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

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 in

CHAPTER 9

Biotransformation, Detoxification, and

BiodegradationINTRODUCTION

As mentioned in Chapter 5, following the entry into a living organism andtranslocation, 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 toxicaction, 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 withoutexhibiting direct harmful effects DDT may be considered as an example Accumu-lation or buildup of free chemicals may be prevented, until the storage sites aresaturated 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, asexemplified 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 thechemical This metabolic conversion process is known as biotransformation andoccurs in any of several tissues and organs such as the intenstine, lung, kidney, skin,and liver

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 factornot 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 liverplays a major role in biotransformation because it contains a number of nonspecificenzymes responsible for catalyzing the reactions involved As a result of the processxenobiotics are converted to more water-soluble and more readily excretable forms.While the purpose of such metabolic processes is probably to reduce the toxicity ofchemicals, this does not prove to be always the case Occasionally the metabolicprocess converts a xenobiotic to a reactive electrophile that is capable of causinginjuries 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 reactionsare hydroxylation, dealkylation, deamination, and sulfoxide formation, whereasreduction reactions include azo reduction and addition of hydrogen Such reactions

as splitting of ester and amide bonds are common in hydrolysis During Phase I, achemical may acquire a reactive group such as OH, NH2, COOH or SH

Phase II reactions, on the other hand, are synthetic or conjugation reactions Anenvironmental chemical may combine directly with an endogenous substance, ormay be altered by Phase I and then undergo conjugation The endogenous substancescommonly involved in conjugation reactions include glycine, cysteine, glutathione(GSH), glucuronic acid, sulfates, or other water-soluble compounds Many foreigncompounds sequentially undergo Phase I and Phase II reactions, whereas othersundergo 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 pound is first oxidized so that a functional group (usually a hydroxyl group) isintroduced into the molecule This functional group is then coupled by conjugatingenzymes to a polar molecule so that the excretion of the foreign chemical is greatlyfacilitated

com-Figure 9.1 The two phases of xenobiotic metabolism.

The NADPH-cytochrome P-450 system, commonly known as the mixed-functionoxygenase (MFO) system, is the most imporant enzyme system involved in thePhase I oxidation reactions Cytochrome P-450 system, localized in the smoothendoplasmic reticulum of cells of most mammalian tissues, is particularly abundant

Figure 9.2 Detoxification pathways.

in the liver This system contains a number of isozymes which are versatile in thatthey catalyze many types of reactions including aliphatic and aromatic hydroxylationsand 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 complexwith an absorption spectrum peak at 450 nm This is the origin of the name of theenzyme 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 thenoccurs, with an electron being transferred from NADPH via NADPH cytochrome P-450

Figure 9.2 (continued)

reductase This reduced (Fe2+) 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 b5 and NADH cytochrome b5 reductase.The enzyme-substrate-oxygen complex splits into water, oxidized substrate, and theoxidized 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 molecularoxygen is reduced to water and the other is incorporated into the substrate Therequirements for this enzyme system are oxygen, NADPH, and Mg2+ ions.Contrary to the cytochrome P-450 system, most hepatic Phase II enzymes arelocated 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+O2+NADPH+H+→SOH+H2O+NADP+

adequate intracellular contents of cofactors such as NADPH, NADH, O2, onate, ATP, cysteine, and GSH are required for one or more reactions.

glucur-Consequence of Biotransformation

Although hepatic enzymes that catalyze Phase I and II reactions convert thelipid-soluble xenobiotic to a more water-soluble metabolite, they also participate inthe metabolism or detoxification of endogenous substances For example, the hor-mone testosterone is deactivated by cytochrome P-450 The S-methylases detoxifyhydrogen 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 byPhase I and II reactions might be expected to produce a stable, water-soluble, andreadily excretable compound However, there are examples of hepatic biotransfor-mation mechanisms by which xenobiotics are converted to reactive electrophilicspecies Unless detoxified, these reactive electrophiles may interact with a nucleo-philic site in a vital cell constituent, leading to cellular damage There is evidencethat many of these reactive substances bind covalently to various macromolecularconstituents of liver cells For example, carbon tetrachloride, known to be hepatotoxic,covalently binds to lipid components of the liver endoplasmic reticulum (Reynoldsand 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.)

Although liver cells are dependent on the detoxification enzymes for protectionagainst 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 radicalscavenger Its main role is to protect the lipid constituents of membranes againstfree radical-initiated peroxidation reactions Experimental evidence has shown thatlivers of animals fed diets deficient in vitamin E were more vulnerable to lipidperoxidation following poisoning with CCl4 (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 (VanBladeren et al 1980) Glutathione also can donate its sulfhydryl hydrogen to areactive free radical (GS) The glutathione radical formed can then react with anotherglutathione 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 areimportant in the defense against free radical-mediated cellular damage These includesuperoxide dismutase (SOD), catalase, and GSH peroxidase Figure 9.4 shows theinterrelationship between these enzymatic components

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 H2O GSH peroxidase also ifies peroxide by reducing it to H2O 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.)

detox-O2−.

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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 isnot 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 cellscontain prokaryotic endosymbionts, some of which contain their own set of plasmids.Given the variety of eucaryotic microorganisms, they have been labeled protistssince they are often a mixing of algal and protozoan characteristics within apparentlyrelated 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 ascribethis capability to the entire microbial community since often more than one type oforganism is responsible for the stages of microbial degradation

Microorganisms often contain a variety of genetic information In prokaryoticorganisms the chromosome is a closed circular DNA molecule However, othergenetic information is often coded on smaller pieces of closed circular DNA calledplasmids The chromosomal DNA codes the sequences that are responsible for thenormal maintenance and growth of the cell The plasmids, or extrachromosomalDNA, often code for metal resistance, antibiotic resistance, conjugation processes,and frequently the degradation of xenobiotics Plasmids may be obtained through avariety of processes including conjugation, infection, and the absorption of free DNAfrom the environment (Figure 9.5)

Eucaryotic microorganisms have a typical genome with multiple chromosomes

as mixtures of DNA and accompanying proteins Extrachromosomal DNA also existswithin 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.

Many microbial also contain prokaryotic and eucaryotic symbionts that can beessential to the survivorship of the organism The ciliate protozoan Paramecium bursaria contains symbiotic chlorella that can serve as a source of sugar when givensufficient light Several of the members of the widespread species complex, Para- mecium aurelia, contain symbiotic bacteria that kill paramecium not containing theidentical bacteria Apparently this killing trait is coded by plasmid DNA containedwithin the symbiotic bacteria Protists generally reproduce the asexual fission but sexualreproduction 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 thematerial results in a material able to enter the tricarboxylic acid or TCA cycle.Molecules that are essentially simple chains are readily degraded since they canenter this cycle with relatively little modification Aromatic compounds are morechallenging 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 catechuic acid The regulation of the resultant metabolic pathway is dependent uponthe group and basic differences that exist between bacteria and fungi

proto-Often the coding process for degradation of a xenobiotic is contained on boththe extrachromosomal DNA, the plasmid, and the chromosome Often the initialsteps that lead to the eventual incorporation of the material into the TCA cycle arecoded by the plasmid Of course, two pathways may exist, a chromosomal and aplasmid pathway Given the proper DNA probes, pieces of DNA with complimentarysequences to the degradation genes, it should be possible to follow the frequency andthereby the population genetics of degradative plasmids in procaryotic communities

In procaryotic mechanisms the essential steps allowing an aromatic or substitutedaromatic 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 areavailable Extrachromosomal DNA can be obtained through a variety of mechanismsand can be very infectious The rapid transmission of extrachromosomal DNA hasthe potential to enhance genetic recombination and result in rapid evolutionarychange In addition, the availability of the pathways on relatively easy-to-manipulategenetic material enhances our ability to sequence and artificially modify the codeand perhaps enhance the degradative capability of microorganisms

Simple disappearance of a material does not imply that the xenobiotic wasbiologically degraded There are two basic methods of assessing the biodegradation

of a substance The first is an examination of the mass balance or materials balanceresulting from the degradative process This is accomplished by the recovery of theoriginal substrate or by the recovery of the labeled substrate and the suspected

radiolabled metabolic products Mineralization of the substrate also is a means ofassessing the degradative process Production of CO2, methane, and other commoncongeners derived from the original substrate can be followed over time Withcompounds that have easily identified compounds such as bromide, chloride, orfluoride, these materials can be analyzed to estimate rates of degradation One of

Figure 9.6 The 3-ketoadipic acid pathway.

the crucial steps is to compare these rates and process with sterilized media or mediacontaining specific metabolic inhibitors to test whether the processes measured arebiological in nature.

Although the specific determination of the fate of a compound is the best means

to establish the degradation of a compound, nonspecific methods do exist that can

be used when it is difficult or impossible to label or analytically detect the substrate.Measurement of oxygen uptake as the substrate is introduced in the culture is ameans of confirming the degradation of the toxic mateiral Biological oxygendemand, as determined for waste water samples, can be used but it is not particularlysensitive Respirometry with a device such as the Warburg respirometer is moresensitive and can be used to measure the degradation rates of suspected intermediates.Often it is possible to grow the degradative organism using only the xenobioticsubstrate as the sole carbon source, additionally confirming the degradative process.Controls using sterilized media or inhibitors are again important since microorgan-ism are able to grow on surprisingly minimal media and with only small amounts

of materials that may be present as contaminants

A wide variety of aromatic organics are degraded by a variety of microorganisms.Table 9.1 provides a compilation from a recent review giving both the compoundand the strains that have so far been found that are responsible for the degradation.Only a few examples will be discussed below

Substituted benzenes are commonly occurring xenobiotics In Figure 9.7 thebiodegradation pathway for toluene is diagrammed The process begins with thehydroxylation of the toluene In one case the hydroxylation of the substituent, themethyl group occurs to form benzyl alcohol Additional steps result in catechol, amaterial readily incorporated into the 3-ketoadipic acid pathway Another set ofspecies hydrolyze the ring itself producing a substituted catechol as the end process.The degradation mechanism of materials such as naphthalene by fungi has beenfound comparable in a broad sense to the detoxification methanisms found in theliver in vertebrates Fungi use a monooxygenase system that incorporates an atom

of oxygen into the ring as the other atom is incorporated to water (Figure 9.8) Theresulting epoxide can be further hydrolyzed to form an intermediate ultimatelyending with a transhydroxy compound The epoxide also can isomerize to form avariety of phenols Both of these mechanisms occur in the degradation of naphthalene

by the fungus Cunninghamella elagans.

A particularly widespread environmental contaminant is the pesticide rophenol (PCP) PCP has been used as a bactericide, insecticide, fungicide, herbicide,and mulloscicide in order to protect a variety of materials from decomposition.Although it has bactericidal properties, PCP has been found to be degraded in avariety of environments by both bacteria and fungi In some instances degradationoccurs with PCP being used as an energy source

pentachlo-A proposed pathway for the degradation of PCP by two bacterial strains isrepresented in Figure 9.9 Cultures of Psuedomonas were found to transform PCPinto tetrachlorocatechol and tetrachlorohydroquinone (TeCHQ) These materials arethen metabolized and radiolabled carbon can be found in the amino acids of thedegradative bacteria Mycobacterium methylates PCP to pentachloroanisole but doesnot use PCP as an energy source Fungi also metabolize PCP to a less toxic metabolite

Table 9.1 Examples of Organic Compounds and Degradative Bacterial Strains

Aniline Frateuria sp ANA-18

Cunninghamella elegans Psuedomonas sp.

Pseudomonas putida 199

Pseudomonas sp.

Pseudomonas aeruginosa Pseudomonas putida

Benzoic acid Alcaligenes eutophus

Aspergillus niger Azotobacter sp.

Bacillus sp.

Pseudomonas sp.

Pseudomonas acidovorans Pseudomonas testosteroni Pseudomonas sp strain H1

Pseudomonas PN-1

Pseudomonas sp WR912

Rhodopseudomonas palustris Streptomyces sp.

By consortia of bacteria 2-Chlorobenzoic acid Aspergillus niger

3-Chlorobenzoic acid Acinetobacter calcoaceticus Bs5

(grown on succinic acid and pyruvic acid)

Alcaligenes eutrophus B9

Arthrobacter sp (grown on benzoic acid)

Aspergillus niger Azotobacter sp (grown on benzoic acid)

Bacillus sp (grown on benzoic acid)

Arthrobacter globiformis Azotobacter sp (grown on benzoic acid)

Pseudomonas sp CBS 3

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 acid Pseudomonas sp WR912

By consortia of bacteria 2,3,6-Trichlorebenzoic acid Brevibacterium sp (grown on benzoic acid)

Table 9.1 (Continued) Examples of Organic Compounds and Degradative Bacterial

By consortia of bacteria

4-Chlorocatechol Achromobacter sp.

3,5-Dichlorocatechol Achromobacter sp.

Chlorobenzene Pseudomonas putida (grown on toluene)

unidentified bacterium, strain WR1306 Chlorocatechol Pyrocatechases

3,5-Dichlorocatechol Achromobacter sp (grown on benzoic acid)

Chlorophenol Arthrobacter sp.

2-Chlorophenol Alcaligenes eutrophus

Nocardia sp (grown on phenol)

Pseudomonas sp B13 3-Chlorophenol Nocardia sp (grown on phenol)

Chlorotoluene Pseudomonas putida (grown on toluene)

Gentisic acid Trichosporon cutaneum

Guaiacols Arthrobacter sp.

(o-methoxyphenol)

3,4,5-Trichloroguaiacol Arthrobacter sp 1395

Homoprotocatechuic acid Trichosporon cutaneum

Naphthalene Cunninghamella elegans

Oscillatoria sp.

Pseudomonads Pentachlorophenol (PCP) Arthrobacter sp.

Coniophora pueana Mycobacterium sp.

Pseudomonas sp.

Saprophytic soil corynebacterium KC3 isolate

Mutant ER-47 Mutant ER-7

Table 9.1 (Continued) Examples of Organic Compounds and Degradative

4-amino-3,5-dichlorobenzoic acid By consortia of bacteria

2,4,5-Trichlorophenoxyacetic acid Psuedomonas cepacia AC1100

List Compiled from: Rochkind, M.L., J.W Blackburn, and G.S Sayler 1986 Microbial

Decomposition of Chlorinated Aromatic Compounds Environmental Protection Agency

160012-861090, pp 45-98.

Figure 9.7 Alternate pathways for the degradation of a substituted benzene, toluene, (Adapted

from Rochkind et al 1986.)

Figure 9.8 Biodegradation of naphthalene by Cunninghamella elagans (Adapted from

Roch-kind et al 1986.)

Figure 9.9 Possible mechanisms for the degradation of pentachlorophenol by Pseudomonas

sp (Adapted from Rochkind et al 1986.)

Given the ability of many organisms to degrade toxic materials within the

environment, a practical application would be to use these degradative capabilities

in the removal of xenobiotics from the environment In the broadest sense this might

entail the introduction of a specifically designed organism into the polluted

environ-ment to ensure the degradation of a known pollutant Other examples of attempts

at using biodegradation for remediation is the addition of fertilizers to enhance

degradation of oil spills and the construction of biological reactors, bioreactors,

through which contaminated water or a soil slurry can be passed In some instances

these attempts have appeared successful, in others the data are not so clear

The most important design criteria for attempting bioremediation is the

com-plexity of the environment and the comcom-plexity and concentration of the toxicants

Controlled and carefully defined waste streams such as those derived from a specific

synthesis at a manufacturing plant may be especially amenable to degradation A

reactor, such as the one schematically depicted in Figure 9.10, could be developed

using a specific strain of bacteria or protist that has been established on a substrate

Nutrients, temperature, oxygen concentration, and toxicant concentration can be

carefully controlled to offer a maximum rate of degradation As the complexity of

the effluent or the site to be remediated increase, a consortia of several organisms

or of an entire degradative community may be necessary Consortia also can be

established in a bioreactor-type setting

Figure 9.10 Schematic of a bioreactor for the detoxification of a waste stream or for inclusion

in a pump and water treatment process.

Concentration of the toxicant is essential in determining the success of the

bioremediation attempt As shown in Figure 9.11, too low a concentration will not

stimulate growth of the degradative organism At too high a concentration the toxic

effects become apparent and the culture dies The shape of the curve is dependent

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 Biotic factors, such as

competitors and predators, also become important as the process is taken out of the

bioreactor and placed in a more typical environment Not only do the degradative

organisms have to be able to degrade the toxicant, they must be able to compete

effectively with other microflora and escape predation

To enhance degradation frequent plowing and fertilization of a terrestrial site

may be done to ensure proper aeration of the soil Ground water is often

nutrient-and oxygen-limited nutrient-and both of these materials can be introduced Often hydrogen

peroxide is pumped into ground water as an effective means of delivering oxygen

as the hydrogen peroxide decomposes

Figure 9.11 Degradative growth curve At low concentrations, degradation may not occur due

to the lack of nutritive content of the xenobiotic as substrate Eventualy a maximal

rate of degradation and also growth may occur with a plateau Eventually the

concentration of the toxic material overwhelms the ability of the organism to

detoxify the material and death ensues.