BIOCHEMISTRY: A TEXTBOOK FOR UNIVERSITIES - E. S. Severin - 2004

SECTION 12. DETOXIFICATION OF TOXIC SUBSTANCES IN THE BODY

The Liver is the largest gland in the digestive tract. It acts as the body's biochemical laboratory and plays a vital role in protein, carbohydrate, and Lipid METABOLISM (see below). The liver synthesizes essential Blood Plasma Proteins, including albumin, fibrinogen, prothrombin, ceruloplasmin, transferrin, and angiotensinogen. Through these proteins, the liver mediates such critical processes as maintaining oncotic pressure, regulating blood pressure and circulating blood volume, blood clotting, and iron metabolism.

A crucial function of the liver is its detoxification (or barrier) role, which is essential for survival. The liver neutralizes substances such as bilirubin and products of intestinal Amino Acid Catabolism, while also inactivating medications, exogenous toxins, and NH₃—a Nitrogen metabolism byproduct that enzymatic reactions convert into non-toxic urea, Hormones, and biogenic amines.

Substances that enter the body from the environment and are neither used to build Tissues nor utilized as Energy Sources are referred to as foreign substances, or xenobiotics. These compounds can enter the body via food, through the Skin, or by inhalation.

Xenobiotics are classified into two groups:

✵ products of human industrial, agricultural, and transport activities;

✵ household chemicals, including detergents, insecticides, and cosmetics.

Hydrophilic xenobiotics are excreted unchanged in the urine, whereas hydrophobic compounds can accumulate in tissues by binding to proteins or forming complexes with Cell Membrane Lipids. Over time, the cellular accumulation of these foreign substances disrupts normal function. To eliminate such unwanted compounds, evolutionary mechanisms for detoxification and clearance have developed.

Main Functions of the Liver

Carbohydrate Metabolism

Gluconeogenesis

Glycogen Synthesis and Breakdown

Lipid and Derivative Metabolism

Synthesis of Fatty acids and fats from CARBOHYDRATES

Synthesis and excretion of Cholesterol

Lipoprotein assembly

Ketogenesis

Synthesis of Bile acids and 25-hydroxylation of Vitamins

Protein metabolism

Synthesis of Blood Plasma proteins (including certain clotting factors)

Urea synthesis (ammonia detoxification)

Hormone Metabolism

Metabolism and excretion of Steroid Hormones

Metabolism of polypeptide hormones

Bilirubin Metabolism and Excretion

Storage

glycogen

vitamin A

vitamin B₁₂

iron

Drugs and Foreign Compounds

Metabolism and excretion

I. Mechanisms of Xenobiotic Detoxification

The detoxification of most xenobiotics occurs via chemical modification and proceeds in 2 phases (Fig. 12-1). As a result of this series of reactions, xenobiotics become more hydrophilic and are excreted in the urine. Substances that are more hydrophobic or have a high molecular weight (>300 kDa) are more frequently eliminated into the intestine with bile and subsequently removed in the feces.

Class="center">Fig. 12-1. Metabolism and elimination of xenobiotics from the body. RH — xenobiotic; K — group used in conjugation (Glutathione, glucuronyl, etc.); M — molecular mass. Of the numerous cytochrome P₄₅₀-dependent reactions, only one is shown in the figure — the scheme of xenobiotic hydroxylation. During the first phase, a polar OH^- group is introduced into The Structure of the RH substance. This is followed by a conjugation reaction; depending on solubility and molecular weight, the conjugate is eliminated either by the Kidneys or in the feces.

The detoxification system includes A wide variety of Enzymes, under the action of which virtually any xenobiotic can be modified.

Microsomal enzymes catalyze C-hydroxylation, N-hydroxylation, O-, N-, and S-dealkylation, Oxidative Deamination, sulfoxidation, and epoxidation reactions (Table 12-1).

Table 12-1. Possible modifications of xenobiotics in The first phase of detoxification

The microsomal oxidation (monooxygenase oxidation) system is localized in the ER membranes of virtually all tissues. In experiments on Cell Fractionation, the ER membrane breaks down into fragments, each forming a closed vesicle called a microsome—hence the name, microsomal oxidation. This system provides the first phase of detoxification for most hydrophobic substances. Enzymes of the kidneys, Lungs, skin, and GI tract can take part in xenobiotic metabolism, but they are most active in the liver. The group of microsomal enzymes includes specific oxidases, various Hydrolases, and conjugation enzymes.

The second phase consists of conjugation reactions, as a result of which the foreign substance, modified by the ER enzyme systems, binds to endogenous substrates—glucuronic acid, sulfuric acid, Glycine, or glutathione. The resulting conjugate is then eliminated from the body.

A. Microsomal Oxidation

Microsomal oxidases are enzymes localized in the membranes of the smooth ER, functioning in conjunction with two extramitochondrial Electron Transport Chains (ETCs). Enzymes that catalyze the reduction of one atom of an O₂ molecule to form Water and the incorporation of the other oxygen atom into the substance being oxidized are called microsomal mixed-function oxidases or microsomal Monooxygenases. Oxidation involving monooxygenases is usually studied using microsomal preparations.

1. Main Enzymes of Microsomal Electron Transport Chains

The microsomal system does not contain protein components soluble in the Cytosol; all enzymes are Membrane Proteins whose active centers are localized on the cytoplasmic surface of the ER. The system includes several proteins that make up electron transport chains (ETCs). There are two such chains in the ER: the first consists of two enzymes—NADPH-P₄₅₀ reductase and cytochrome P₄₅₀; the second includes NADH-cytochrome b₅ reductase, cytochrome b₅, and another enzyme, stearoyl-CoA desaturase.

Electron Transport Chain — NADPH-P₄₅₀ reductase — cytochrome P₄₅₀. In most cases, the electron (e) donor for this chain is NADPH, which is oxidized by NADPH-P₄₅₀ reductase. The enzyme contains two Coenzymes as a prosthetic group—flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Protons and electrons from NADPH are sequentially transferred to the coenzymes of NADPH-P₄₅₀ reductase. Reduced FMN (FMNH₂) is oxidized by cytochrome P₄₅₀ (see scheme below).

Cytochrome P₄₅₀ is a hemoprotein containing a heme prosthetic group and binding sites for oxygen and a substrate (xenobiotic). The name cytochrome P₄₅₀ indicates that the absorption maximum of the cytochrome P₄₅₀ complex lies in the 450 nm region.

The oxidizable substrate (electron donor) for NADH-cytochrome b₅ reductase is NADH (see the diagram above). Protons and electrons from NADH are transferred to the FAD coenzyme of the reductase, and the next electron acceptor is Fe³⁺ of cytochrome b₅. In some cases, cytochrome b₅ can serve as an electron (e) donor for cytochrome P₄₅₀ or for stearoyl-CoA desaturase, which catalyzes The formation of double bonds in fatty acids by transferring electrons to oxygen with the formation of water (Fig. 12-2).

Fig. 12-2. Electron transport chains of the ER. RH is the cytochrome P₄₅₀ substrate; arrows indicate Electron transfer reactions. In one system, NADPH is oxidized by NADPH-cytochrome P₄₅₀ reductase, which then transfers electrons to an entire family of Cytochromes P₄₅₀. The second system involves The oxidation of NADH by cytochrome b₅ reductase, with electrons transferred to cytochrome b₅; the reduced form of cytochrome b₅ is oxidized by stearoyl-CoA desaturase, which transfers electrons to O₂.

NADH-cytochrome b₅ reductase is a two-domain protein. The globular cytosolic domain binds the prosthetic group, the FAD coenzyme, while a single hydrophobic "tail" anchors the protein in the membrane.

Cytochrome b₅ is a heme-containing protein that features a domain localized On the surface of the ER membrane and a short helical domain anchored in The Lipid Bilayer.

NADH-cytochrome b₅ reductase and cytochrome b₅, being anchored proteins, are not strictly fixed to specific sites of the ER membrane and can therefore change their localization.

2. Functioning of cytochrome P₄₅₀

It is known that molecular oxygen in its triplet state is inert and unable to interact with Organic compounds. To make oxygen reactive, it must be converted into singlet oxygen using enzymatic reduction systems. Among these is the monooxygenase system containing cytochrome P₄₅₀. The binding of the lipophilic substance RH and an oxygen molecule to the active center of cytochrome P₄₅₀ increases the oxidative activity of the enzyme. One oxygen atom accepts 2 e and is converted into the O^2- form. The electron donor is NADPH, which is oxidized by NADPH-cytochrome P₄₅₀ reductase. O^2- interacts with protons: O^2- + 2H⁺ —> H₂O, forming water. The second atom of the oxygen molecule is incorporated into the RH substrate, forming the R-OH hydroxyl group of the substance (Fig. 12-3).

Fig. 12-3. Electron transport during monooxygenase oxidation involving P₄₅₀. Binding (1) of substance RH in the active center of cytochrome P₄₅₀ activates the reduction of iron in the heme — the first electron is attached (2). The change in iron valence increases the affinity of the P₄₅₀-Fe²⁺•RH complex for the oxygen molecule (3). The appearance of an O₂ molecule in the binding center of cytochrome P₄₅₀ accelerates The addition of the second electron and the Formation of the P₄₅₀-Fe²⁺O₂--RH complex (4). At the next stage (5), Fe²⁺ is oxidized, and the second electron is attached to the oxygen molecule of P₄₅₀-Fe³⁺O₂^2-. The reduced oxygen atom (O^2-) binds 2 protons, forming 1 water molecule. The second oxygen atom goes into forming the OH group (6). The modified substance R-OH separates from the enzyme (7).

The overall equation for the hydroxylation reaction of substance RH by microsomal oxidation enzymes:

RH + O₂ + NADPH + H⁺ —> ROH + H₂O + NADP⁺.

The substrates of P₄₅₀ can be many hydrophobic substances of both exogenous (drugs, xenobiotics) and endogenous (Steroids, fatty acids, etc.) origin.

Thus, as a result of the first phase of detoxification involving cytochrome P₄₅₀, substances are modified with the formation of functional groups that increase the solubility of the hydrophobic compound. This modification may result in the loss of the molecule's biological activity or even the formation of a more active compound than the precursor from which it was formed.

3. Properties of the microsomal oxidation system

The MOST IMPORTANT PROPERTIES of microsomal oxidation enzymes are broad substrate Specificity, which allows the detoxification of structurally diverse substances, and The regulation of activity via an induction mechanism.

Broad substrate specificity. P₄₅₀ isoforms

To date, about 150 cytochrome P₄₅₀ genes encoding various enzyme isoforms have been described. Each P₄₅₀ isoform has many substrates. These substrates can be either endogenous lipophilic substances, whose modification is part of the normal metabolic pathway of these compounds, or hydrophobic xenobiotics, including drugs. Certain isoforms of cytochrome P₄₅₀ are involved in the metabolism of low-molecular-weight compounds such as ethanol and acetone.

Regulation of the activity of the microsomal oxidation system

The regulation of The activity of the microsomal system is carried out at the level of Transcription or post-transcriptional modifications. Induction of synthesis makes it possible to increase The amount of enzymes in response to the Introduction or formation in the body of substances whose elimination is impossible without the participation of the microsomal oxidation system.

Currently, more than 250 chemical compounds that cause the induction of microsomal enzymes have been described. These Inducers include barbiturates, polycyclic aromatic Hydrocarbons, alcohols, ketones, and certain steroids. Despite The Diversity of their chemical structures, all inducers share A number of common features; they are classified as lipophilic compounds and serve as substrates for cytochrome P₄₅₀.

B. Conjugation — the second phase of substance detoxification

The second phase of substance detoxification consists of conjugation reactions, during which other molecules or groups of endogenous origin are attached to the functional groups formed in The First stage, increasing the hydrophilicity and decreasing the toxicity of xenobiotics (Table 12-2).

Table 12-2. Main enzymes and metabolites involved in conjugation

1. Involvement of transferases in conjugation reactions

All enzymes functioning In the second phase of xenobiotic detoxification belong to the transferase class and are characterized by broad substrate specificity.

UDP-glucuronosyltransferases

Localized primarily in The Endoplasmic reticulum, uridine diphosphate (UDP)-glucuronosyltransferases attach a glucuronic acid residue to molecules produced during microsomal oxidation (Fig. 12-4).

Fig. 12-4. Uridine diphosphoglucuronic acid (UDP-C₆H₉O₆).

The general reaction involving UDP-glucuronosyltransferase can be written as follows:

ROH + УДФ-С₆Н₉О₆ = RO-C₆H₉O₆ + УДФ.

Sulfotransferases

Cytoplasmic sulfotransferases catalyze conjugation reactions in which a sulfuric acid residue (-SO₃H) from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is transferred to phenols, alcohols, or Amino Acids (Fig. 12-5).

Fig. 12-5. 3'-Phosphoadenosine-5'-phosphosulfate (PAPS-SO₃H).

The general reaction involving sulfotransferase can be written as follows:

ROH + ФАФ-SO3H = RO-SO₃H + ФАФ.

Sulfotransferases and UDP-glucuronosyltransferases play a key role in xenobiotic detoxification, as well as the inactivation of drugs and endogenous biologically active compounds.

Glutathione transferases

Glutathione transferases (GSTs) hold a special place among enzymes involved in xenobiotic detoxification and the inactivation of normal metabolites and drugs. GSTs are present in all tissues and play a vital role in inactivating endogenous metabolites, including certain steroid hormones, Prostaglandins, bilirubin, bile acids, and Lipid Peroxidation products.

A multitude of GST isoforms with diverse substrate specificity are known. While predominantly localized in the cytosol, Various Forms of these enzymes are also found in The Nucleus and Mitochondria. GST activity requires glutathione (GSH) (Fig. 12-6).

Fig. 12-6. Glutathione (GSH).

Glutathione is a tripeptide Glu-Cys-Gly (in which the glutamic acid residue is linked to Cysteine via its side-chain carboxyl group).

GSTs exhibit broad substrate specificity, acting on over 3,000 different compounds. They bind a wide range of hydrophobic molecules to inactivate them, though only those possessing a polar group undergo chemical modification via glutathione. In other words, the substrates must feature both an electrophilic center (such as an -OH group) and hydrophobic regions. Detoxification—meaning the chemical modification of xenobiotics mediated by GSTs—can occur via three distinct mechanisms:

✵ via conjugation of substrate R with glutathione (GSH):

R + GSH —> GSRH,

✵ as a result of nucleophilic substitution:

RX + GSH —> GSR + НХ,

✵ reduction of organic peroxides to alcohols:

R-HC-O-OH + 2 GSH —> R-HC-OH + GSSG + Н₂О.

In the reaction: ООН is the hydroperoxide group, and GSSG is oxidized glutathione.

The GST- and glutathione-mediated detoxification system plays a unique role in building organismal resistance to a wide variety of harmful factors and serves as The Cell's most vital defense mechanism. During the biotransformation of certain xenobiotics, GST action yields thioethers (RSG conjugates) which are subsequently converted into mercaptans, some of which prove to be toxic. However, GSH conjugates with the majority of xenobiotics are less reactive and more hydrophilic than the parent compounds, making them less toxic and easier for the body to excrete (Fig. 12-7).

Fig. 12-7. Detoxification of 1-chloro-2,4-dinitrobenzene involving glutathione.

Through their hydrophobic sites, GSTs can non-covalently bind a vast array of lipophilic compounds (physical detoxification), preventing them from embedding into the lipid bilayer of membranes and disrupting cellular functions. For this reason, GSTs are sometimes referred to as intracellular albumin.

GSTs can covalently bind xenobiotics that act as strong electrolytes. The attachment of such substances is a form of "suicide" for GST, yet it provides an additional protective mechanism for the cell.

Acetyltransferases, Methyltransferases

Acetyltransferases catalyze conjugation reactions—The transfer of an acetyl group from acetyl-CoA to the nitrogen of the -SO₂NH₂ group, as seen, for example, in sulfonamides. Membrane-bound and cytoplasmic methyltransferases, utilizing SAM, methylate the -Р = O, -NH₂, and SH groups of xenobiotics.

2. Role of Epoxide Hydrolases in Diol Formation

Certain Other Enzymes also participate in the second phase of detoxification (conjugation reactions). Epoxide hydrolase (epoxide hydratase) adds water to the epoxides of benzene, benzpyrene, and other polycyclic hydrocarbons generated during the first phase of detoxification, converting them into diols (Fig. 12-8). Epoxides produced via microsomal oxidation are known carcinogens. They possess high chemical reactivity and can participate in non-enzymatic alkylation reactions involving DNA, RNA, and proteins (see Section 16). Chemical modifications of these molecules can drive the malignant Transformation of a normal cell into a tumor cell.

Fig. 12-8. Detoxification of benzanthracene. Е₁ — enzyme of the microsomal system; Е₂ — epoxide hydratase.

C. Putrefaction of amino acids in the intestine. Detoxification and excretion of putrefactive products from the body

Amino acids that are not absorbed by intestinal Cells are utilized as nutrients by the microflora of the Large Intestine. Bacterial enzymes break down amino acids, converting them into amines, phenols, indole, skatole, hydrogen sulfide, and Other Compounds toxic to the Organism. This process is sometimes referred to as intestinal protein putrefaction. At the core of putrefaction lie AMINO ACID DECARBOXYLATION and deamination reactions.

Formation and Detoxification of p-Cresol and Phenol

Driven by bacterial enzymes, phenol and cresol can be formed from The amino acid Tyrosine through microbial breakdown of its side chains (Fig. 12-9).

Fig. 12-9. Bacterial catabolism of tyrosine. Е — bacterial enzymes.

The absorbed products travel via the portal vein to the liver, where the detoxification of phenol and cresol can occur through conjugation with a sulfate group (PAPS) or with glucuronic acid as part of UDP-glucuronate. The conjugation reactions of phenol and cresol with PAPS are catalyzed by the enzyme sulfotransferase (Fig. 12-10).

Fig. 12-10. Conjugation of phenol and cresol with PAPS. Е — sulfotransferase.

The conjugation of glucuronic acids with phenol and cresol proceeds with the participation of the enzyme UDP-glucuronosyltransferase (Fig. 12-11). The conjugation products are highly water-soluble and are excreted in the urine via the kidneys. An elevated level of glucuronic acid conjugates with phenol and cresol in the urine indicates an increase in protein putrefaction products within the intestine.

Fig. 12-11. Involvement of UDP-glucuronosyltransferase in the detoxification of cresol and phenol. E — UDP-glucuronosyltransferase.

Formation and Detoxification of Indole and Skatole

Microorganisms in the intestine produce indole and skatole from the amino acid Tryptophan. Bacteria break down the side chain of tryptophan while leaving its ring structure intact.

Indole is formed as a result of the Cleavage of the side chain by bacteria, potentially in the form of Serine or Alanine (Fig. 12-12).

Fig. 12-12. Bacterial catabolism of tryptophan. E — bacterial enzymes.

Skatole and indole are detoxified in the liver in two stages. First, they acquire a hydroxyl group through microsomal oxidation. Thus, indole is converted into indoxyl and then undergoes conjugation with PAPS to form indoxyl sulfuric acid, the potassium salt of which is known as animal indican (Fig. 12-13).

Fig. 12-13. Participation of sulfotransferase in indole detoxification. E — sulfotransferase.

Detoxification of Benzoic Acid

The synthesis of hippuric acid from benzoic acid and glycine occurs predominantly in the liver in humans and most animals (Fig. 12-14). The rate of this reaction reflects the functional state of the liver.

Fig. 12-14. Formation of hippuric acid from benzoic acid and glycine. E — glycine transferase.

In clinical practice, the rate of hippuric acid formation and excretion is determined following the administration of the xenobiotic benzoic acid (sodium benzoate) to the body—known as the Quick's test.

G. Binding, Transport, and Excretion of Xenobiotics

In blood plasma, numerous both endogenous and exogenous lipophilic substances are transported by albumin and other proteins.

Albumin — the primary blood plasma protein that binds various hydrophobic substances. It can function as a carrier protein for bilirubin, xenobiotics, and pharmaceutical drugs.

In addition to albumins, xenobiotics can be transported through the bloodstream as part of Lipoproteins, as well as in a complex with acidic α₁-glycoprotein. A distinct feature of this glycoprotein is that it is an inducible protein involved in the body's response to stress-induced changes, such as myocardial infarction or inflammatory processes; its concentration in plasma increases alongside other proteins. By binding xenobiotics, acidic α₁-glycoprotein inactivates them and transports them to the liver, where the protein complex dissociates, and the foreign substances are detoxified and eliminated from the body.

Role of P-glycoprotein in Xenobiotic Excretion

A crucial mechanism for the cellular efflux of hydrophobic xenobiotics is the function of P-glycoprotein (a transport ATPase). P-glycoprotein is a phosphoglycoprotein with a molecular mass of 170 kDa, present in The Plasma Membrane of cells in many tissues, particularly the kidneys and intestines. The polypeptide chain of this protein contains 1280 amino acid residues, forming 12 transmembrane domains and two ATP-binding sites (Fig. 12-15). Under normal conditions, its function is to excrete chloride ions and hydrophobic toxic compounds from cells.

Fig. 12-15. Structure of P-glycoprotein. P-glycoprotein is an integral protein featuring 12 transmembrane domains that span the lipid bilayer of the cytoplasmic membrane. The N- and C-termini of the protein face the cytosol. The extracellular regions of P-glycoprotein are glycosylated. The region between the sixth and seventh domains contains sites for ATP binding and autophosphorylation.

When a hydrophobic substance (e.g., an antitumor drug) enters a cell, it is actively pumped out by P-glycoprotein utilizing energy (Fig. 12-16). A reduction in the intracellular drug concentration diminishes the efficacy of Cancer Chemotherapy.

Fig. 12-16. MECHANISM OF ACTION of P-glycoprotein. The shaded oval represents an antitumor drug (a hydrophobic compound).

D. Induction of defense systems

Many enzymes involved in phase I and phase II detoxification are inducible proteins. As early as antiquity, King Mithridates knew that Acute Poisoning could be avoided by systematically taking small doses of poison. The "Mithridates effect" is based on the induction of specific defense systems (Table 12-3).

Table 12-3. Induction of defense systems against xenobiotics

Cytochrome P₄₅₀ is more abundant (20%) in the membranes of the liver endoplasmic reticulum (ER) than other membrane-bound enzymes. The drug phenobarbital activates the synthesis of cytochrome P₄₅₀, UDP-glucuronosyltransferase, and epoxide hydrolase. For example, in animals treated with phenobarbital as an inducer, the surface area of ER membranes increases, reaching up to 90% of all Cellular Membrane Structures, which consequently leads to an elevation in the levels of enzymes involved in detoxifying xenobiotics or endogenous toxic substances.

During chemotherapy for malignant tumors, the initial efficacy of a drug often gradually declines. Furthermore, multidrug resistance develops, meaning resistance not only to the therapeutic agent in question but also to a wide range of other drugs. This occurs because antitumor drugs induce the synthesis of P-glycoprotein, glutathione transferase, and glutathione. The administration of agents that inhibit or activate the synthesis of P-glycoprotein, as well as enzymes involved in glutathione synthesis, enhances the efficacy of chemotherapy.

Metals act as inducers for the Synthesis of Glutathione and the low-molecular-weight protein metallothionein, both of which contain SH-groups capable of binding these metals. As a result, cellular resistance to toxins and drugs increases.

Elevated levels of glutathione transferases enhance the body's ability to adapt to increasing environmental pollution. This enzyme induction explains the absence of an anticarcinogenic effect when certain pharmaceutical drugs are administered. In addition, inducers of glutathione transferase synthesis include normal metabolites such as Sex Hormones, iodothyronines, and cortisol. Catecholamines phosphorylate glutathione transferase via the adenylate cyclase system, thereby increasing its activity.

A number of substances, including certain drugs (e.g., heavy metals, polyphenols, S-alkylglutathiones, and specific herbicides), inhibit glutathione transferase.