Biotransformation, Metabolism
12 Temmuz 2007
Biotransformation, Metabolism
and Biologic Defenses
Defense Mechanisms
Respiratory
Mucociliary blanket
impaction, sedimentation
Irritant cough reflex
Phagocytosis - tissue macrophages
Immunologic
Gastrointestinal - Digestion
Poor absorbtion - Pb, Ba, As
Vomiting
Peristalsis - secretions
Gut bacterial
Reduction of R-O-NO2 ® R-O-NO
Nitrates ® Nitrites
Methylation of Mercury
Many processes in common with liver biotransformations
Blood Brain Barrier
Tight capillary endothelial junctions
Glial cells, astrocytes, about capillaries
Less protein in CNS interstitium
3BB less developed in newborns
Placental Barrier
Anatomic vs functional?
Ca++, Fe++, sugars, A.A.Â’s, vitamins are concentrated to fetus
Physicochemical
Storage depots -
ÂÂ Volumes of distribution
ÂÂ T 1/2, elimination time
¯¯ Bioavailability
Bone - Ca++, F– ® crystalline
Plasma Proteins – Ligands
Albumin, transferrin, ceruloplasmin, metallothionein, alpha & beta lipoproteins
(Not protective when binding sites are receptors in target organs radium and strontium in bone.)
GENERAL ASPECTS OF XENOBIOTIC BIOTRANSFORMATION AND METABOLISM
The enzymes involved with biotransformation or metabolism occur in almost all tissues of the body, although certain tissues are much more active in metabolism than are others. In order of quantitative importance, one can list liver > kidney > GI tract, lung > other tissues.
The enzymes responsible for biotransformation are also involved in the metabolism of natural substances. These enzymes are located in membranes (e.g., endoplasmic reticulum, or microsomes) and are also found in the cytoplasm of cells. The oxidation (e.g., hydroxylation) of lipid-soluble substances most often results from an action of a family of hemoprotein oxygenases called cytochrome P-450. These hemoproteins require NADPH and molecular oxygen and exhibit some, but not strict, substrate specificity. These enzymes can be induced to become more active through the action of some other foreign compounds. In this way, the rate of drug metabolism can be increased due to environmental factors.
A group of transferases are involved in biotransformation. These enzymes add a water-soluble, endogenous substance (e.g., glucuronic acid, sulfate, glutathione) to relatively lipid-soluble substances. The water-soluble metabolites, called conjugates, are usually readily excreted. The activity or amounts of transferases can also be increased by foreign compounds, but this does not occur as readily as does stimulation of cytochrome P-450)
Some agents are metabolized at a high rate while others are metabolized slowly. The rate of metabolism is usually quite dependent on chemical structure and physical properties. The activity of metabolizing enzymes in individuals appears to be under genetic control.
Drugs and other foreign compounds can be converted to metabolites which are chemically reactive. Once formed, epoxides, N-hydroxy compounds, etc., can react with nucleophilic groups on cellular macromolecules (proteins and nucleic acids) and thereby alter cell function. Cell death can result, or genetic material may be altered. The formation of reactive metabolites is the basis for some severe drug toxicities and also can be the cause of chemically-induced carcinogenesis.
Biotransformation, then, is a “double-edged sword.” On one hand, it is often the major factor in terminating the pharmacologic action of a drug. On the other hand, drugs may be converted into toxic compounds that can markedly limit their usefulness.
Lipid-soluble compounds would be retained for a long time in the body unless converted to more water-soluble metabolites which can be readily excreted. Metabolites may be pharmacologically or toxicologically active or they may be biologically inactive. A high degree of water solubility usually imparts less activity because of limited distribution to tissues and rapid-excretion. Relatively lipid-soluble metabolites may be inactive because of an inability to fit receptors.
Biotransformations
Primarily in liver, but occurs in all organs
Teleologically = Detoxification
Make xenobiotics more hydrophyllic
Phase 1. Catabolic reactions breakdown, create or expose - OH, -SH, -NH2, -COOH through
oxidation, reduction or hydrolysis
Mono oxygenases - a variety of cytochrome P450/NADPH reductase systems.
RH + CP450 Fe+3 (ferric) - reduced to Fe+2 by NADPH, oxygen ion created – ROH
oxidation - monooxygenases
epoxides from phenols
ketones or aldehydes and acids from alcohols
reduction - NADPH CP450 reductases
hydrolysis - esterases (cholinesterase), amidases
epoxide hydrolases
Phase 2. Biosynthetic
Covalent bonding of additional moiety–enhancing solubility
Requires energy and cofactors
Glucuronosyl transferase - UDPGA
Sulfotransferase - PAPS
Methylation - SAM
N-Acetyl transferase - Acetyl-CoA
Glutathione-S-transferase
(Mechanism by which acetaminophens toxic metabolite is handled.)
Hepatotoxicity — when system is saturated
Mercapturic acids
OXIDATIONS
Aliphatic Alcohols
//
CH3CH2OH ¾¾® CH3CH ¾¾® CH3COOH
1 2
Ethanol Acetaldehyde Acetic Acid - (KrebbÂ’s Cycle)
//
H3COH ¾¾® CH2 ¾¾® HCOOH
1 2
Methanol Formaldehyde Formic Acid
O O O
// \ //
HO-CH2CH2OH ¾¾® HOCH2C H ¾¾¾¾® HOC-C-OH
1 2
Ethylene Glycol Glycoaldehyde Oxalic Acid
1)Â Â Â Â Â Alcohol Dehydrogenase
2)Â Â Â Â Â Aldehyde Dehydrogenase
Aliphatic side chain Hydroxylation
CH3 CH3 OH
½ ½ ½
R-CH-CH2-CH2-CH3 —- R-CH-CH2-CH-CH3
OH OH O O
O2 ½ ½ \ //
CH3CH2CH2CH2CH2CH3 ¾® CH3CHCH2CH2CHCH3 ¾® CH3CHCH2CH2CHCH3
MFO
Hexane 2,5 Hexanediol 2,5 Hexanedione
Oxidation products, responsible for hexane neuropathy
[Frontiers in Bioscience 2, d427-437, September 15, 1997]
Reprints
PubMed
CAVEAT LECTOR
MULTIPLE TRANSPORT PROTEINS INVOLVED IN THE DETOXIFICATION OF ENDO- AND XENOBIOTICSÂ
Yogesh C. Awasthi1,2, Sanjay Awasthi3, and Piotr Zimniak4
Departments of 2Human Biological Chemistry & Genetics and 3Internal Medicine, University of Texas Medical Branch, Galveston, Texas; and 4Department of Internal Medicine and Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, and McClellan VA Hospital, Little Rock, Arkansas
Received 9/3/97 Accepted 9/8/97
2. INTRODUCTION
Living organisms defend themselves from the toxicants present in the environment through biotransformation of these compounds to relatively non-toxic metabolites and their subsequent elimination through transport. Most cells are equipped with a multitude of phase I and phase II biotransforming enzymes (1). In phase I, reactive groups such as -OH, -NH2, or >O are introduced/exposed on relatively hydrophobic xenobiotics so that they can be conjugated to hydrophilic-compounds such as GSH, glucuronate, sulfate, etc. by the phase II enzymes, and the resultant products (usually less toxic, more hydrophilic) can be excreted through active/facilitated transport processes across the cellular membrane. Likewise, electrophiles (both hydrophobic and water soluble) can be conjugated to the abundant nucleophiles such as GSH by phase II enzymes, and the conjugation products can be transported out of cells. Whereas extensive studies have been conducted on biotransformation enzymes resulting in the identification of numerous isozymes belonging to the superfamilies of phase I (2,3) and phase II enzymes (4-8) in mammalian tissues including humans, much less information is available on the enzymes which are involved in the active transport of xenobiotics and/or their metabolites. A concept that the transport of xenobiotic metabolites may be considered as phase III of the detoxification mechanisms has been propagated recently (9-11).
Cells resist chemical aggression of environmental toxins by, 1) warding off the aggression through repulsion by force, 2) capturing or converting the toxins into relatively harmless entities and, 3) expulsion of the converted entities in order to avoid any possible long term harmful effects. In general, all cells are equipped with defense mechanisms involving these three discrete steps. Bacteria and parasites are known to defend against toxins by exclusion mechanisms as exemplified by the acquisition of drug resistance by the malaria parasite, Plasmodium falciparum (12). In humans, the significance of these exclusion mechanisms was not fully recognized until the relatively recent discoveries of drug efflux pumps, including the P-glycoproteins (Pgp) and multidrug resistance associated proteins (MRP), which are overexpressed in multidrug-resistant cancer cells (13,14). Capturing of toxicants is carried out in cells through their binding to certain abundant proteins such as albumin and ligandin (15, review) which trap and often inactivate these compounds, while their conversion to relatively harmless molecules is catalyzed by a multitude of phase I (2,3) and phase II (4-8,15-18) enzymes. The efflux of the metabolites of xenobiotics is carried out by plasma membrane transporter proteins, thus ensuring a safe environment for the cellular components. As expected, all these steps require energy. The drug exclusion pumps require ATP while phase I reactions require the reducing equivalents of NADPH. Some of the phase II enzymes (e.g. glucuronosyl and sulfotransferases) require energy for the activation of substrates, while others, such as glutathione (GSH) S-transferases (GST), require energy for the synthesis of GSH. Phase I and phase II enzymes are often induced by the invading chemicals. Overexpression of the drug efflux pumps, Pgp (13,19) and MRP (14,20), in cancer cells exposed to gradually increasing drug concentrations suggests that the transporters are also induced by xenobiotics. When the toxicants evade or overwhelm the cellular defense mechanisms, they cause toxicity which may eventually result in cell death.
As pointed out above, studies on cellular defense mechanisms have largely focused on phase I and phase II biotransforming enzymes. Multiple forms of these enzymes (e.g. CYP450s, glutathione S-transferases, UDP-glucuronosyltransferases, and sulfotransferases) are expressed in mammals in a tissue-specific manner. The structural and functional properties of these enzymes have been extensively studied. In particular, a large number of isozymes belonging to the gene superfamilies? of CYP450s (2,3) and GSTs (4,5,8,15-18) have been well characterized. The transport mechanisms which flank the biotransformation processes in the simplified description of cellular defense mechanisms given above are rather poorly understood at present. The importance of the transport mechanisms in cellular defense against xenobiotics has been underscored by the discoveries of drug transporters overexpressed in multidrug resistant cancer cells. These drug efflux pumps have been covered in detail in several excellent review articles (13,19,20). In this mini-review, our current understanding of the structure and function of transport mechanisms for xenobiotics and their metabolites in mammalian tissues including humans is summarized.
Ontwikkeling en evaluatie van genotyperings- technieken voor klinisch geneesmiddelenonderzoek
Titel van het onderzoek
Ontwikkeling en evaluatie van genotyperings- technieken voor klinisch geneesmiddelenonderzoek (GPR.4084).
Omschrijving van het onderzoek
Biotransformation of exogenous compunds like drugs is often catalyzed by hepatic enzymes. Some of these enzymes shows different isoforms or are easily induced or inhibited by other substances. This may lead to reduced drug activity or an increase in adverse drug reactions. CYP2D6 and CYP2C19 are members of the cytochrome P450 family and are involved in the oxidative biotransformation (phase I) of many common drugs. N-acetyltransferase (NAT-2) is involved in phase II metabolism of some drugs. All these three enzymes are subjected to genetic polymorphism causing reduced or absent enzyme activity. The incidence of this polymorphism has shown wide interracial differences. In Caucasians absence of enzyme activity (poor metabolism; PM) occurs in about 50% for NAT-2, 8% for CYP2D6 and 2-5% for CYP2C19. Enzyme activity can be assessed by phenotyping using an enzyme specific substrate (probe) or can be predicted by genotyping on the enzyme coding genes. For the above mentioned iso-enzymes deficient enzyme activity has showed Mendelian inheritance in an autosomal recessive trait.
The aim of the project is to develop, implement and evaluate genotyping techniques to predict the metabolic capacity for the CYP2D6, CYP2C19 and NAT-2.
Resultaten van het onderzoek
Assay methods have been developed for genotyping on the most common mutant alleles for CYP2D6, CYP2C19 and NAT-2. All methods are based on PCR: for CYP2C19 mutant alleles were detected by restriction enzymes for CYP2D6 and NAT-2 allele specific PCR methods are developed. At this moment methods for other genetic defects (deletion of the gene and gene amplification) are in development. All developed methods are validated and shown to be robust. For CYP2C19 and CYP2D6 excellent correlation between genotype and phenotype was observed: no false positives were found and the number of false negative was below 1%. For NAT-2 the correlation was found to be good: about 0.8% fase positive and about 9% false negative. Further sutdies are planned on optimization of of analytical methods of especially NAT-2. Both phenotyping and genotyping results in the Dutch population were evaluated and shown to be inaccordance with other data on Caucasians. The incidence of subject with impaired metabolism was found to be 2% for CYP2C19, 8% for CYP2D6 and 52% for NAT-2 in the Dutch population using our volunteer phenotype database. Genotyping in both volunteers (approximately 500 subjects) and pshychiatric patients (about 300 patients) showed similar results. Genotyping data of pshychiatric patients will be evaluated using medication history as obtained from pharmacy records. For CYP2C19 oral contraceptive (OC) related gender differences were observed: the use of OCs significatnly decreased CYP2C19 activity. The clinical significance is not clear yet and is subjected to further studies.
Projectleider
Dr. J. Wemer
Pharma Bio-Research International BV
Postbus 200
9470 AE Zuidlaren
Begeleider namens de universiteit
Prof.dr. R.A. de Zeeuw, RUG.
Status van het project
Gestart
: 01-12-1996
Einddatum
: 01-10-2000
Trefwoorden
Enzymen; Farmacie; Geneesmiddelen; Klinische evaluatie.
© STW, 1999
Laatste wijziging: 27-05-1999
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Kategori: Biyoloji