Textbook - BIOLOGICAL CHEMISTRY - Hubsky Y.I. - 2000

Chapter VI. BIOCHEMISTRY OF PHYSIOLOGICAL FUNCTIONS AND SPECIALIZED TISSUES

CHAPTER 31. BIOCHEMICAL FUNCTIONS OF THE LIVER. DETOXIFICATION PROCESSES

31.2. BIOTRANSFORMATION OF XENOBIOTICS AND ENDOGENOUS TOXINS. MICROSOMAL OXIDATION

Compounds that exert adverse, toxic effects on both individual Cells and the higher Organism as a whole include:

(1) foreign chemical compounds that enter the Human and Animal body in the course of life activity; these include Pharmaceuticals, household chemicals, cosmetics, food additives (antioxidants, preservatives, colorants), pesticides, industrial poisons, etc. These substances are termed xenobiotics (from Greek xenos — alien, bios — life) and in recent years have become the subject of detailed research in xenobiochemistry — a research field that studies the regularities of transformation (xenobiokinetics — Y.I. Hubsky, 1989) and MOLECULAR MECHANISMS OF the Physiological effects of these compounds;

(2) end-products of METABOLISM possessing potentially toxic properties (Bile Pigments and Their transformation products in the intestine; oxidation products of Steroid Hormones and catecholamines, which undergo conjugation in the Liver as a stage preceding their excretion). It should be noted that microsomal oxygenases (see below) and liver amine oxidases effect the inactivation of the physiologically active hormone compounds themselves, counteracting their excessive accumulation in the body;

(3) products of microbial degradation (putrefaction) of Proteins (Amino Acids) in the Large Intestine (substituted phenols, indoles, biogenic amines).

Class="center">Types of biotransformation reactions for xenobiotics and endogenous toxins

As already noted, the detoxification of toxic substances in hepatocytes involves converting them into a molecular form with altered biological properties (as a rule, less toxic) that can be excreted from the body via urine or bile.

These processes of biotransformation of xenobiotics and endogenous toxic compounds consist of two phases.

Phase 1 consists of oxidation-reduction and hydrolytic Reactions Catalyzed by membrane-bound Enzymes of the hepatocyte Endoplasmic reticulum ("microsomal enzymes"). As a result of Phase 1 reactions, functional groups such as -OH, -COOH, -SH, -NH₂ are formed within the biotransformation substrates; thus, these reactions (functionalization or preconjugation) lead to an increase in the polarity of the xenobiotic or endogenous substrate (steroid) molecule.

Phase 2 comprises synthetic or conjugation reactions based on the attachment of glucuronic acid, sulfuric acid, Glycine, glutamine, Glutathione, methyl, or acetyl radicals to the molecular products of Phase 1 (or to starting substrates that already possessed polar functional groups).

In some cases, the detoxification of chemical substances involves only one of the specified biotransformation phases — either the first or the second.

Microsomal oxidation reactions

The primary role among Phase 1 reactions of xenobiotic and endogenous compound biotransformation belongs to the enzyme systems of The endoplasmic reticulum membranes, which function with the participation of cytochrome P-450. Because the biochemical equivalent of liver Cell endoplasmic reticulum membranes is the microsomal fraction obtained by differential centrifugation, this type of reaction has become known in the scientific literature as "microsomal oxidation reactions," and the corresponding enzymes as "microsomal oxygenases" (A.I. Archakov, 1975).

Reactions catalyzed by these enzymes belong to the monooxygenase type, meaning they catalyze the incorporation of a single oxygen atom directly into the molecule of the substrate being oxidized (Chapter 9):

Such a reaction (oxidative hydroxylation) is fundamental in the metabolism of hydrophobic compounds within the membranes of the hepatocyte Endoplasmic reticulum and requires NADPH as an electron donor:

Since one atom of the oxygen molecule is incorporated into a Water molecule and the other into the hydroxylated substrate molecule during this process, the enzyme systems catalyzing these reactions are also referred to as "mixed-function microsomal oxygenases."

Cytochrome P-450

The enzyme systems catalyzing microsomal oxidation reactions of hydrophobic substrates are Electron Transport Chains localized in the membranes of the hepatocyte endoplasmic reticulum (as well as cells of certain other Organs that also participate in detoxification reactions). The components of these enzyme chains include an FAD-containing flavoprotein, cytochrome b₅, and the terminal monooxygenase — cytochrome P-450:

A similar cytochrome P-450-dependent Electron Transport Chain catalyzes the oxidative hydroxylation reactions of Steroids (synthesis and biotransformation) present in the Mitochondria of the adrenal cortex and Gonads.

Cytochrome P-450 is an enzyme first discovered in 1958 by American researchers D. Garfinkel and M. Klingenberg. It is a family of Hemoproteins with a Molecular Weight of approximately 50 kDa; more than 300 isoforms of cytochrome P-450 have been identified across various BIOLOGICAL OBJECTS AND Tissues, differing in substrate Specificity and Primary Structure. The physiological significance of cytochrome P-450 isoforms lies in protecting the animal body from numerous low-molecular-weight xenobiotics entering the internal environment. This system complements the immune defense system (the "second immune system"), which counteracts The entry of foreign high-molecular-weight compounds of biological origin.

The Biosynthesis of various cytochrome P-450 isoforms is encoded by multiple Gene families, designated in mammals as CYP genes (CYTOCHROME P-450). In The Human Genome, more than 100 genes are responsible for the synthesis of this hemoprotein, and their expression leads to The production of isoforms with distinct substrate specificities (CYP1, CYP2, CYP3, etc.), which are further subdivided into enzyme subfamilies. Of particular clinical interest are individual isoforms that catalyze the metabolism (biotransformation) reactions of many common pharmacological drugs (CYP1A2, CYP2C8-10, CYP2C-19, CYP2D6, CYP2E, CYP3A4).

The catalytic cycle driving the oxidative hydroxylation of substrates (SH) mediated by cytochrome P-450 consists of several partial reactions, as shown in Fig. 31.5.

Fig. 31.5. Catalytic cycle of cytochrome P-450 function.

Types of Microsomal Oxidation Reactions

The discussed scheme of redox biotransformation of xenobiotics involving cytochrome P-450 is carried out through several types of microsomal oxidation reactions, depending on the Chemical Nature of the substrate and process conditions. These include:

1) oxidative hydroxylation of aliphatic compounds:

1a — alkanes and alkenes:

(the substrates of this reaction are Hydrocarbons that may enter The Human Body during occupational activities);

1b — alkyl side chains of cyclic compounds, for example:

(such reactions occur, in particular, during the biotransformation of numerous barbiturate drugs such as hexobarbital, phenobarbital, and pentobarbital);

2) oxidative hydroxylation of cyclic compounds via a benzene-like hydroxylation pathway:

This type of biotransformation applies to monocyclic hydrocarbons (aniline, acetanilide), chlorinated cyclic hydrocarbons (such as the pesticides heptachlor, aldrin, etc.), and polycyclic hydrocarbons with carcinogenic properties (benzpyrene, dimethylbenzanthracene);

3) oxidative dealkylation, specifically:

- N-dealkylation:

(substrates for this reaction include widely used medications such as aminopyrine (amidopyrine), chlorpromazine, ephedrine, and imipramine);

- O-dealkylation:

(substrates for this reaction include, among others: the narcotic drug codeine, yielding morphine as a reaction product; phenacetin, etc.);

4) reduction reactions (proceeding without the participation of oxygen), specifically:

- reduction of nitro and azo compounds, for example:

- reductive dehalogenation, for example:

Intermediate metabolites in the reductive dehalogenation reactions of industrial poisons (carbon tetrachloride) or volatile anesthetics (halothane, methoxyflurane, etc.) can be free radicals (R˙) that trigger the activation of free-radical peroxidation reactions of Biomolecules (Lipids, proteins, Nucleic Acids), leading to severe necrodystrophic lesions of the liver, Kidneys, and myocardium.

Induction of Microsomal Monooxygenases

A biologically crucial feature of cytochrome P-450 is its capacity for induced synthesis (enzyme induction) upon the influx into the animal organism of low-molecular-weight hydrophobic compounds, which serve as substrates for microsomal oxidation. Currently, several hundred chemical substances are known to act as cytochrome P-450 Inducers; the administration of these compounds into the body triggers the activation of specific genes responsible for the synthesis of certain hemoprotein isoforms that are specific to the given substrate or a group of chemically related substrates.

The physiological Significance of the cytochrome P-450 induction phenomenon lies in enhancing the capacity of liver cells for the biotransformation of foreign chemical compounds, i.e., significantly increasing its detoxification function. This same mechanism is responsible for the reduction or loss of specific pharmacological effects of many drugs upon their prolonged administration (The Development of "tolerance" to physiologically active compounds). A widely known inducer of microsomal oxidation used in medical practice is the sedative and anticonvulsant agent Phenobarbital (5-phenyl-5-ethylbarbituric acid).

Conjugation Reactions in Hepatocytes

Conjugation reactions resulting in The formation of "paired" compounds represent a pathway for the detoxification of most xenobiotics containing -OH, -COOH, -NH₂, and -SH functional groups (or those that acquire them during phase 1 biotransformation reactions). This same mechanism is utilized to generate molecular forms destined for excretion from the body from such endogenous substrates as the bile pigment bilirubin, products of bacterial breakdown of cyclic amino acids in the intestine (phenol, cresol, indoxyl), steroid hormones and their hydroxylation products, as well as monoamine oxidase breakdown products of catecholamines, serotonin, and other biogenic amines.

The most common conjugation reactions include:

1. Glucuronidation reactions (the primary type of conjugation in humans and animals for both xenobiotics and endogenous substrates), which involve the active form of glucuronate — UDP-glucuronic acid (UDPGA). UDPGA is formed via The oxidation of UDP-glucose (UDPG) by an NAD-dependent UDPG dehydrogenase:

The enzyme that catalyzes glucuronidation reactions, UDP-glucuronosyltransferase, is localized in the membranes of the endoplasmic reticulum of hepatocytes and certain other organs and tissues involved in detoxification reactions (the gastrointestinal tract, kidneys, and Skin).

Depending on the chemical Nature of the substrate, O-, N-, and S-glucuronidation reactions are distinguished, for example:

2. Sulfonation reactions, in which the donor of sulfate radicals is the biologically active form of sulfuric acid — 3'-phosphoadenosine-5'-phosphosulfate (PAPS).

An example of a sulfonation reaction is the formation in the liver of a conjugate based on indoxyl, a product of the microbial breakdown of The amino acid L-Tryptophan in the large intestine. Free indole is produced through the action of microbial enzymes on L-tryptophan (one of the reactions of "protein putrefaction in the intestine"); subsequent oxidation of indole to indoxyl provides the substrate for interaction with PAPS. The resulting indoxyl sulfate is excreted in the urine as a potassium salt known as "animal indican":

The level of animal indican excretion is considered in clinical practice as an indicator of The activity of protein putrefaction in the gut and the functional state of the liver.

3. Methylation and Acetylation reactions are a widespread type of conjugation involving both xenobiotics and endogenous substrates; these reactions utilize S-adenosylmethionine (O-methylation) and acetyl-CoA (N-acetylation):

An important example of N-acetylation is the acetylation of sulfonamides, which are common chemotherapeutic agents; the intensity of this reaction serves as an indicator of drug biotransformation activity in the human body.

4. Glycine conjugation reactions; a clinically important example of this reaction is the formation of hippuric acid through the interaction of endogenous glycine with benzoic acid administered into the body:

Determining the intensity of the reaction (The amount of hippuric acid excreted in the urine following an oral administration of a standard dose of benzoate) underlies the Assessment of the liver's antitoxic function (Quick's test).