The amount of free plasma toxicant is a function of the toxicant's absorption, distribution, and elimination (Figure 9.1). In Chapter 8, a variety of factors that govern the absorption of toxicants in the gastrointestinal (GI) tract were discussed. This chapter focuses on factors that influence distribution and elimination of toxicants.
Toxicants and other foreign compounds (xenobiotics) undergo metabolic transformation in the body. In many situations, the rate of metabolism is the primary determinant of the substance for both duration and intensity of action. Compounds that are deactivated by metabolism tend to be more active and linger in the body when the metabolic rate is slow compared with compounds that are rapidly metabolized. Any substance if not eliminated could eventually reach a toxic level. A feature characteristic of most toxicants is that the subsequent metabolic products are more polar than the original compound. Thus, metabolism of toxicants decreases biological activity and increases polarity or reduces lipid solubility. The implication of increased toxicant polarity is that such compounds are more likely to be excreted by renal or biliary processes. On the other hand, compounds with high lipid-water partition coefficients pass effortlessly across membranes and diffuse back freely from the tubular urine through the renal tubular cells into the plasma, and such compounds thus tend to have a low renal clearance and a long endurance in the body. But if a toxicant is metabolized to a more water-soluble compound or one with a lower partition coefficient, the compound's tubular reabsorption is greatly reduced. The terms metabolism and biotransformation are often used synonymously, particularly when applied to xenobiotic compounds such as drugs. Sometimes the connotation associated with metabolism is to describe the total fate of a compound, including absorption, distribution, biotransformation, and elimination. But more often, metabolism is used to mean biotransformation, because metabolites are the products of xenobiotic biotransformation. Metabolism has evolutionary significance because if humans had not evolved with such capabilities, compounds such as pentobarbital would be pharmacologically active for hundreds of years. Toxicant-metabolizing systems have developed as adaptations to terrestrial life. It is likely
Ingestion of toxicant
Plasma protein bound toxicant
Free plasma toxicant
Site of action
that toxicants have been metabolized by living systems since the first cells were formed in the primordial slime. Marine organisms often lack the major toxicant-metabolizing systems found in mammals; however, they excrete lipid-soluble compounds directly into the surrounding water across the gill membranes. Conversion of a methyl group to a carboxyl group could reduce the biological half-life of a compound from many hours to a few minutes. Enzymatic biotransformation of xenobiotics facilitates the elimination of such compounds from the body. Without such enzymes, lipophilic xenobiotics would stay in the body for a prolonged duration following exposure. These compounds would accumulate in the body with repeated exposures, reaching toxic levels. Animals, especially herbivores, which consume a wide variety and amount of plant material laden with unusual secondary products, need to be able to biotransfer xenobiotics.
Some compounds that undergo metabolism become more toxic, e.g., carbon tetrachloride or benzo(a)pyrene. Decreased lipid solubility of a toxicant does not necessarily mean increased water solubility, e.g., acetylsulfathiazole transformed from sulfathiazole. The reduced water solubility of acetylsulfathiazole results in a compound with serious toxicity, which results from precipitation in renal tubules.
Overall, two scenarios can be mediated by biotransformation reactions: (1) xenobiotics (toxic) converted to intermediates (toxic or nontoxic) converted to products (nontoxic), or (2) xenobiotics (nontoxic) converted to intermediates (toxic) converted to products (nontoxic). The main site of toxicant metabolism is the liver, but other tissues may play an active part too. Table 9.1 summarizes the relative tissue distribution of toxicant metabolism. The hepatopancreas and the fat bodies are major organs involved in biotransformation in lobsters and insects, respectively.
Thus, the liver is the richest source of enzymes for metabolizing toxicants, but there is ample evidence that enzyme systems are ubiquitous, which can be rationalized on the basis of the importance of such enzymes in detoxifying various compounds. Intestinal microflora plays an important role in the biotransformation of
Organs, Tissues, and Cells Involved in Toxicant Metabolism and Their Percent Distribution
Intestine (Gut flora)
xenobiotics because of the impact of evolution on the survival of the species. Ruminants may be able to cope with relatively high intakes of fungal toxins, e.g., aflatoxin, because of their breakdown by the rumen bacteria.
Within the liver and other organs, the microsomes or endoplasmic reticulum and the cytosol or soluble fractions of the cytoplasm are the principal sites of xenobiotic metabolism. The lipid-rich endoplasmic reticulum is a strong attractant for lipophilic xenobiotics. To a lesser extent, metabolism occurs in the mitochondria, nuclei, and other subcellular organelles.
Although some extrahepatic sites contain relatively high levels of enzymes systems for xenobiotic metabolism, their size minimizes their overall contribution to the metabolism of such compounds. Tissue differences in their capacity to metabolize toxicants can have important toxicological consequences, such as in tissue-specific chemical injury.
One of the unique facets of toxicant metabolism is that even though structures of these potentially toxic products, be they natural or synthetic, are so tremendously varied, the body seems to have evolved detoxifying processes that can cope with almost any of the many different compounds. Animals possess enzymes that can metabolize drugs, pesticides, secondary plant metabolites, and synthetic compounds as defense mechanisms, which are likely because of evolution in response to selective pressures for protection against many naturally occurring toxic products. There are two categories of animal enzyme systems: (1) those for the transformation of normal endogenous chemicals in tissue, such as nutrients and metabolic by-products of nutrients; and (2) those that alter structure of many foreign compounds and essentially have no established normal endogenous substrates. The first category of enzyme systems has been studied in detail for their general biochemistry. These are enzymes of intermediary metabolism and are characterized by high turnover numbers and enormous rate enhancements over uncatalyzed reactions. Also, these reactions demand a precise chemical fit between substrate and enzyme (lock and key model),
Catalytic Specificity and Efficiency of Enzymes
Intermediary Metabolism Xenobiotic Metabolism
High efficiency (high turnover) Broad substrate specificity
High substrate specificity Low catalytic efficiency and the fit dictates stringent substrate specificity. Typically, enzyme systems involved with intermediary metabolism have low KM values or are tightly bound and high Kcal values or high efficiency and rapid turnover. Examples include cholinesterase and the hydrolysis of acetylcholine, and monoamine oxidase acting on epinephrine, tyramine, and short-chain amines.
The genetic capacity and thus the metabolic ability of an organism limits its ability to produce numerous distinct enzymes to detoxify all the foreign compounds that it may come in contact with. Thus, enzymes systems involved in xenobiotic metabolism exhibit broad substrate specificity and low catalytic efficiency. Such enzyme systems represent a metabolic compromise. To offset specificity or the cost of reduced precision of substrate binding to enzymes, these enzymes can metabolize diverse substrates. Table 9.2 lists the differences in these enzyme systems.
The ultimate goal of xenobiotic metabolism is to increase the hydrophilic property of the compound. Such metabolism is delineated in Figure 9.2, showing the integration of phase I and phase II biotransformation reactions. When an organism deals with a toxicant or an inactive compound that might be converted to a toxicant, biotransformation usually proceeds by a two-phase process. The aim of the enzymatic reactions responsible for biotransformation is to form hydrophilic products that are less toxic and can be excreted by the body.
These biotransformation reactions involve oxidation, reduction, and hydrolysis of foreign compounds. Table 9.3 to Table 9.5 give a detailed description of phase I reactions.
In addition, the hydration of epoxides and dehydrohalogenations can be considered as phase I reactions. Many foreign compounds that enter the body are lipophilic, because of which they can easily penetrate lipid membranes and be transported by lipoproteins in body fluids. Therefore, the essence of such reactions is to introduce or expose a functional group that decreases the lipid solubility of the compound. They promote insertion, addition, or exposure of functional groups on the structure of the lipophilic compound to form electrophilic compounds (Figure 9.1). Such action gives the compound a polar group, making it a suitable substrate for phase II reactions. Phase II reactions alter compounds by combining them with endogenous substrates to produce a water-soluble conjugated products that can readily be excreted.
The predominant biotransformation enzyme systems in phase I reactions are cytochrome P450 and mixed-function amine oxidases. Other phase I enzymes
Ingestion of Toxic Foreign Compound
Phase I Reactions Oxidation Reduction Hydrolysis
Phase II Reactions Conjugation
FIGURE 9.2 Metabolism of a toxicant.
TABLE 9.3 Oxidative Reactions
Hydroxylation Oxidative dealkylation
Direct insertion of a hydroxyl functional group Cleavage of alkyl groups and aromatic groups from amines or ethers Loss of amino groups Oxidation of the nitrogen
TABLE 9.4 Reductive Reactions
Azo reduction Nitro reduction Keto reduction
Compounds possessing ester linkages (amides and esters) Formation of alcohol and acids include flavin-containing monooxygenase (FMO), prostaglandin synthetase (PGS; carries out cooxidation), molybdenum hydroxylases, alcohol dehydrogenase, aldehyde dehydrogenase, esterases and amidases, epoxide hydrolase, DDT-dehydrocho-rinase, and glutathione reductase. Cytochrome P450 and the flavin-containing monooxygenase are located in the microsome.
Cytochrome P450 (CYP) Enzymes
Cytochrome P450 enzymes of microsomal enzyme systems are embedded in phos-pholipid, which is important because the phospholipid facilitates interaction between the enzymes NADPH-cytochrome P450 reductase and cytochrome P450 (Figure 9.3). Cytochrome P450 derives its name from the fact that reduced cytochrome P450 (Fe2+) forms a ligand with carbon monoxide (CO), which can be observed at a spectral absorption of 450 nm. It has been determined that cytochrome P450 is more than one enzyme and the location of the gene on a particular chromosome has been determined for cytochrome P450 isoenzymes. A system of nomenclature based on such findings has been used since 1987. Since the most recent update, P450 human genes are designated as CYP. Each designation is followed by an arabic numeral designating the individual gene, followed by a letter designating the subfamily, and finally an arabic numeral designating the individual gene.
Cytochrome P450 microsomal monooxygenase reactions are similar, but the enzyme classes differ in their substrates and products. Hence, these activities are classified on the basis of chemical reactions. Classes can overlap and the same substrate may undergo more than one oxidative reaction.
• Aliphatic and aromatic hydroxylations (Figure 9.4). Alkyl side chains of aromatic compounds are easily oxidized to alcohols. Epoxides of aromatic rings are intermediates in aromatic hydroxylations. Oxides of polycyclic hydrocarbons (arene oxides) are known to be involved in carcinogenesis. The proximate carcinogens derived from the metabolic activation of benzo(a)pyrene are isomers of benzo(a) 7,8-diol-9,10-epoxide.
NADPH-cytochrome P450 reductase
Fe2+ OOH RH
FIGURE 9.3 The cytochrome P450 enzyme system.
• Dealkylation. The reaction can involve O-, N-, and S-dealkylation. An example of O-dealkylation is the demethylation of p-nitroanisole. Many drugs and insecticides undergo N-dealkylation (Figure 9.5).
• N-oxidation. These reactions can result in hydroxylamine formation, oxime formation, and N-oxide formation (Figure 9.6). Several amines can undergo N-oxidation to form hydroxylamine. The classical example is aniline. Imines and primary amines can undergo N-hydroxylation. Noxide formation is mostly a function of flavin-containing monooxygenase.
• Oxidation (S and P). Both microsomal monooxygenases and flavin-containing monooxygenase act on thioethers to oxidize them to sulfox-ides and trisubstituted phosphines to phosphine oxides (Figure 9.7). Sulfoxides are further metabolized to sulfones, a common reaction for insecticides and drugs, chlorinated hydrocarbons and chlorpromazine, respectively.
• Desulfuration and ester cleavage. These reactions can convert the P-S double bond to P-O double bond. When cholinesterases are converted, powerful cholinesterase inhibitors are produced, e.g., paraoxon from par-athion (Figure 9.7).
These microsomal enzymes are involved in the oxidation of several inorganic compounds and organic compounds containing nitrogen, sulfur, or phosphorus. The catalytic cycle of FMO is shown in Figure 9.8, and it requires NADPH and oxygen
as cytochrome P450 does. Many of the reactions catalyzed by FMOs can be catalyzed by cytochrome P450 too. Several techniques can determine whether compounds are metabolized by FMOs or by cytochrome P450. FMO is inactivated in the absence of NADPH by subjecting the microsomes to 50°C for 1 min, with no effect on cytochrome P450. Cytochrome P450 can be inactivated with nonionic detergents, which have no effect on FMO. Antibodies for cytochrome P450 can identify the particular P450 enzyme that catalyzes the reaction. Substrates oxidized by the FMO include inorganic compounds (HS-, I-, IO-, I2, and CNS-); organic nitrogen compounds (acyclic and cyclic amines, N-alkyl and N, N-dialkylarylamines, hydrazine, primary amines); organic sulfur compounds (thiols and disulfides, cyclic and acyclic sulfides, mercaptopurines, pyrimidines, imidazoles, dithio acids and dithiocarbam-ides, thiocarbamides and thioamides); organic phosphorus compounds (phosphines, phosphonates); and selenides and selenocarbamides.
These oxidoreductases are located in either the mitochondrial fraction or the cyto-solic fraction (soluble supernatant) of the tissue homogenate.
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