver, eg. Drug isoniazid for tuberculosis) 2. Methylation (using S-adenosylmethionine as methyl donor & methyltransferase as the enzyme)

Notes for glutathione:• Other important function of glutathione:

– Participate in the decomposition of potentially toxic hydrogen peroxide catalyzed by glutathione peroxidase

– Intracellular reductant, helping to maintain essential SH groups of enzymes in their reduced state

– Transport of certain amino acids across membrane in the kidney

Factors affecting xenobiotic-metabolizing enzymes

• Species different among species• Individual different among

individuals within same species (particularly in human:– Genetic factors– Age & sex

• Diseases eg. cirrhosis of the liver can affect activities of drug metabolizing enzymes

Xenobiotic Reactive metabolite Nontoxic metabolite

Covalent bonding To macromolecules

Hapten MutationCell injury

CancerAntibody production

Cell injury

Cyp450 GSH S-transferase or epoxide hydrolase

Spectrum of toxic effects of xenobiotics