Detoxifying Enzymes and Insect Symbionts Samuel K. hen' and Patrick F. Dowd Mycotoxin Research Unit, National Center for Agricultural Utilization Research Peoria, iL 61604 U.S.D.A., Agricultural Research The Toxic Environment Toxins are both naturallv nroduced as defensive substances and artificially made Gy man. Toxins serve a widespread defensive role, especially in plants and microorganisms, which cannot readily flee. Toxins may also be nroduced to reduce comnetition from other species. For exHmple, plants with elevated levels of certain toxins are often bred for their resistance to insects or plant pathogens. Humans also rely on microbial toxins as antigiotics. When the need for these toxins is over, as when the producing organism dies, the toxins themselves are typi&lly broken down by natural processes. The presence of toxins in the environment has been increased by humans both inadvertently (e.g., industrial byproducts) and intentionally (e.g., production of pesticides). Because many artificial toxins are analogous in both structure and function to those produced by natural sources, these tend to degrade rapidly by natural means. However, other toxins are quite persistent in the environment and exert unwanted effects. Typically, such toxins were manufactured before the adverse effects of any residues had been rewgnized. Dealing with Toxins Because many organisms consume and wmpete snccessfully with toxin producers, they must be capable of dealing with these toxins. Some oreanisms avoid toxins bv usine barriers that prevent toxins from moving into areas that would be affected bv them. For example. toxins that dissolve in water (hydrophilie)will not &ipihly penetrate fat or oil barriers, while toxins that dissolve in oils or orzanic solvents (lipol;hilic) will not rapidly penetrate water,-acid, or alkali barriers. When toxins are consumed, they may move through the digestive system so quickly that harmful quantities can not penetrate. The extreme pH levels of an organism's digestive system may also inactivate toxins. Highly acid or alkaline conditions will hydrolyze, and typically inactivate, taxic esters. Lipophilic toxins may be stored (sequestered) in fat deposits and rendered harmless, although these toxins may be released in small amounts over time. Other passive means of toxin avoidance, frequently occurring in combination, may also be involved. Enzymatic Detoxification by Metabolic Conversion One of the more nervasive means of detoxification involves metabolic conversion. In the following discussion, lipophilic toxins will be emphasized, because enzymatic conversion is one of the most common means of dealing with these toxins. Manv different enzvme svstems mav be there are t h e e involved in toxin degradation. main types of enzymatic detoxification: hydrolytic, oxidative-reductive, and conjugating. (See the figure).
ow ever,
'Present adaress Zoonotc D seases -awratory U S D A . Agr cuhural Research Sew ce. BARC-East Beltsv l e MD 20705 2The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.
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Oxidative-Reductive Enzymes The oxidative-reductive enzymes involved in detoxification are represented mainly by the unspecific monooxygenases. also known as nolvsubstrate monooxveenases or mixed-function oxidases (see the figure, parti&. The activity ofthese enzymes is tied to NADPH-ferrihemoprotein reductase, which catalyzes the reduction (regeneration) of the unspecific monooxygenases through the oxidation of NADPH. (This second reaction can be used as an indicator of monooxygenase activity provided appropriate controls are used because NADPH oxidation can be readily monitored spectrophotometrically.) The unspecific monooxygenases can catalyze such reactions a s deamination, N-, S-, and O-dealkylation, desulfuration, epoxidation, hydroxylation, N-oxidation, and sulfoxidation (I).Orieinallv it was thought that one enzyme of broad substrate~~ecifficity was involved in all of these reactions. It now appears that several isozymes are involved that have different but often overlapping substrate specificity, inducibility, and inhibitor susceptibility (2).These enzymes are particularly important in attacking underivatized toxins, such as those containing double bands. Hydrolytic Enzymes
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Hvdrolvtic enzvmes (nart IB of fieure) are hiehlv " " " effective in detoxifying appropriately structured esters, althoueh the relative imnortance of this role is still unclear (3). They typically req&re only water a s a cofactor, which is ubiquitous in most environments. Carboxylic and other esters are substrates for various carboxyl ester hydrolases. Epoxides are the substrates for epoxide hydrolases, and glucosides are the substrates for for a- and P-glncosidases. Toxicity may be enhanced by these enzymes, depending on the substrate. Phosphate esters, such as many insecticides, are hydrolyzed by alkaline phosphatases, acid phosphatases, or other related enzymes. Other hydrolytic enzymes, such as proteinases, may also hydrolyze toxic esters, but this is apparently a coincidental reaction. Conjugating Enzymes Coniueatine enzvmes (Part IC of fieure) are a third groupbf;deto&yingenzymes. ~ l t h o n g hk e s e enzymes are usuallv thoueht to a d on nroducts of hvdrolvtic or oxidative e&e< they can actbn the initiaitoxin as well, provided appropriate functional groups are present. One of the most widely studied types are the glutathione transferases. These enzvmes coniueate a substrate with glutathione and eliminate a port:on-of the molecule after complexing it with a proton. Groups removed may include halides, nitrogen, or sulfur. Additional reactions include disulfide interchanpes. - . isomerizations. and additions to epoxides (1). As the term " elutathione transferase imnlies. these enzymes require glutathione as a cofactor. Depending on the organism, the conjugate may be excreted as the glutathione conjugate, or it may be processed further. A
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Other conjugating enzymes include thiosulfate sulfurtransferase,which ads on cyanide groups aryl sulfotransferase 'phenol P-glueosyl transferase, which conjugates aromatic alcohols
The relative rates of activity of these different enzymes for a particular compound may vary in different tissues and organisms. Designing'Toxins for Increased Selectivity
Knowledge about these pathways can be exploited to make toxins that are more selective than the original. A Detoxification Pathways prime example of this selective design is the organophosAny particular toxin may undergo several detoxification phorus insecticides that wntain the P=S moiety. These inreactions at the same time, and a particular product may secticides are generally poor inhibitors of acetylcholinesbe further processed until the products are ultimately exterase, a n important enzyme in the nervous system and a creted or otherwise utilized (4). In some cases, these enpotential target site that is found in both mammals and zymes may make a compound more toxic (activation) (5). insects. Due to the differing prevalence of pathways, insects I. Widely Distributed tend to convert this moiety to P=O, which produces a very efA. Oxidative-unspecified monooxygenase fective acetylcholinesterase inhibitor, while mammals primarily hydrolyze these insecticides (6). Thus, although most organisms share wmmon detoxification pathways, relative rates 0 OH I epoxidationl and substrate specificity may RrC=C-F$ R-CH--CH-b hy,jmxylation vary widely. In addition to these I widely distributed detoxificaOH COUDS tion ~athwavs. . . some .. . of organisms also have specialized pathways of detoxification.
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C. Conjugating-glutathione fransferase R
A
+
G S H
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RS-G
+ AH
II. Group Prevalent A. Mammalian-glucumnosyl transferase R--OH
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+ UPE-p-D-glucuronosyl
R-C-p-D-glucuronoside
Detoxification in Different Organisms Detoxifying enzymes are present in many different classes of organisms, including microorganisms, invertebrates, vertebrates, and plants. The ability of plants to detoxify can be important in agricultural situations (7). The plants themselves may be involved in breaking down insecticides, antimicrobials, and herbicides. Thus, plants are important in determining the residuality of A = sulfate, nitrite or halide insecticides and antimicrobials and the efficacy of herbicides. However, for the sake of the following discussion, we will consider only mammals, insects, and microorganisms.
+ UPD
6. Insect-phenol-P-D-gluwsyl transferase
Ar--OH + UPWP-D-glucose
Ar--0--p-D-glucoside C. Microorganism-laccase
Common detoxifying methods.
+ UPD
Mammals
Mammals have been the most widely studied group of organisms in relation to detoxification mechanisms, such a s hydrolytic, oxidative, and conjugating enzymes. Mammals can detoxify many toxins, including plant and microbial derivatives, pesticides, and man-made waste products. An important additional conjugation reaction in mammals is glucuronidation (see figure), catalyzed by glucuronosyl transferase. Activated UDP-glumronic acid causes the nucleophilic displacement of functional p u p s . These enzymes also act on alcohols, phenols, esters, aromatic amines, carbamates, and thiophenols (8). Other conjugating reactions include methylations, acylations, and amino acid conjugations (with glycine and glutamine). Glutathione transferase products are further shunted through the mercapturic acid pathway, which regenerates amino acids (8). Volume 69 Number 10 October 1992
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Insects
Insects perform manv of the same detoxifvine reactions as mammals. 1f trends-continue, further sirhil&ties may be determined over time. These enzymes are particularly important in allowing herbivorous insects to feed on plant hosts, and various detoxifviw reactions are known for the natural pesticides they t h i s encounter (9). Of course. insecticide detoxification bv insects has also been intensively studied. In many cases i t appears that insecticide resistance is due to enhanced rates of detoxification (10). Hydrolytic and glutathione transferase enzymes are widespread in insects (11). Glucose conjugation is more common in insects than in mammals. It is catalyzed by phenol P-glucosyl transferase (part IIB of figure) and involves intermediates and substrates that are analogous to those of the glucuronidation reaction in mammals (11).Glycine is the most commonly used amino acid in conjugation reactions in insects (11). Microorganisms
The three main classes of detoxifvine enzvmes are also present in microorganisms, althouih &ch iess is known about them than the enzvmes in higher organisms (12). Because the microorganiHms comm~nly&died includk both prokaryotes (such as bacteria) and eukaryotes (such as fungi) generalization is more difficult. More often, information exists on the toxins that are utilized or the products that are formed, and the types of enzymes involved are inferred from the respective structures. Due to the less specialized nature of microorganisms, compounds generally considered toxic to vertebrates may have little effect on~mi~roor~anisms (e.g., neuroactive compounds). Nevertheless, monooxygenase enzymes have been reported in microorganisms. The reactions they catalyze are similar to those catalyzed by unspecific monooxygenases in mammals and insects. However, microbial monooxygenases in general appear to be more specific. Examples include the camphor-hydroxylating enzyme of Pseudomonas putida (13)and the enzymes that catalyze the alkane hydroxylations carried out by alkane-utilizing yeasts, such as Candida tropicalis (14). 11~drol~tic enzymes also appear to be present in microorganisms. The primary evidence for this comes from studyine oesticides 1121. . . ORen fields that have been treated for many years with, for example, an organophosphorus or carbamate insecticide. will contain microoreanisms that are very effective in hydrolyzing these compounds. This can be a narticular nroblem for soil-iucomorated insecticides, for h c h efficacy may be severely rLduced. Conjugating enzymes have been studied in even less detail in microorganisms. A recent survey of glutathione transferase activity in microorganisms indicates that they are fairly widespread, although relatively inactive (15). However, their importance in detoxification has apparently not been demonstrated. Enzymes Specific to Microorganisms
Microorganisms also may contain enzymes capable of detoxification that are rarely present in higher organisms, for example, the laccases (see figure) that act on halogenated phenols. Also, catechol oxidases (polyphenol oxidases) are among the most important enzymes in polymerizing t h e a n a l i n s or phenolics t h a t originate with herbicides (12). Although these enzymes have not been investigated as thoroughlv as in mammals, evidence from demadation studies-indicates that detoxifying enzyme activity in microorganisms is diverse, even within a particular species. For example, many species of Pseudomonas degrade vari798
Journal of Chemical Education
ous cyclic hydrocarbons (16) and pesticides (17). A white rot fungus (Phanerochaete chrysosporium) is known to degrade over 50 toxic substances, including phenols, phenolic acids, and chlorinated compounds such as DDT and polychlorinated biphenyls (PCB's) (18). Thus, although detoxifying enzymes have been studied less thoroughly in microorganisms than in mammals, detoxifying capabilities appear to be comparable. Because micmorgani.ims may beeiposed to assoried toxins in their various environments, they too have a need for detoxifying enzvmes. Of course. the soectrum of toxins encountered bv microrganisms may differ from the spectrum encountered bv higher organisms due to the inherent differences in ootintial target sites. When microorganisms occur in assmiation with higher organisms, detoxification reactions can be more involved. However. the interrelationshins have not been studied until rec&tly.
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Insect Symbionts and Detoxification We have alreadv discussed that nlant-feedine insects are exposed to toxin; in the form of plant met&olites that have a defensive role. Many of these insects also contain microorganisms that interaEt in a mutually beneficial relationship (symbionts). These insects include most sucking insects (such as aphids and stink bugs) and beetles (such as long-horned beetles, weevils, and stored-product beetles). As mentioned earlier, microorganisms appear to have a widespread and diverse ability to detoxify. Jones (19) suggested that the microorganisms that act as symbionts of insects mav also contribute to the survival of their insect hosts by aGgmenting the hosts ability to detoxify Our recent work has concentrated on exploring this possibility. Initial work dealt with demonstrating that symbionts will produce detoxifying enzymes while in the insect. Histochemical techniques were used to visualize this activity, mainlv relvine on the abilitv of diazo reagents to react with the aromakc &ohols that are produced&om the toxins by detoxifvine " -enzvmes. " For example, aromatic alcohol derivatives were apparentlv produced bv columbine anhid (Kakima essiei) svmbi" onti&om a vareity of organic &emicals (20).
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the aromatic ester, I-naphthyl acetate plant toxins: tannie acid and 6-methoxy-2-benzaxazolinone 'the insecticide, diazinon Apparent detoxification of 1-naphthyl acetate and tannic acid by symbionts was also seen in the maize weevil (Sitophilus zeamais) and the boll weevil (Anthonomus grandis) (20). The Cigarette Beetle and its Symbionts Ayeast-like symbiont (Symbiotaphrina koehii) of the cigarette beetle (Lasioderma serricorne) was apparently found to break down several compounds (20,21).
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1-naphthylacetate tannie acid 6-methoxy-2-benzoxazolinone
the fungal toxin, oehratoxin A These symbionts of the cigarette beetle can be readily cultured independent of the host, and the beetles can be rendered symbiont-free by surface-sterilizing their eggs. For these reasons, the cigarette beetle and its symbiont were good subiects for further exploration concerning - the role of symbioits in detoxificatio
