Introduction to ENVIRONMENTAL TOXICOLOGY Impacts of Chemicals Upon Ecological Systems - CHAPTER 9 potx

CHAPTER 9 Biotransformation, Detoxification, and Biodegradation INTRODUCTION As mentioned in Chapter 5, following the entry into a living organism and translocation, a foreign chemical may be stored, metabolized, or excreted (Figure 5.2). When the rate of entry is greater than the rate of metabolism and/or excretion, storage of the chemical often occurs. Storage or binding sites may not be the sites of toxic action, however. For example, lead is stored primarily in the bone, but acts mainly on the soft tissues of the body. If the storage site is not the site of toxic action, selective sequestration may be a protective mechanism, since only the freely circu- lating form of the foreign chemical produces harmful effects. Some chemicals that are stored may remain in the body for a long time without exhibiting direct harmful effects. DDT may be considered as an example. Accumu- lation or buildup of free chemicals may be prevented, until the storage sites are saturated. Selective storage limits the amount of foreign chemicals to be excreted, however. Since bound or stored toxicants are in equilibrium with their free forms, a chemical will be released from the storage site as it is metabolized or excreted. On the other hand, accumulation may result in illnesses that develop slowly, as exemplified by fluorosis and lead and cadmium poisoning. METABOLISM OF ENVIRONMENTAL CHEMICALS: BIOTRANSFORMATION Subsequent to the entry of an environmental chemical into an organism such as a mammal, chemical reactions occur within the body to alter the structure of the chemical. This metabolic conversion process is known as biotransformation and occurs in any of several tissues and organs such as the intenstine, lung, kidney, skin, and liver. © 1999 by CRC Press LLC By far the largest number of these chemical reactions are carried out in the liver. The liver metabolizes not only drugs but also most of the other foreign chemicals to which the body is exposed. Biotransformation in the liver is thus a critical factor not only in drug therapy but also in the body’s defense against the toxic effects of a wide variety of environmental chemicals (Kappas and Alvares 1975). The liver plays a major role in biotransformation because it contains a number of nonspecific enzymes responsible for catalyzing the reactions involved. As a result of the process xenobiotics are converted to more water-soluble and more readily excretable forms. While the purpose of such metabolic processes is probably to reduce the toxicity of chemicals, this does not prove to be always the case. Occasionally the metabolic process converts a xenobiotic to a reactive electrophile that is capable of causing injuries through interaction with liver cell constituents (Reynolds 1977). Types of Biotransformation The process of xenobiotic metabolism includes two phases commonly known as Phases I and II. The major reactions included in Phase I are oxidation, reduction, and hydrolysis, as shown in Figure 9.1. Among the representative oxidation reactions are hydroxylation, dealkylation, deamination, and sulfoxide formation, whereas reduction reactions include azo reduction and addition of hydrogen. Such reactions as splitting of ester and amide bonds are common in hydrolysis. During Phase I, a chemical may acquire a reactive group such as OH, NH 2 , COOH or SH. Phase II reactions, on the other hand, are synthetic or conjugation reactions. An environmental chemical may combine directly with an endogenous substance, or may be altered by Phase I and then undergo conjugation. The endogenous substances commonly involved in conjugation reactions include glycine, cysteine, glutathione (GSH), glucuronic acid, sulfates, or other water-soluble compounds. Many foreign compounds sequentially undergo Phase I and Phase II reactions, whereas others undergo only one of them. Several representative reactions are shown in Figure 9.2. Mechanisms of Biotransformation In the two phases of reactions shown in Figure 9.1, the lipophilic foreign com- pound is first oxidized so that a functional group (usually a hydroxyl group) is introduced into the molecule. This functional group is then coupled by conjugating enzymes to a polar molecule so that the excretion of the foreign chemical is greatly facilitated. Figure 9.1 The two phases of xenobiotic metabolism. © 1999 by CRC Press LLC The NADPH-cytochrome P-450 system, commonly known as the mixed-function oxygenase (MFO) system, is the most imporant enzyme system involved in the Phase I oxidation reactions. Cytochrome P-450 system, localized in the smooth endoplasmic reticulum of cells of most mammalian tissues, is particularly abundant Figure 9.2 Detoxification pathways. © 1999 by CRC Press LLC in the liver. This system contains a number of isozymes which are versatile in that they catalyze many types of reactions including aliphatic and aromatic hydroxylations and epoxidations, N-oxidations, sulfoxidations, dealkylations, deaminations, dehaloge- nations and others (Wislocki et al. 1980). These isozymes are responsible for the oxi- dation of different substrates or for different types of oxidation of the same substrate. Carbon monoxide binds with the reduced form of the cytochrome, forming a complex with an absorption spectrum peak at 450 nm. This is the origin of the name of the enzyme. As a result of the complex, inhibition of the oxidation process occurs. At the active sites of cytochrome P-450 is an iron atom that, in the oxidized form, binds the substrate (SH) (Figure 9.3). Reduction of this enzyme-substrate complex then occurs, with an electron being transferred from NADPH via NADPH cytochrome P-450 Figure 9.2 (continued) © 1999 by CRC Press LLC reductase. This reduced (Fe 2+ ) enzyme-substrate complex then binds molecular oxygen in some unknown fashion, and is then reduced further by a second electron, possibly donated by NADH via cytochrome b 5 and NADH cytochrome b 5 reductase. The enzyme-substrate-oxygen complex splits into water, oxidized substrate, and the oxidized form of the enzyme. The overall reaction is therefore: (9.1) where SH is the substrate. As shown in the above equation, one atom from molecular oxygen is reduced to water and the other is incorporated into the substrate. The requirements for this enzyme system are oxygen, NADPH, and Mg 2+ ions. Contrary to the cytochrome P-450 system, most hepatic Phase II enzymes are located in the cytoplasmic matrix. In order for these reactions to occur efficiently, adequate activity of the enzymes involved is essential. In addition, it is clear that Figure 9.2 (continued) SH O 2 NADPH H + SOH H 2 O NADP + ++→++ + © 1999 by CRC Press LLC adequate intracellular contents of cofactors such as NADPH, NADH, O 2 , glucur- onate, ATP, cysteine, and GSH are required for one or more reactions. Consequence of Biotransformation Although hepatic enzymes that catalyze Phase I and II reactions convert the lipid-soluble xenobiotic to a more water-soluble metabolite, they also participate in the metabolism or detoxification of endogenous substances. For example, the hor- mone testosterone is deactivated by cytochrome P-450. The S-methylases detoxify hydrogen sulfide formed by anaerobic bacteria in the intestinal tract. It can be seen, therefore, that chemicals or conditions that influence the activity of the Phase I and II enzymes can affect the normal metabolism of endogenous substances. As mentioned previously, the biotransformation of lipophilic xenobiotics by Phase I and II reactions might be expected to produce a stable, water-soluble, and readily excretable compound. However, there are examples of hepatic biotransfor- mation mechanisms by which xenobiotics are converted to reactive electrophilic species. Unless detoxified, these reactive electrophiles may interact with a nucleo- philic site in a vital cell constituent, leading to cellular damage. There is evidence that many of these reactive substances bind covalently to various macromolecular constituents of liver cells. For example, carbon tetrachloride, known to be hepatotoxic, covalently binds to lipid components of the liver endoplasmic reticulum (Reynolds and Moslen 1980). Some of the reactive electrophiles are carcinogenic as well. Figure 9.3 The cytochrome P-450 monoxygenase system. P-450 3+ : cytochrome P-450 with heme iron in oxidized state (Fe 3+ ); P-450 2+ : cytochrome P-450 with iron in reduced state; S: substrate; e: electron. (Adapted from J.A. Trimbrell. 1982. Principles of Biochemical Toxicology. Taylor and Francis Ltd., London.) © 1999 by CRC Press LLC Although liver cells are dependent on the detoxification enzymes for protection against reactive electrophilic species produced during biotransformation, endoge- nous antioxidants such as vitamins C and E and glutathione also provide protection. As mentioned in Chapter 5, these substances are widely known as a free radical scavenger. Its main role is to protect the lipid constituents of membranes against free radical-initiated peroxidation reactions. Experimental evidence has shown that livers of animals fed diets deficient in vitamin E were more vulnerable to lipid peroxidation following poisoning with CCl 4 (Reynolds and Moslen 1980). Glu- tathione, on the other hand, is a tripeptide and has a nucleophilic sulfhydryl (SH) group that can react with and thus detoxify reactive electrophilic species (Van Bladeren et al. 1980). Glutathione also can donate its sulfhydryl hydrogen to a reactive free radical (GS). The glutathione radical formed can then react with another glutathione radical to form stable oxidized GSSG. The GSSG can be reduced back to GSH through an NADPH-dependent reaction catalyzed by glutathione reductase. The NADPH is generated in reactions involved in the pentose phosphate pathway. In addition to vitamin E and C and GSH, there are enzymatic systems that are important in the defense against free radical-mediated cellular damage. These include superoxide dismutase (SOD), catalase, and GSH peroxidase. Figure 9.4 shows the interrelationship between these enzymatic components. MICROBIAL DEGRADATION Microbial degradation of xenobiotics is crucial in the prediction of the lon- gevity and thereby the long-term effects of the toxicant and also may be crucial in the actual remediation of a contaminated site. Utilization of the propensity of microorganisms to degrade a wide variety of materials may actually provide an opportunity for environmental toxicologists to not only diagnose and provide a Figure 9.4 The four important enzymatic components of the cellular antioxidant defense system. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide to peroxide. Catalase reduces peroxide to H 2 O. GSH peroxidase also detox- ifies peroxide by reducing it to H 2 O. GSH reductase re-reduces the oxidized glutathione (GSSG) to GSH. The NADPH required for the reduction of GSSG to GSH is primarily supplied by the oxidation of glucose via the pentose phosphate pathway. (Based on N.K. Mottet, Ed. Environmental Pathology. Oxford University Press, New York, 1985.) O 2 − . () © 1999 by CRC Press LLC prognosis, but also to prescribe a treatment to assist the ecosystem in the removal of the xenobiotic. Microbial cell structure is varied with a tremendous diversity in size and shape. Prokaryotic cells typically contain a cell wall, 70s ribosomes, a chromosome that is not membrane bound, various inclusions and vacuoles, and extrachromosomal DNA or plasmids. Eucaryotic microorganisms are equally varied with a variety of forms, many are photosynthetic or harbor photosynthetic symbionts. Many eucaryotic cells contain prokaryotic endosymbionts, some of which contain their own set of plasmids. Given the variety of eucaryotic microorganisms, they have been labeled protists since they are often a mixing of algal and protozoan characteristics within apparently related groups. Many of these microorganisms have the ability to use xenobiotics as a carbon or other nutrients source. In some instances it may be more appropriate to ascribe this capability to the entire microbial community since often more than one type of organism is responsible for the stages of microbial degradation. Microorganisms often contain a variety of genetic information. In prokaryotic organisms the chromosome is a closed circular DNA molecule. However, other genetic information is often coded on smaller pieces of closed circular DNA called plasmids. The chromosomal DNA codes the sequences that are responsible for the normal maintenance and growth of the cell. The plasmids, or extrachromosomal DNA, often code for metal resistance, antibiotic resistance, conjugation processes, and frequently the degradation of xenobiotics. Plasmids may be obtained through a variety of processes including conjugation, infection, and the absorption of free DNA from the environment (Figure 9.5). Eucaryotic microorganisms have a typical genome with multiple chromosomes as mixtures of DNA and accompanying proteins. Extrachromosomal DNA also exists within the mitochondria and the chloroplasts that resembles prokaryotic genomes. Figure 9.5 Schematic of a typical prokaryote. Genetic information and thereby coding for the detoxification and degradation of a xenobiotic may be available from a variety of sources. © 1999 by CRC Press LLC Many microbial also contain prokaryotic and eucaryotic symbionts that can be essential to the survivorship of the organism. The ciliate protozoan Paramecium bursaria contains symbiotic chlorella that can serve as a source of sugar when given sufficient light. Several of the members of the widespread species complex, Para- mecium aurelia , contain symbiotic bacteria that kill paramecium not containing the identical bacteria. Apparently this killing trait is coded by plasmid DNA contained within the symbiotic bacteria. Protists generally reproduce the asexual fission but sexual reproduction is available. Often during sexual reproduction an exchange of cyto- plasm takes place, allowing cross infection of symbionts and their associated DNA. Microorganisms are found in a variety of environments, such as aquatic, marine, ground water, soil, and even in the Arctic. many are found in extreme environments, from tundra to the superheated smokers at sites of seafloor spreading. The adapt- ability of microorganisms extends to the degradation of many types of xenobiotics. Many organic xenobiotics are completely metabolized under aerobic conditions to carbon dioxide and water. The essential criteria is that the metabolism of the material results in a material able to enter the tricarboxylic acid or TCA cycle. Molecules that are essentially simple chains are readily degraded since they can enter this cycle with relatively little modification. Aromatic compounds are more challenging metabolically. The 3-ketoadipic acid pathway is the generalized path- ways for the metabolism of aromatic compounds with the resulting product acetyl- CoA ad succinic acid, materials that easily enter into the TCA cycle (Figure 9.6). In this process the aromatic compound is transformed into either catechol or proto- catechuic acid. The regulation of the resultant metabolic pathway is dependent upon the group and basic differences that exist between bacteria and fungi. Often the coding process for degradation of a xenobiotic is contained on both the extrachromosomal DNA, the plasmid, and the chromosome. Often the initial steps that lead to the eventual incorporation of the material into the TCA cycle are coded by the plasmid. Of course, two pathways may exist, a chromosomal and a plasmid pathway. Given the proper DNA probes, pieces of DNA with complimentary sequences to the degradation genes, it should be possible to follow the frequency and thereby the population genetics of degradative plasmids in procaryotic communities. In procaryotic mechanisms the essential steps allowing an aromatic or substituted aromatic to enter the 3-ketoadipic acid pathway are often, but not always, encoded by plasmid DNA. In some cases both a chromosomal and plasmid pathway are available. Extrachromosomal DNA can be obtained through a variety of mechanisms and can be very infectious. The rapid transmission of extrachromosomal DNA has the potential to enhance genetic recombination and result in rapid evolutionary change. In addition, the availability of the pathways on relatively easy-to-manipulate genetic material enhances our ability to sequence and artificially modify the code and perhaps enhance the degradative capability of microorganisms. Simple disappearance of a material does not imply that the xenobiotic was biologically degraded. There are two basic methods of assessing the biodegradation of a substance. The first is an examination of the mass balance or materials balance resulting from the degradative process. This is accomplished by the recovery of the original substrate or by the recovery of the labeled substrate and the suspected © 1999 by CRC Press LLC radiolabled metabolic products. Mineralization of the substrate also is a means of assessing the degradative process. Production of CO 2 , methane, and other common congeners derived from the original substrate can be followed over time. With compounds that have easily identified compounds such as bromide, chloride, or fluoride, these materials can be analyzed to estimate rates of degradation. One of Figure 9.6 The 3-ketoadipic acid pathway. © 1999 by CRC Press LLC [...]... consortia of bacteria Psuedomonas cepacia AC1100 List Compiled from: Rochkind, M.L., J.W Blackburn, and G.S Sayler 198 6 Microbial Decomposition of Chlorinated Aromatic Compounds Environmental Protection Agency 16001 2-8 61 090 , pp 4 5 -9 8 Figure 9. 7 Alternate pathways for the degradation of a substituted benzene, toluene, (Adapted from Rochkind et al 198 6.) © 199 9 by CRC Press LLC Figure 9. 8 Biodegradation of. .. enzymatic-hydrolysis and nerve function Science 172: 1243 Hoskin, F.C.G 197 6 Distribution of diisopropylphosphorofluoridate hydrolyzing enzyme between sheath and axoplasm of squid giant-axon J Neurochem 26: 104 3-1 045 Hoskin, F.C.G 198 5 Inhibition of soman- and di-isopropylphosphorofluoridate (DFP)-detoxifying enzyme by mipafox Biochem Pharmacol 34: 206 9- 2 072 Hoskin, F.C.G 198 9 An organophosphorus detoxifying... 198 7 Bacterial detoxification of diisopropylfluorophosphate Appl Env Micro 53: 168 5-1 6 89 Awasthi, Y.C., D.D Dao, and R.P Saneto 198 0 Interrelationships between anionic and cationic forms of glutathione s-transferases of human liver Biochem J 191 : 1-1 0 Bianchi, M.A., R.J Portier, K Fujisaki, C.B Henry, P.H Templet, and J.E Matthews 198 8 Determination of optimal toxicant loading for biological closure of. .. Walker 198 8 Soman hydrolyzing and detoxifying properties of an enzyme from a therophilic bacterium Fund Appl Toxicol 11: 37 3-3 80 © 199 9 by CRC Press LLC Chiang, T., M.C Dean, and C.S McDaniel 198 5 A fruit-fly bioassay with phosphotriesterase for detection of certain organophosphorus insecticide residues Bull Environ Contam Toxicol 34: 80 9- 8 14 Crawford, R.L and W.W Mohn 198 5 Microbiological removal of pentachlorophenol... (Adapted from Rochkind et al 198 6.) Figure 9. 9 Possible mechanisms for the degradation of pentachlorophenol by Pseudomonas sp (Adapted from Rochkind et al 198 6.) © 199 9 by CRC Press LLC Figure 9. 10 Schematic of a bioreactor for the detoxification of a waste stream or for inclusion in a pump and water treatment process BIOREMEDIATION Given the ability of many organisms to degrade toxic materials within the... insensitive to ammonium sulfate (Dumas et al 198 9) Molecular weight as determined by analysis of the gene sequence is 35,418 D (McDaniel et al 198 8) However, disassociated from the membrane using a Triton-X-100 or Tween 20, the apparent molecular weight is estimated to be 60,000 to 65,000 D These data raise the possibility that the active enzyme is dimeric © 199 9 by CRC Press LLC Figure 9. 14 Structures of several... F.C.G Hoskin 197 9 Stereospecificity and active-site requirements in a diisopropylphorofluoridate-hydrolyzing enzyme Biochem Pharmacol 128: 125 9- 1 261 Haley, M.V and W.G Landis 198 7 Confirmation of multiple organofluorophate hydrolyzing activities in the protozoan Tetrahymena thermophila CRDEC-TR-880 09 Harper, B.G., L.P Midgley, I.G Resnick, and W.G Landis 198 6 Scale-up production and purification of diisopropylfluorophosphatase... not only upon the degradative system of the organism, but also upon the availability of nutrients, temperature, and the other factors essential for microbial growth One of the advantages of the bioreactor system is that all of these factors can be carefully controlled In a situation where it may be necessary to attempt the in situ remediation of a toxicant these factors are more difficult to control... pp 5 3-6 4 Hoskin, F.C.G., M.A Kirkish, and K.E Steinman 198 4 Two enzymes for the detoxification of organophosphorus compounds—sources, similarities, and significance Fund Appl Toxicol 4: 516 5-5 172 Hoskin, F.C.G and R.J Long 197 2 Purification of DFP-hydrolyzing enzyme from squid head ganglion Arch Biochem Biophys 150: 54 8-5 55 © 199 9 by CRC Press LLC Hoskin, F.C.G., P Rosenberg, and M Brzin 196 6 Re-examination... acetyl-B-methylcholine chloride Can J Biochem Physiol 3: 96 3 -9 69 Jakoby, W.B 197 8 The glutathione s-transferases: a group of multifunctional detoxification proteins Adv Enzymol 46: 38 3-4 14 Kappas, A and A.P Alvares 197 5 How the liver metabolizes foreign substances Sci Am 232(6): 2 2-3 1 Kitteridge, J.S and E Roberts 196 9 A carbon-phosphorus bond in nature Science 164: 3 7-4 2 Landis, W.G., R.S Anderson, N.A Chester, . these materials can be analyzed to estimate rates of degradation. One of Figure 9. 6 The 3-ketoadipic acid pathway. © 199 9 by CRC Press LLC the crucial steps is to compare these rates and process. reactions. Cytochrome P-450 system, localized in the smooth endoplasmic reticulum of cells of most mammalian tissues, is particularly abundant Figure 9. 2 Detoxification pathways. © 199 9 by CRC. Pseudomonas sp. WR912 4-Chloro- Chlamydomonas sp. A2 3,5-Dinitrobenzoic acid 2,5-Dichlorobenzoic acid By consortia of bacteria 3,4-Dichlorebenzoic acid By consortia of bacteria 3,5-Dichlorobenzoic