Organic Toxicants and Plants

Ecotoxicology and Environmental Safety 47, 1}26 (2000) REVIEW Environmental Research, Section B doi:10.1006/eesa.2000.1929, available online at http://www.idealibrary.com on

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REVIEW Organic Toxicants and Plants F. Korte,* G. Kvesitadze, D. Ugrekhelidze, M. Gordeziani, G. Khatisashvili, O. Buadze, G. Zaalishvili, and F. CoulstonDurmishidze Institute of Plant Biochemistry, Academy of Sciences of Georgia, Digomi, Tbilisi 380059, Georgia; *Institute of Chemistry Weihenstephan, TU Munich, Schulstrasse 10, D-85356 Freising, Germany; and -Coulston Foundation, Alamogordo, New Mexico 88310 Received August 11, 1999 Table of Contents 1. Introduction 2. Absorption and Transport 3. Excretion 4. Conjugate Formation 4.1. Glycosylation of Hydroxylic groups of alcohols and Phenols 4.2. Glycosylation of Carboxyl Groups of Organic Acids 4.3. Glycosylation of Amino Groups 4.4. Conjugation of Carboxyl Groups with Amino Acids 4.5. Conjugation of Xenobiotics with Peptides 5. Oxidative Degradation 5.1. Hydroxylation 5.2. Reactions of the Hydrolytic Cleavage Type

6. Deep Oxidation 7. Enzymatic Reactions 7.1. Cytochrome P450-Dependent Monooxygenation 7.2. Components of Monooxygenase System 7.3. The Induction of Cytochrome P450 7.4. Inactivation and Transformation of Cytochrome P450 8. Action on the Cell Structure 8.1. The Movement of Toxicants in the Plant Cells 8.2. Alkanes and Alkenes 8.3. Compounds Containing an Aromatic Nucleus 9. Conclusions 10. References

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Organic xenobiotics absorbed by roots and leaves of higher plants are translocated by di4erent physiological mechanisms. The following pathways of xenobiotic detoxication have been observed in higher plants: conjugation with such endogenous compounds as peptides, sugars, amino acids, and organic acids; oxidative degradation and consequent oxidation of xenobiotics with the 5nal participation of their carbon atoms in regular cell metabolism. The small parts of xenobiotics are excreted maintaining their original structure and con5guration. Enzymes catalyze oxidative degradation of xenobiotics from the initial hydroxylation to their deep oxidation. The wide intracellular distribution and inductive nature of oxidative enzymes lead to the high detoxication ability. With plant aging, transformation of the monooxygenase system into peroxidase takes place. Once in the cells, xenobiotics are incorporated into di4erent cell organelles. All xenobiotics examined are characterized by a negative e4ect on cell ultrastructure. The penetration of high doses of xenobiotics into plant cells leads to signi5cant deviations from the norm and, in some cases, even to the complete cell destruction and plant death.  2000 Academic Press

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1. INTRODUCTION

All ecological activities have the following basic goals: to expose the sources and determine the degree of contamination of any particular part of the biosphere; to develop e!ective technologies to prevent or signi"cantly decrease the level of contamination; to predict possible ecological catastrophes. Though, in practice, the synthetic ecotechnologies of air, water, and soil resource puri"cation have already been used, the main detoxication potential is still provided by nature. Microorganisms, higher plants, and algae are the main instruments of nature in regulating ecological conditions of the biosphere. The ecological problems caused by contamination of soil and water may be partially solved by phytoremediation. These technologies are valuable and cost e!ective, using the plants' abilities to accumulate and transform toxicants. Phytoremediation e!ectively works in the cases of heavy metals and organic toxicants (Salt et al., 1998).

 To whom correspondence should be addressed. 1

0147-6513/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.

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As was found in the last two decades, the huge detoxication potential of higher plants, still occupying vast areas, could be e!ectively used as the basis to create the modern ecological biotechnologies. Elaboration of the &&green "lter'' biotechnology and its proper realization could delay for long the feasible ecological catastrophe that might be provoked by industrialization of the planet. With respect to the ability to assimilate toxic substances, plants signi"cantly di!er. For example, under the same conditions, per 1 kg of green leaf mass, common maple (Acer campestre) and elaeagnus (Elaeagnus angustifolia) absorb a hundred times more benzene from the atmosphere than alder (Alnus barbata) and elm (;lmus foliacea), and a thousand times more than white mulberry (Morus alba) and linden (¹ilia platyphyllos). The ability to assimilate aerosols and chemical compounds from air and liquid solutions is an important industrial characteristic of higher plants. For instance, polycyclic hydrocarbons exist in the atmosphere as solid and liquid aerosols, and among these are such strong carcinogens as benzo[a]pyrene, benz[a]anthracene, and dibenzanthracene. One more extremely signi"cant ecological feature of higher plants is their ability to take up great amounts of carcinogenic hydrocarbons from soil and water. For example, English ryegrass (¸olium perenne) absorbs from the soil signi"cant amounts of benzo[a]pyrene, and being cultivated in water, accumulates a thousand times more benzo[a]pyrene as compared with the surrounding water. At present, su$cient information has been accumulated to estimate the detoxication potential of plants. In this review, analyses of the existing data are directed to expose the ability of plants to assimilate and metabolize di!erent kinds of organic toxicants. Attempts have been made to describe the enzymatic systems participating in consecutive oxidative transformations of xenobiotics, and "nally, the common regularities of the in#uence of exogenous compounds on structure and resistance of the plant cell have been elucidated. 2. ABSORPTION AND TRANSPORT

Absorption of xenobiotics by roots and leaves di!ers: to get into roots the substances must penetrate only through unsuberi"cated cell walls free of cuticle, but to get into the inner part of the leaf the substances must penetrate either through stomata or through the cuticle of epidermis. Due to the more simple mechanism of penetration, leaves absorb substances more selectively than roots. Absorption of organic xenobiotic by roots from water solutions is carried out, as a rule in two stages: the "rst step is the fast di!usion into free space and the second, the subsequent slow accumulation. Temperature is an essential factor in the absorption of organic xenobiotics by roots (Nandihalli and Bhowmik,

1989; Kristich and Schwarz, 1989). Another important factor determining the absorption of xenobiotics is their concentration in soil (MuK ller, 1976; Dean-Raymond and Alexander, 1976; Motooka et al., 1977; Kristich and Schwarz, 1989). Absorption of exogenous compounds by roots signi"cantly depends on the pH of the nutrient area or soil. Since the pH of the medium determines the dissociation of ionogenic molecules it considerably in#uences the penetration capacity of xenobiotics (Leroux and Gredt, 1975; Djula, 1975; Ugrekhelidze et al., 1986). A very important factor also in#uencing the penetration into the plant cell is the molecular weight of the xenobiotic. It was found that absorption and accumulation of polyethylene glycol by pepper (Capsicum annuum) from nutrient area is inversely proportional to its molecular weight (Janes, 1974). While testing with young plants of snapbean (Phaseolus vulgaris) and cotton (Gossypium hirsutum) a very important conclusion was made: polyethylene glycol is absorbed and then translocated through the plant without changes in its molecular characteristics (Andreopulos-Renaud et al., 1975). Foreign compounds penetrate a leaf in two ways: through stomata or through the cuticle of epidermis. Both pathways function simultaneously in plants. Penetration through the cuticle seems to be of less importance for inorganic gases; however, for organic molecules characterized by high lipophilicity, it is rather important. Such molecules penetrate leaves through the stomata but much more intensively through the cuticle (Ugrekhelidze, 1976). It was found that absorption of methane and vapors of benzene was performed by leaves of vine (
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synthesis stops just after the complete development of a leaf (Kolattukudy, 1980). This explains why young leaves absorb foreign compounds more intensely than mature ones. Thus, for example, retardant 2,2-dimethylhydrazide of succinic acid penetrates through the surface of younger leaves of snapbean more intensely (SchoK nherr and Bukovac, 1978). Young leaves of olive (Olea europaea) absorb indole acetic acid from solution more actively than old leaves (Epstein and Lavee, 1977). Hypostomatous leaves of hedge maple, apple, and vine absorb benzene and toluene vapors by the adaxial side more intensively than by the abaxial side (Ugrekhelidze et al., 1997). It should be noted that the thickness of the cuticle is not always an index of waxiness. For example, according to Leece's (1978) data, the adaxial surface of leaves of two varieties of garden plum has a thicker cuticle (1.6}2.0 lm) than the abaxial surface (1.2 lm), but contains less surface wax (34}35 and 47}52 lm/cm, respectively) (Leece, 1978). Some authors have supposed that trichome cells also take part in the process of foreign compound absorption by plants (Hull et al., 1975). According to the data of King and Radosevich (1979) leaves of tanoak (¸ythocarpus densi-orus) absorb more [C]triclopyr if more trichomes are on their outer surface. In particular, the largest quantity of starry trichomes was found on the adaxial surface of young leaves characterized by maximum absorption capacity. One of the probable ways xenobiotic molecules penetrate a leaf is through ectodesmata. Ectodesmata are considered to serve as polar ways during the absorption and excretion of substances by leaves (Franke, 1975). In cell walls, these channels may serve as conductive ways during absorption of water-soluble substances by leaves. Another more possible way foreign lipophilic molecules penetrate leaves is by the migration of separate compounds of wax from cuticle to epidermal cells and back (Cassagne and Lessire, 1975). The penetration of xenobiotics through the cuticle greatly depends on its structure. Thus, for example, pyrazon penetrates beet (Beta vulgaris) leaves comparatively easily, but phenmedipham and benzthiazuron penetrate rather slowly and in negligible amounts (Merbach and Schilling, 1977). Leaf cuticle is also penetrable by large molecules such as surface-active substances (Eynard, 1974), long-chain fatty acids (Cassagne and Lessire, 1975), longchain alkanes (Cassagne and Lessire, 1975) and peptides (Shida et al., 1975). The intensity of xenobiotic absorption by plants and their further active transport are greatly determined by the physiological activity of foreign compounds. Plants placed in an atmosphere containing low concentrations of alkanes (C }C ), cyclohexane, benzene, and toluene absorb these   substances, causing their deep oxidation. As a result of incubation of numerous plants (55 representatives of annual and perennial plants) with labeled carbohydrates, it was demonstrated that all of them absorb and metabolize al-

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kanes and arenes with di!erent intensities (Durmishidze et al., 1974a, b; Durmishidze and Ugrekhelidze, 1975). The products of transformation of hydrocarbons absorbed by leaves #ow along the stem to the roots, and from the roots the intermediates of absorbed and metabolized hydrocarbons migrate to leaves. Benzo[a]pyrene (MuK ller, 1976) and benz[a]anthracene (Devdariani and Kavtaradze, 1979) are actively absorbed and transported by plants from nutrient solutions. Aryloxyalkylcarbonic acids, penetrating through the cuticle in the form of undissociated molecules, are absorbed by living parenchymal cells. As a result of #ow along the symplast, molecules reach the phloem and, after penetration into sieve tubes, #ow with other assimilates from leaves to actively growing tissues and reproductive organs. 2,4-Dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids, after injection into shoots of kidney bean, moved basipetally and acropetally (Long and Basler, 1974). Herbicide mecoprop #ows from leaves to roots with equal intensity in sensitive and resistant biotypes of common chickweed (Stellaria media) (Coupland et al., 1990). Pesticides, derivatives of urea, are easily absorbed from nutrient solution by plant root systems, and most are quickly translocated into shoots and leaves with the transpiration stream. Cotton and snapbean seedlings absorb [C]#uometuron from nutrient solution, and the herbicide absorbed by roots is quickly translocated acropetally with the transpiration stream. When applied to leaves the herbicide moved basipetally and acropetally. These data indicate symplastic translocation of herbicide along the phloem (Rubin and Eshel, 1977). Another urea herbicide, tebuthiuron, is characterized by an analogous migration (Steinert and Stritzke, 1977). Chlorimuron, applied to leaves of soybean (Glycine max), peanut (Arachis hypogaea), and some other weeds, showed weak symplastic and apoplastic migration (Wilcut et al., 1989). It is interesting to note that chlorimuron, absorbed by the root system of yellow nutsedge (Cyperus esculentus) and purple nutsedge (Cyperus rotundus), #ows to shoots, but because it was absorbed by tubers, the herbicide was not translocated at all (Reddy and Bendixen, 1989). For carbamates acropetal translocation is typical. Good examples are translocations of carbofuran in seedlings of soybean and mung bean (
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of resistant maize (Zea mays), only basipetally (Hatzios and Penner, 1980). The same herbicide did not translocate at all or #owed slightly along the apoplast in leaves of soybean (Haderlie, 1980). 4,4-Methylene bis(2-chloroaniline), after application to leaves of di!erent plants, was absorbed by tissues, but was not translocated. It should be noted that some attempts at uni"cation of exogenous substance translocation in plants and foundation of a common theory have been made (Jacob and Noiman, 1975; Bromilow et al., 1990; Devine and Hall, 1990). Mainly for this reason the data on transport of ionogenic molecules were analyzed. Thus, for xenobiotic weak acids, a theory of phloem translocation based on the di!erent membrane permeabilities of electroneutral molecules and of their charged forms was suggested (Chamberlain et al., 1986). The re"ned model of phloem translocation of xenobiotics capable of existing in di!erent ionic forms has also been suggested (Hsu and Kleier, 1990).

As it was mentioned above, if a substance applied to plant leaves is excreted through roots, it indicates the ability of a xenobiotic to #ow quickly along the phloem. The study of xenobiotic excretion into nutrient solution indicates that the process is not always determined by the gradient of xenobiotic concentration between the root and nutrient solution. For example, [C]alachlor applied to leaves of soybean and wheat (¹riticum aestivum) is excreted through the roots into nutrient solution despite the higher herbicide concentration in nutrient solution than in roots, i.e., against the concentration gradient (Chandler et al., 1974). From these data, it may be proposed that xenobiotic excretion by the root system is a functional process characteristic of higher plants. Thus, translocating along the pholem with a stream of assimilates, phloemomobile and ambimobile xenobiotics, as most of assimilates, are excreted through the root system by a mechanism of active excretion. 4. CONJUGATE FORMATION

3. EXCRETION

Absorbed xenobiotics are partially excreted by plants through leaves or the root system. Excretion of xenobiotics absorbed through roots via leaves is a rather rare process and, due to the low concentrations of excreted compounds in the phyllosphere it cannot be always detected. The most evident example is the excretion of phenol absorbed by the root system via leaves in cane (Scirpus lacustris) (Seidel and Kickuth, 1965). It was found that detached leaves of tobacco and radish are capable of absorbing 1,2-dibromoethane from water solutions and then quickly excreting it (Isaacson, 1986). According to these data, it was suggested that volatile halogenic hydrocarbons could be taken up by plants from soil and soil water and excreted into the atmosphere. As a rule, phloemomobile, and sometimes ambimobile, substances are excreted through the root system. Excretion from roots is characteristic of phenoxyacetic acids, 2,4-D applied to leaves of intact plants is excreted through the roots in leafy spurge (Euphorbia esula) (Lingle and Suttle, 1985), rape (Brassica oleifera), and sun#ower (Helianthus annuus) (Hallmen, 1974). 2,4,5-Trichlorophenoxyacetic acid applied to leaves of snapbean is excreted through the roots (Long and Basler, 1974). 2,4-D excretion through roots of hemp dogbane (Apocynum cannabinum) seedling is carried out more intensively in the case of herbicide application to the lower than the upper leaves (Schultz and Burnside, 1980). Dicamba, absorbed by above-ground parts of plants, is partially excreted by the roots. It is interesting to mention that more intensive absorption of dicamba by leaves is accompanied by its increased excretion through roots. Picloram absorbed through the leaves of sun#ower and rape is excreted by roots (Hallmen, 1974).

4.1. Glycosylation of Hydroxylic Groups of Alcohols and Phenols Glycosylation of foreign molecules is one of the main detoxication mechanisms of higher plants. Alcohols and phenols very often undergo such transformations in plants. Thus, formation of ethyl-b-glucoside was observed during cultivation of seedlings of mung bean (Phaseolus aureus) in an ethanol-containing area (Middleton et al., 1978). After injection into apple (Malus sylvestris) geraniol was subjected to glucosylation, forming geranyl-b-D-glucoside (Wills and Scriven, 1979). The example of glycosylation of a foreign alcohol hydroxyl group could be the glucoside formation from saligenin. Thus, in experiments with broadbean seedlings, it was found that o-hydroxybenzyl-b-glucoside is formed in tissues from introduced saligenin (Pridham, 1958). Note that not the phenolic hydroxyl, but exclusively the alcohol hydroxyl group was glucosylated. The study of saligenin transformation by a suspension culture of datura (Datura innoxia) demonstrated that the main metabolite of phenol is the product of alcohol hydroxyl group glucosylation and the product of phenol hydroxyl group glucosylation is formed only in trace amounts (Tabata et al., 1976). The herbicide N-hydroxymethyl dimethoate is also subjected to glucosylation via a free primary alcohol hydroxyl group (Garner and Menzer, 1986). Pentachlorophenol was glucosylated in wheat and soybean plants, and then formed glucoside combined with malonic acid. So, b-D-glucoside and o-malonyl-b-D-glucoside conjugates of pentachlorophenol were found in tissues simultaneously (Schmitt et al., 1985). Pridham (1958), in experiments with broadbean seedlings, found that foreign mono-, di-, and triatomic phenols are converted into corresponding b-monoglucosides. See Scheme 1.

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Note that in some cases phenols are glycosylated with formation of di- and triglucosides. In wheat (¹riticum vulgare) embryo, diglucoside (gentiobioside) and triglucoside are formed from foreign hydroqinone (Harborne, 1977). Very often, during transformation of foreign substances in plant tissues, a hydroxy derivative is formed as one of the primary products and is further subjected to fast glucosylation. Thus, the example of glycosylation of an in vivo formed hydroxyl group is formation of a conjugate from the oxidation product of the systematic fungicide etirimol (Harborne, 1977). In leaves of barley (Hordeum vulgare), the aliphatic side chain (butyl group) is oxidized and the alcoholic hydroxyl formed is glucosylated. The herbicide diphenamid assimilated from nutrient medium is oxidized (N-methyl group is hydroxylated) in pepper seedlings (Hodgson and Ho!er, 1977) and in callus tissue of tobacco grown in areas containing this herbicide (Burrows and Leworthy, 1976).

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The study of 2,4-D metabolism intermediates in leaves of nine species of plants proved that the 2,4-D glucose ester was formed in large amounts in herbicide-resistant plants of wild wheat (¹riticum dicoccum), timothy (Phleum pratense), and snapbean (Chkanikov et al., 1976). In plants, besides carboxyl groups, other acidic groups are also subjected to glycosylation. Thus, in bark cuts of Hevea brasiliensis, the plant growth regulator ethephon is glucosylated by formation of b-D-glucopyranoside-1-(2chloroethyl)-phosphonate (Audley, 1979). It must be mentioned that in some cases, besides glucose, other sugars also participate in the esteri"cation reaction of carboxyl group. Thus, e.g., nicotinic acid forms arabinoside in suspensions of cultures of parsley (Petroselinum sativum) (Leienbach et al., 1975). 4.3. Glycosylation of Amino Groups

In plants, carboxyl groups of exogenous acids often undergo glycosylation. The formation of esters with glucose

Glycosylation is the widespread way of blocking free exogenous amino groups. Thus in roots, shoots, and hypocotyls of Setaria sp., 3-amino-2,5-dichlorobenzoic acid, in addition to glucose ester, forms N-glucoside (Frear et al., 1978). See Scheme 2.

is characteristic of phenoxyacetic acids. Thus, during cultivation of rice (Oryza sativa) root callus tissues in a liquid nutrient area containing 2,4-[C]D, the glucosyl ester of 2,4-D is the main product isolated. At the same time, it was found that amino acid conjugates of 2,4-D widely spread metabolite in callus tissues of some other plants were not formed. Hence, the authors concluded that the main pathway of 2,4-D metabolism in rice root callus tissue is the formation of ester with glucose (Feung et al., 1976).

During study of the glycosylation process of cytokinin synthetic analogues in rootless seedlings of radish (Raphanus sativus), it was found that the amides of 4-(purin6-yl-amino)butanoic acid, 6,(34,-dimethoxybenzyl-amino)purine, and 6-benzylaminopurine are converted into corresponding 7-glucopyranosides. Adenine and methylaminopurine were not glucosylated under these conditions (Letham et al., 1978). As a result of absorption of 6-benzylaminopurine through roots in snapbean seedlings,

4.2. Glycosylation of Carboxyl Groups of Organic Acids

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riboside was formed (Ramina et al., 1979). In wheat plants and in suspensions of cultures of wheat and soybean, 4chloroaniline and 3,4-dichloroaniline were subjected to glucosylation, and simultaneously the formation of malonic conjugates took place (Wilkner and Sandermann, 1989). The herbicide metribuzin in tomato (¸ycopersicon esculentum) after glucosylation, forms a conjugate with malonic acid (Frear et al., 1983b). According to other data, during a study of metribuzin metabolism in tomato biotypes with high, medium, and low sensitivity to herbicide, it was reported that in all biotypes, N-glucoside is the dominant metabolite formed (Smith et al., 1989). 4.4. Conjugation of Carboxyl Groups with Amino Acids Conjugation with amino acids is a wide spread reaction of carboxyl groups of exogenous compounds. A study of 2,4-D metabolism in Glycine species demonstrated that, in resistant species, the primary metabolite is the glucoside conjugate of 4-oxy-2,4-D, but, in sensitive species, conjugates with amino acids are formed (White et al., 1990). It was found that in callus and the di!erentiated root tissues of soybean (Davidonis et al., 1978), in tissue cultures of maize endosperm, and in medullar parenchyma of tobacco, carrot, and sun#ower (Feung et al., 1975), 2,4-D forms conjugates with glutamic and aspartic acids. See Scheme 3.

4.5. Conjugation of Xenobiotics with Peptides One of the most important detoxication abilities of higher plants is the conjugation of xenobiotic molecules with tripeptide glutathione. This detoxication pathway is most characteristic of symmetric triazines, chloroacetamides, and other halogen-containing xenobiotics. A study of atrazine transformation in 53 representatives of herbaceous plants

(Festucaeae, Avenae, ¹riticeae, Paniceae, Andropogenae, Eragrosteae, Chlorideae) revealed that in all of them, the herbicide formed conjugates with glutathione (Jensen et al., 1977). Atrazine transformation product analysis in herbicide-resistant and -sensitive herbs revealed that in the resistant plants big bluestem (Andropogon qerardii vitman) and switch-grass (Panicum virgatum), the major metabolite was atrazine conjugate with glutathione. In plants sensitive to atrazine, such as Indian grass (Sorghastrum nutans) and sideoats grama (Bouteloua curti pendula Michx. Torr.) mainly the product of herbicide N-deethylation was formed (Weimer et al., 1988). Metabolism of atrazine in suspensation cultures of wheat and potato indicated that in wheat cells, the herbicide was transformed by N-deethylation, but in potato cells it was transformed by conjugation with glutathione. In potato cells, the enzyme glutathione-S-transferase, capable of using atrazine as a substrate, was found (Edwards and Owen, 1989). Conjugation with glutathione is characteristic of chloroacetamide herbicides (Le Baron et al., 1988). For example, acetochlor forms conjugates with glutathione in seedlings of maize, morning-glory (Ipomoea purpurea), "eld bindweed (Convolvulus arvensis), cocklebur (Xanthium pensylvanicum), and velvetleaf (Abutilon theophrasti) (Breaux 1987). Applied to coleoptiles of maize seedlings, alachlor and metolachlor formed glutathione conjugates. Isolated from seedlings of maize, glutathioneS-transferase, catalyzing xenobiotic conjugation with

glutathione, had three times higher activity when alachlor was used as a substrate (O'Connel et al., 1988). In rice seedlings, active transformation of pretilachlor into glutathione conjugate was observed (Han and Hatzios, 1991). Substances such as benzyl chloride and propachlor form conjugates with glutathione both ways, enzymatically and nonenzymatically (Han and Hatzios, 1991). See Scheme 4.

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Another plant tripeptide, homogluthatione, also participates in conjugation reactions with xenobiotics. It di!ers from glutathione in containing P-alanine instead of glycine. Formation of homoglutathione conjugates is characteristic mainly of soybean plant. Thus, the herbicide propachlor in soybean seedlings forms a conjugate with homoglutathione (Lamoureux and Rusness, 1989). Homoglutathionic conjugate, as the main product of chlorimuron-ethyl transformation, is formed in soybean plants (Brown and Neighborns 1987; Brown et al., 1990). Homoglutathionic conjugates of acetochlor are formed by other plants, in particular by soybean, mung bean, and alfalfa (Medicago sativa) (Breaux, 1987). Glutathione and homoglutathione conjugate with a xenobiotic hydroxyl group formed in vivo. Thus, acifluorofen, the derivative of diphenyl ether, is cleaved in soybean seedlings into 2-nitro-5-oxybenzoic acid, which couples to homoglutathione via a hydroxyl group (Frear et al., 1983a). One more mechanism characteristic of xenobiotic binding with glutathione and homoglutathione is the reaction with aklylthio groups. In maize seedlings S-ethyldipropyl thiocarbamate conjugates with glutathione via an ethyl group (Lay and Casida, 1976; Carringer et al., 1978). It is supposed that in this particular case the herbicide is oxidized into the corresponding sulfoxide, but the latter conjugates with glutathione catalyzed by glutathione-Stransferase. In soybean plants, metribuzin combines with homoglutathione via a methylthio group (Frear et al., 1985). Microsomes from cell suspension cultures of parsley (Petroselinum hortense) and, soybean and from primary leaves of pea seedlings oxidize benzo[a]pyrene by conjugation with glutathione (Trenck and Sandermann, 1980). Numerous data indicates that phenol (oxybenzene) is not glycosylated in intact plants. A study of [1,6-C]phenol metabolism in sterile seedlings of maize, pea, and pumpkin (Cucurbita pepo) demonstrated that phenols form conjugates with low-molecular-weight peptides in plants (Chrikishvili et al., 1977; Ugrekhelidze et al., 1997). Other monoatomic phenols also form peptide conjugates in plants: a-naphthol in maize, pea, and pumpkin seedlings (Ugrekhelidze and Arziani, 1980; Ugrekhelidze et al., 1983); onitrophenol in pea seedlings (Ugrekhelidze and Arziani, 1980; Ugrekhelidze et al., 1983); 2,4-dinitrophenol in maize, pumpkin, and pea seedlings (Arziani et al., 1983). Phenols are covalently bound to peptides via hydroxyl groups. The amino acid composition of peptides participating in conjugation with phenols varies. In plants treated with phenol, the content of low-molecular-weight peptides increases (Ugrekhelidze et al., 1983). In some plants, conjugation with low-molecular-weight peptides seems to be an important detoxication pathway for exogenous monoatomic phenols. Phenoxyacetic acids introduced into plant tissues form peptide conjugates. In sterile seedlings of maize and snap-

bean, phenoxyacetic and 2,4-dichlorophenoxyacetic acids form conjugates with low-molecular-weight peptides. In vine, the conjugates of these acids with peptides are formed (Mithaishvili et al., 1979; Kakhniashvili et al., 1979). As a result of hydrolysis of phenoxyacetic and 2,4-dichlorophenoxyacetic acids peptide conjugates, from 6 to 10 amino acids are formed (Kakhniashvili et al., 1979; Kakhniashvili, 1988; Durmishidze et al., 1982). In cereals, peptides/proteins participating in conjugation with phenoxyacetic acid contain from 2 to 220 amino acid residues (Chkanikov, 1985; Chkanikov et al., 1982). 5. OXIDATIVE DEGRADATION

5.1. Hydroxylation Introduction of a hydroxyl group into a xenobiotic molecule increases its polarity and hydrophilicity. In some cases, hydroxylation is the primary detoxication reaction, followed by the processes of profound oxidation and conjugation. Study of metabolic products of exogenous alkanes and N-alkali derivatives indicates that oxidative degradation of these molecules often starts with hydroxylation of alkyl groups. Though it is not always possible to isolate and identify the corresponding hydroxy derivatives, the products of their further metabolism indicate formation of intermediates. It was found that low-molecular-weight [C }C C]alkanes absorbed by leaves are subjected to   oxidative transformation to CO (Durmishidze and Ug rekhelidze, 1967; 1968a, b, 1975). Analyses of the products of metabolism enable us to suppose that these hydrocarbons are oxidized monoterminally, with intermediate formation of corresponding primary alcohols, by their following oxidation to carbonic acids. Hydroxylation of alkyl groups is a characteristic reaction of urea herbicide transformations in plants. In this case, an urea herbicides, alkyl (N-alkyl) groups bound to nitrogen atoms are subjected to hydroxylation. Fast oxidation of the hydroxylalkyl groups formed is accompanied by the process of hydroxylation, as a result of which dealkylated product is formed. It is supposed that N-dealkylation is the primary metabolic transformation of N-methylphenyl urea herbicides. In some cases the hydroxylic groups formed are immediately glucosylated. So, in gosspium cotton leaves the b-D-glucoside of the hydroxymethyl derivative of monuron is formed from [C]monuron (Frear and Swanson, 1972). Enzymatic cleavage or acidic hydrolysis of this glucosidic bond leads to the formation of corresponding demethylated products. Simultaneously, the formation of labeled formaldehyde is observed. An analogous glucoside of intermediate products of durone hydroxymethyl derivative was isolated from sugar cane (Saccharum o.cinarum) (Liu et al., 1978). Products of hydroxylation of methyl groups (hydroxymethyl derivatives) are formed during the transformation of urea herbicides in plants: buturon in wheat (Hague

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et al., 1977), monolinuron in spinach (Spinacia oleracea) (Schuphan and Ebing, 1978), terbuthiuron in sugar cane (Loh et al., 1978), chlorotoluron in wheat (Gross et al., 1979), Chlorotoluron is hydroxylated in two positions: hydroxylation of the N-methyl group leads to demethylation, and hydroxylation of the methyl group bound to the aromatic ring leads to the formation of stable products (in contrast to the N-hydroxymethyl group, the C-hydroxymethyl group is stable (Gross et al., 1979). Both products are formed in herbicide-resistant and -sensitive varieties of wheat (Cabanne et al., 1985). After 24 h of treatment the product of N-demethylation (about 5.8%) as the dominant metabolite is formed; the C-hydroxymethyl derivative presents as a minor component (about 1.4%). In rice plants, absorbed 2-secbutylphenol-N-methylcarbamate is subjected to hydroxylation by sec-butyl as well as by N-methyl groups (Ogawa et al., 1976). sym-Triazines are subjected to N-dealkylation in plants. It is supposed that in the case of triazine herbicides, N-dealkylation is proceeded by hydroxylation of a side chain (alkali group), but attempts to isolate the corresponding hydroxy derivatives are not always successful. For example, atrazine and symazine are easily dealkylated, though corresponding hydroxy derivatives were not isolated (Wichman and Byrnes, 1975; Pillai et al., 1977; Weimer et al., 1988). In suspension cultures of potato and wheat the hydroxy derivative of atrazine was not found, but the product of hydroxylation of another sym-triazine, terbutryne was identi"ed, and appeared to be the basic metabolite (Edwards and Owen, 1989). In alfalfa plants treated with [2C]terbacil, among other herbicide metabolites, the product of its hydroxylation via the methyl group was found (Rhodes, 1977). Hydroxylation of the methylene group of xenobiotic molecules has also been reported. Thus, carbofuran is hydroxylated at the C -atom in barley (Hordeum vulgare),  maize (Penner and Early, 1973), and strawberry (Fragaria vesca) (Archer et al., 1977). The metabolism of [C]cyclohexane in plants indicates that the ring of this hydrocarbon is cleaved with the formation of aliphatic products. It was supposed that in plants the "rst step of cyclohexane transformation is its hydroxylation into cyclohexanol (Ugrekhelidze, 1976). The "rst step in the metabolism of aromatic hydrocarbons is the formation of hydroxy derivatives. In plants [1,6-C]benzene is cleaved with the formation of aliphatic products (muconic and fumaric acids) (Durmishidze et al., 1974a). The same products are formed from benzene in fruits too (Durmishidze et al., 1974c). From sterile seedlings of maize, pea, and pumpkin to which a solution of labeled benzene, was applied, the labeled phenol was isolated. The phenol was present in tissues in negligible amounts, though the degree of benzene label incorporation into aliphatic products was much higher (Ugrekhelidze et al., 1977).

Benzo[a]pyrene absorbed by plants is subjected to oxidative degradation and a signi"cant portion of the carbon atoms are incorporated into the aliphatic compounds (Devdariani and Durmishidze, 1983, Devdariani, 1988). The analogous transformation of this toxicant was determined in cell suspension cultures (Harms, 1975; Harms et al., 1977; Trenck and Sandermann, 1978). For such polycyclic hydrocarbons as benz[a]anthracene and dibenzanthracene, the same kind of transformation is observed (Devdariani and Kavtaradze, 1979; Devdariani et al., 1979; Devdariani, 1988). It is supposed that hydroxylation is the primary reaction in the transformation of polycyclic hydrocarbons in plants (Devdariani, 1988). Hydroxylation of the aromatic ring is an important step in phenoxyacetic acid deep transformation in plants; at the same time, the hydroxyl group introduced is often subjected to glycosylation. Phenoxyacetic acid is hydroxylated mainly at position 4 of the aromatic ring. A 16-fold increase in hydroxylase activity was observed during the formation of phenoxyacetic acid hydroxylated metabolite (4-hydroxyphenoxyacetic acid) in oat (Avena sativa) seed embryos (Hutber et al., 1978). Phenoxyacetic acids halogenated in the aromatic ring are hydroxylated at unsubstituted carbon atoms of the benzene ring. But hydroxylation of 2,4-D often occurs at position 4 and an atom of chlorine moves to positions 5 or 3. For example, identi"cation of hydroxylated 2,4-D compounds in di!erent plants such as wild buckwheat (Polygonum convolvulus), leafy spurge (Euphorbia esula), yellow foxtail (Setaria glauca), wild oat (Avena fatua), wild mustard (Brassica caber) perennial sowthistle (Sonchnus arvensis), kochia (Kochia scoparia) has revealed that 2,5-dichloro-4-hydroxyphenoxyacetic acid is the dominant metabolite in all plants studied (Fleeker and Steen, 1971). The study of the transformational ability of 2,4-D in herbicide-sensitive andresistant Glycine sp. demonstrates that the 4-hydroxy derivative of 2,4-D, in particular, was intensively formed in resistant species, exclusively in the form of glucoside (White et al., 1990). The herbicide diclofop (Shimabukuro et al., 1987) and its methyl ether (diclofopmethyl) (Tanaka et al., 1990) are similarly hydroxylated in plants, though in some plants, the product formed by glucosylation of carboxyl group is the dominant metabolite (Jacobson and Shimabukuro, 1984). The tolerance of di!erent biotypes of ryegrass resistant and sensitive to diclofop does not depend on its metabolic products, as in stems and roots of both biotypes; a considerable amount of phytotoxic diclofop as well as its conjugates and hydroxylated ring derivatives are formed (Shimabukuro and Ho!er, 1991). The enzyme catalyzing diclofop transformation into 2-b-(2,5-dichloro-4-hydroxyphenoxy)phenoxy propionic acid was isolated and puri"ed from etiolated wheat seedlings (McFadden et al., 1989).

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Benzoic acid and its derivatives are hydroxylated by di!erent plants. Benzoic acid is hydroxylated simultaneously in o- and p-positions and sometimes both hydroxy acids are present in tissues simultaneously. Dicamba is hydroxylated at position 5 and this product is the main herbicide metabolite in many plants (Chang and Vanden Born, 1971; Robocker and Zamora, 1976). Isopropyl carbanilate and isopropyl m-chlorocarbanilate are also subjected to hydroxylation of the aromatic ring at di!erent positions with subsequent conjugation of the hydroxy derivatives formed with glucose. Thus, in alfalfa plant absorbed isopropyl carbanilate is transformed with the formation of the main component isopropyl-4-hydroxycarbanilate (Still and Mansager, 1975b). Among wheat, sugar beet, and alfalfa plants cultivated in nutrient solution and in soil, formation of isopropyl-4-hydroxycarbanilate was observed only in wheat, and, in addition, 4-hydroxy and 2hydroxy derivatives were formed (Burt and Corbin, 1978). Isopropyl-3-chlorocarbanilate is hydroxylated with the formation of isopropyl-3-chloro-2-hydroxycarbanilate or isopropyl-3-chloro-4-hydroxycarbanilate (Still and Mansager, 1975b). The products of aromatic ring hydroxylation in most cases are immediately subjected to glycosylation via the hydroxyl group formed. That is why isolation of hydroxylation products is not always possible. Thus, the herbicide bentazon is hydroxylated into 6-hydroxybentazon or 8hydroxybentazon, followed by glucosylation, though in plant tissues treated with bentazon together with glucosides the initial herbicide hydroxy derivatives are also found (Connelly et al., 1988; Leah et al., 1989a,b). Plants resistant to bentazon intensively transform this herbicide, whereas in sensitive plants this process occurs slowly. A similar picture is observed during bentazon transformation by suspension cultures of the same plant cells (Sterling and Blake, 1988, 1989, 1990). The herbicide chlorosulfuron is hydroxylated by the aromatic ring in wheat seedlings and the hydroxy derivative formed undergoes immediate glucosylation (Sweetser et al., 1982). On the other hand, in seedlings of "ber #ax (¸inum usitatissimum), the same herbicide is hydroxylated exclusively by the methyl group of the heterocyclic ring (Hutchinson et al., 1984). Herbicides of the sulfonylurea type, as a rule, are initially subjected to hydroxylation at the aromatic or heterocyclic ring or at the aliphatic radical, and consequently the hydroxy derivatives formed are glycosylated (Beyer et al., 1988). Thus, the sulfonylurea herbicide primisulfuron is hydroxylated by the pyrimidine ring, but benzene ring hydroxylation does not take place in plants (Echinochloa cross galli) (Neighbors and Privalle, 1990). On the other hand, microsomes from etiolated maize seedlings catalyze hydroxylation of this herbicide by both benzene and pyrimidine rings (Fonne-P"ster et al., 1990).

9

Special attention must be paid to methalaxyl transformation by cell suspension cultures of lettuce (¸actuca sativa) and vine. In these plants, not only the aromatic ring and the methyl group bound to it, but apparently also the methyl group of the N-methoxyacetyl radical, are subjected to hydroxylation simultaneously (Cole and Owen, 1987). Finally, the rather rare hydroxylation at the amide nitrogen must be mentioned. The herbicide phenmedipham undergoes such hydroxylation in leaves of herbicide-resistant and -sensitive types of sugar beet. The transformation rate in resistant leaves was much higher (Davies et al., 1990). 5.2. Reactions of the Hydrolytic Cleavage Type In most cases in xenobiotic molecules primarily the esteric bond is cleaved. For example, transformation of triclopyr esters in resistant wheat, tolerant barley, and sensitive common chickweed plants proved that 3 days after treatment, 94% of both esters absorbed by plants were hydrolyzed. Triclopyr formed as a result of hydrolytic cleavage immediately conjugates with glucose and aspartic acid (Lewer and Owen, 1990). The herbicide assert is a mixture of two isomeric imidazolinonaryl carboxylates. The study of its metabolism in resistant maize and wheat and sensitive wild oat (Avena fatua) proved that in all three plants this herbicide is hydroxylated by the methyl group of the aromatic ring. Metabolism of three derivatives of sulfonylurea in soybean plants indicated that intensities of cleavage of the esteric bonds are di!erent (Brown and Neighbors, 1987; Brown et al., 1990). Thifensulfuron-methyl is intensely hydrolyzed by the formation of the corresponding thifensulfuronic acid. The half-time of decomposition of methyl ester of thifensulfuric acid in tissues was 4}6 h. Another ester, chlorimuron-ethyl, was deesteri"ed too, but less intensely, although the main process was xenobiotic conjugation with homoglutathione. The third ester, metsulfuron-methyl, under the same conditions and in the same soybean seedlings, did not undergo deesteri"cation. If a xenobiotic contains an esteric bond, the latter undergoes primarily transformation. In the absence of an esteric bond, other easily oxidized side groups of the diphenyl ether system are transformed and only in their absence does cleavage of ether bonds take place in wheat seedlings (Jacobson and Shimabukuro, 1984; Tanaka et al., 1990), oats (Jacobson and Shimabukuro, 1984), suspension cultures of oat (Shimbukuro et al., 1987), and ryegrass (Shimabukuro and Ho!er, 1991). Difenopenten-ethyl is deesteri"ed in soybean and wheat seedlings (Shimabukuro et al., 1989). The highly selective diphenyl ether herbicide AKH-7088 is metabolized in soybean plants by complete oxidation of the side chain (Kouji et al., 1990). On the other hand, in soybean plants the aci#uorofen molecule is cleaved at ether bond, forming the corresponding

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phenols, which immediately conjugate with glucose (and latter with malonic acid) and homoglutathione (Frear et al., 1983). In the same way #uorodifen (Eastin, 1971; Shutte and Golfman, 1975), nitrofen (Shutte and Golfman, 1975), and other diphenyl ethers are cleaved, forming the corresponding phenols. All these phenols are immediately transformed into conjugates. See Scheme 5.

(Ugrekhelidze, 1976). Based on the experimental data, it has been supposed that transformation of the methane proceeds according to the following scheme:

6. DEEP OXIDATION

The above-presented data summarize those initial transformations of foreign compounds that penetrate the plant cell. Obviously, the majority of low-molecular-weight substances formed after transformation of exogenous molecules, similarly to secondary metabolites, accumulate in vacules, and their further transformation proceeds slowly; however, this has not been experimentally con"rmed. In experiments devoted to absorption and transformation of xenobiotics labeled with radioactive carbon, the evolution of CO is reported. Therefore, simultaneously with the main transformations, at which the basic structure of xenobiotic molecules is maintained (formation of conjugates), deep oxidation of xenobiotics also proceeds. As was mentioned above, plants absorb alkanes and cycloalkanes from the environment and metabolize them. The experiments with C-labeled hydrocarbons proved that sterile plant seedlings, placed in an atmosphere containing low-molecular-weight alkanes (C }C ) or cyc  lohexane, absorb these compounds, exposing their molecules to deep transformations. In a plant cell, these

Obviously, the long-chain alkanes are subjected to a similar transformation. For instance, after 40 min of incubation of leek leaves with an emulsion of exogenous [C]octadecane in water, 9.6% of the total label is detected in esters, 6.4% in alcohols, and 4% in organic acids (Cassagne and Lessire, 1975). Using benzene and phenol labeled with radioactive carbon, it has been proven that higher plants are able to metabolize exogenous aromatic hydrocarbons and simple phenols via aromatic ring cleavage (Durmishidze et al., 1974d). The carbon atoms of these compounds, as a result of cellular metabolism, are incorporated into the molecules of such endogenous metabolites as carbonic acids and amino acids. Similar data were obtained for toluene (Tkhelidze, 1969; Jansen and Olson, 1969; Durinishidze et al., 1974b), a-naphtol (Ugrekhelidze and Kavtaradze, 1970) and benzidine (Durmishidze et al., 1979). It has been found that during oxidation of benzene and phenol by plant total enzyme preparations, the product of ring cleavage, muconic acid, is formed; it is supposed that during this process, pyrocatechin is formed as an intermediate (Durmishidze et al., 1969). See Scheme 6.

hydrocarbons are oxidized and form the corresponding carbonic acids. Alkanes undergo monoterminal oxidation, while cyclohexane oxidizes via ring cleavage. And the evolution of CO in the dark which can be quantitatively  estimated is observed. Consequently, Krebs cycle acids as well as amino acids are formed. So, the carbon skeleton of exogenous hydrocarbons participates in cell metabolism

Muconic acid is often detected in plants, absorbing benzene or phenol. The further oxidation of muconic acid leads to the formation of fumaric acid, which is almost always labeled in plants incubated with radioactive benzene or phenol. It is interesting that the cleavage of the aromatic ring of endogenous substrates occurs similarly: from 3,4-dihydroxybenzoic acid, 3-carboximuconic acid is formed (Tateoka, 1979).

CH PCH OHPHCHOPHCOOH   Pcell regular metabolism.

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11

Higher phenoxyalkyl-carbonic acids often undergo such oxidation in plants. So, 2,4-dichlorophenoxybutyric acid is converted into 2,4-D (Hawf and Behrens, 1974; Taylor and Wain, 1978; McComb and McComb, 1978). 7. ENZYMATIC REACTIONS

7.1. Cytocrome P450-Dependent Monooxygenation Detoxication of the organic xenobiotic toxicants in plants proceeds basically by enzymatic oxidative degradation. The "rst reaction in this process is monooxygenation (hydroxylation), where the active insertion of oxygen into a toxicant molecule takes place; the source of oxygen is atmospheric air. See Scheme 7.

According to the generation and realization of an active oxygen, oxidases are divided into two groups: (1) using previously activated oxygen (catalase, peroxidase, etc.); (2) activating oxygen themselves and providing for its immediate insertion into the substrate molecule. The second group is divided into two subgroups: enzymes that use the reductive equivalents of cofactors for oxygen activation (NADH and NADPH-dependent monooxygenases) and those, that do not need the cofactors (ascorbatoxidase, phenoloxidases, etc.).

For further hydroxylation, cytochrome P450 uses the products converted by monooxygenation. Every subsequent reaction probably must be less dependent on cytochrome P450 for two reasons: (1) The products of reactions accept the de"nite polarity and their a$nity to enzymes is decreased (Gordeziani et al., 1991a). (2) In some cases, after the reactions, cytochrome P450 is partially inactivated (Karuzina and Archakov, 1994); therefore, the formation of only primary products signi"cantly depends on hemoprotein. Monooxygenation of aromatic amines is accompanied by their N-oxidation. It is important to determine the subsequent transformation of highly toxic N-oxides. Cytochrome P450 participates in the formation of Noxides from primary amines, while secondary and tertiary amines are oxidized by microsomal NADPHspeci"c #avoproteins (Archakov, 1975). The N-oxides formed are transformed into N-demethylated (hydroxylated) compounds again by cytochrome P450. See Scheme 9.

Among the well-known oxidative reactions of toxicants are hydroxylation, deamination, desulfuration, N- and S-oxidation, and oxidative degradation of acyclic and cyclic hydrocarbons. One substance may be oxidized in di!erent ways. It is stipulated by a chemical structure of the toxicant, particularly by the disposition of hydrophobic groups in the molecule and their electronic structure. Microsomal oxidation of N,N-dimethylaniline can be a good example. Dimethylaniline contains two hydrophobic groups, phenyl and methyl radicals; and so microsomal NADPH-dependent monooxygenase has two alternative ways of dimethylaniline oxidation. See Scheme 8.

Cytochrome P450-dependent N-oxidation takes place in the case of aniline too: (Parke, 1973), according to Scheme 10. p-Hydroxylation of aniline is carried out comparatively faster, because the p-aminophenol produced has higher polarity, as compared with N-hydroxyaniline. In plants (soybeans, Glycine hispida, and maize) the N-demethylation reaction proceeds much more intensely than p-hydroxylation of aniline (Gordeziani et al., 1979). The enhancement of the polarity of xenobiotic molecules except N-demethylation is stipulated by O-demethylation. It is well exposed in the case of anisole and its nitro-

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derivative (Ugrekhelidze and Durmishidze, 1984), as shown in Scheme 11.

Hydroxylation of the aromatic ring is extremely important to hydrocarbon biodegradation. Microsomal monooxygenase has the ability to hydroxylate mono- and polycyclic aromatic compounds. In plants, this was con"rmed by the oxidation of benzene, naphthalene, and benzo[a]pyrene (Durmishidze et al., 1974 a}d; Miminoshvili et al., 1986; Trenck and Sandermann, 1980). Further oxidation of aromatic compounds leads to quinone formation, in most cases followed by cyclic cleavage. In compounds that do not contain substituting groups, e.g., benzo[a]pyrene, there always exists an L-section capable of undergoing hydroxylation. For benzo[a]pyrene, such a section is the position at the sixth carbon atom (Ugrekhelidze and Durmishidze, 1984). 7.2. Components of Monooxygenase System In plants, all components of the microsomal monooxygenase complex are identi"ed: the initial section of electron transfer NADPH-cytochrome P450 reductase (EC 1.6.2.4); the intermediate carrier, cytochrome b ; and the  terminal acceptor of electrons, cytochrome P450 (EC 1.14.14.1). In the system when NADH is used as the only source of reductive equivalents, the existence of one additional carrier, NADH-dependent #avoprotein, becomes necessary (West et al., 1974; Lu et al., 1974). It is worthwhile to

underline that NADH may be oxidized also by the NADPH-dependent redox system. In this case the attendance of b , as the medium carrier, is not needed (Hansikova  et al., 1994). Cytochrome P450 and appropriate NADPH-dependent reductases were isolated from tulip (¹ulipa fosteriana) and puri"ed to an electrophoretically homogenous state. Their molecule weights were 54.2 and 77.6 kDa, respectively. In the reconstructed system, they exhibited ability for xenobiotic oxidation (Menting et al., 1994). The existence of cytochrome C (P450) reductase has been reported in microsomes of other plants. A puri"ed preparation of the enzyme from Petunia hyspida contains two chains of di!erent molecular weights (75 and 81 kDa) (Benveniste et al., 1991). Investigations carried out on artichoke (Cynara scolymus) indicate the existence of several forms of reductase. Other data indicate the functioning of di!erent forms of this enzyme in plants (Madyastha et al., 1993). In animal organisms only one molecular form of reductase has been found; in plant microsomes there are at least three functional forms. In microsomes from some plants (Catharanthus roseus), the existence of NADH-cytochrome b reductase has been  established, which in the presence of NADH catalyzes the reduction of cytochrome b . This reductase easily transfers  electrons from NADH to ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome C. In addition, it was demonstrated that the reduction of cytochrome C is carried out via cytochrome b .  For electron transfer from NADPH-cytochrome P450 reductase to cytochrome P450, interactions between these proteins are required. This condition becomes especially important during the regulation of xenobiotic monooxygenation reactions. Reductase contains the membranecoupled domain, which has decisive signi"cance in its binding with cytochrome P450 (Strobel et al., 1989). If this domain is removed from reductase, the transfer of electrons to hemoprotein is stopped. Due to the membrane localization of cytochrome P450 and reductase, their interactions in plants are limited. An important functional characteristic of cytochrome P450-containing monooxygenases, explaining the wide spectrum of their action, is the intracellular distribution of these proteins. Prokaryotic organisms contain this enzyme system in soluble form. In eukaryotic organisms hemoprotein is localized in the microsomal membrane. Cytochrome P450 belongs to those unique proteins that have the ability to build into membrane. All three components of cytochrome P450-containing monooxygenases of liver microsomes are membrane-bound proteins (Archakov, 1983). It was reported that NADPH-cytochrome P450 reductase in lung cells of animals is located in cytosol (Lee and Dinsdace, 1995). Some eukaryotic microorganisms contain cytochrome P450 as a soluble protein (Duppel et al., 1973).

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In microsomes of soybean seedling roots, cytochrome P450 reductase exists in both membrane-bound and soluble forms (Gordeziani et al., 1991b). The importance of the intracellular distribution of oxidative enzyme systems in toxicants deserves special attention. The study of dimethylaniline NAD(P)H-dependent oxidation in the leaves of perennial trees and bushy plants [nut (Juglans regia), willow, poplar (Populus gracilis bossh), and ¸igustrum vulgare] often used as greenery in settlements, show that this process is intense in chloroplasts (Togonidze et al., 1990). With aging, leaf microsomes lose their ability for xenobiotic monooxygenation. It has been found that the chloroplasts have an ability to hydroxylate benzene, phenol, and benzoic acid (Ugrekhelidze, 1976). The hydroxylation of [1,6-C]benzene into [C]phenol in vitro is suppressed by diethyldithiocarbamate. The oxidation is catalyzed by copper-containing phenolase via o-hydroxylation of monophenols and oxidation of diphenols, according to the following mechanism (Koon, 1968): monophenol#O #2ePo-diphenol#O\  o-diphenol#O Po-quinone#H O.    This enzyme system is an oxidase of mixed function. The coexistence of an electron donor is essential to exhibit its activity. Consequency the codes and names of o-diphenol oxidase (EC 1.10.3.1) and p-diphenol oxidase (EC 1.10.3.2) have been changed to monophenol monooxygenase (EC 1.14.18.1). Chloroplasts play a signi"cant role in oxidation of monoatomic phenols (Sato et al., 1968), trans-cinnamic acid (Gestetner and Coon, 1974), and alkanes and arenes (Zenser et al., 1983; Ugrekhelidze and Durmishidze, 1984). Such diamines as benzidine are oxidized by chloroplast peroxidase and catalase. The reaction involves oxidation of amino groups in the toxicant molecule and aromatic ring cleavage, which is preceded by hydroxylation, formation of o-aminophenolic derivative, and then transformation into iminoquinone (Durmishidze et al., 1979). It is important to underline that chloroplasts contain the set of enzymes providing for the complete degradation of toxicants from ohydroxylation to cleavage of the aromatic ring. Cultivation of soybean and ryegrass in light leads to decreases in the activities of all components of the monooxygenase system (Gordeziani et al., 1991b). Rapid reduction of hemoprotein was reported in mung bean seedlings grown in the dark and transferred to light (Hendry et al., 1981). Based on the above-mentioned and existing literature data (Durst, 1991), the following suggestions has been made: the light-dependent reduction of enzyme activity should be provided by the degradation of heme in cytochrome P450.

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A study of the transformation of monoatomic phenols, chlorogenic acid, and p-phenylenediamine in chloroplasts of spinach demonstrated that light signi"cantly intensi"ed the oxidative reactions catalyzed by phenoloxidase (Tomova et al., 1966). In chloroplasts of snapbean, diaminobenzidine is oxidized only in light (Porat et al., 1978; Wrischer, 1978). It seems that hydrocarbon oxidation in light is connected with the function of chloroplasts; for instance, intact chloroplasts of pea much more intensely oxidize such aromatic compounds as benzene and phenol in light. The chloroplasts being incubated with benzene perform aromatic ring hydroxylation in the light. Similar photosensitization is characteristic of the total chlorophilic preparation (Ugrekhelidze and Phiriashvili, 1979). Obviously, chloroplasts play an important role in detoxication of xenobiotics, but the oxidative ability of xenobiotics is not the function of only microsomal monooxygenases. Despite that, the signi"cance of this system cannot be ignored. Cytochrome P450-containing monooxygenase represents an adaptive self-regulating system. It is also important that enzymatically oxidized products can be used for the regulation of cell metabolisms (Kovalev and Malenkov, 1980). In comparison with other eukaryotic organisms, plants have both membrane-bound and soluble forms of monooxygenase system, which signi"cantly increases their detoxication ability. Organic toxicants, according to their biological activities and concentrations, have di!erent in#uence on plant cell metabolism. Small doses of toxicants do not expose visible changes of metabolic processes and cellular reproduction, but are characterized by highly expressed inductive abilities (Durmishidze, 1979). Obviously, penetration of xenobiotic in small doses causes such physiological and biochemical deviations which can be completely restored by the internal potential of the cell. Thus small doses of xenobiotics are metabolic concentrations that, for their complete detoxication, the oxidative power of the cell is enough. In the case of monooxygenation, the NAD(P)H-dependent redox system completely provides the transmission of electrons to cytochrome P450. In the ordinary ecological environment, the intensity of toxicant #ow from biosphere to plant is more or less perpetual and, in the great majority of cases, corresponds to the metabolic dose. Hence, oxidative degradation of small xenobiotic doses is within the abilities of general cell metabolism. For NADPH-dependent monooxygenation, all the necessary components are provided by the cellular metabolism of cytochrome P450 from biosynthetic processes (Gordeziani et al., 1987): NADPH, from photosynthesis and the pentose cycle; electrons, from the respiratory chain (Moldeus et al., 1973; Gordeziani et al., 1986), and free radicals, from the peroxidation of membrane lipids (Dmitriev et al., 1983; Kurashvili and Gordeziani, 1990).

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7.3. The Induction of Cytochrome P450

7.4. Inactivation and Transformation of Cytochrome P450

While plants grow in xenobiotic-containing medium, the quantitative content of cytochrome P450 is increased. Nearly all xenobiotics examined have an inductive nature. The inductive abilities of such xenobiotics as phenobarbital, clo"brat, aminopyrine, and 2,4,-D and herbicides propanil, chloracetamide, thiocarbamate, chlortoluron, bentazon, and others (SalauK n, 1991) have been described. The inductive e!ect of each particular xenobiotic depends on its chemical nature and the inductive abilities of intermediate metabolites. Some of intermediates appear to be reactive and most of them cause the inactivation of cytochrome P450 and its further conversion into cytochrome P420. Good examples of these intermediates are dimethylaniline and benzo[a]pyrene. Growth of soybean and ryegrass in dimethylaniline-containing solution leads to the intensive induction of an active form of hemoprotein. Dimethylaniline oxidation (N-oxidation, N-demethylation, p-hydroxylation) metabolites cannot provide such &&active'' intermediates as benzo[a]pyrene oxidation. The rate of enzymatic oxidation of benzo[a]pyrene is much higher than its autooxidation. The products of photoxidation have a toxic e!ect on plants. Incubation of plants with benzo[a]pyrene causes the formation of dioles, phenols, and polyhydroxylated metabolites. The transformation is carried out by generation of intermediate products*epoxides. Among the products of benzo[a]pyrene oxidation quinones are always present (Kirso et al., 1983; Warshawsky et al., 1983). The &&aggressiveness'' of these substances is expressed by formation of active radicals of oxygen, which cause the irreversible conversion of cytochrome P450 (Khatisashvili et al., 1991). For instance, enhancement of the peroxidation of fatty acids also leads to the generation of active forms of oxygen. Reduction of lipid content is also marked in microsomes of euglena (Euglena gracilis) when ethanol is used as an inducer. The redoubling of content of cytochrome P450 is always accompanied by its conversion into P420 (Trullier-Bruston et al., 1991). The induction of cytochrome P450 is reached not only by xenobiotic toxicants. Substrates of cytochrome P450 of endogenous origin, e.g., cinnamic acid, are characterized by the analogous e!ect. It should be mentioned that the inductive e!ect of xenobiotics is comparatively higher than that of endogenous metabolites (Gordeziani et al., 1989). The study of the enzymatic activities of microsomes of plants grown in inducer-containing solution, demonstrated that the enzyme system of seedlings transforms intensely not only toxicant inducers, but also xenobiotics of di!erent chemical nature. This indicates a wider spectrum of action for plant monooxygenases in comparison with the animal analogues.

As a result of reaction, some oxidases are signi"cantly inactivated. Such enzyme behavior is caused by active forms of oxygen, used as substrates or formed as products of catalyzed reactions. Typical examples of such enzymes are superoxide dismutase (Bray et al., 1974), D-glucose oxidase (Bourdillon et al., 1982), xanthine oxidase (Lynch and Fridovich, 1979), chloroperoxidase (Shahangian and Hager, 1981), and glutathione peroxidase (Blum and Fridovich, 1985). Often, during enzymatic transformation of highly reactive intermediates, inactivation of hemoprotein occurs. This is explained by the chemical modi"cation in the active site of the enzyme or by the covalent binding of modi"ed heme to protein (Ortiz de Montellano and Reich, 1986; Bornheim et al., 1987; Ortiz de Montellano and Stearns, 1989; Manno et al., 1988). After transferring electrons, cytochrome P450 may be inactivated in di!erent ways. One way is based on the following: active intermediates (epoxides, N-oxides, aldehydes, ketones) generated during destruction of the enzyme peroxy complex destruction cause degradation of hemoprotein. Though the real mechanism of inactivation is unknown, the priority in inactivation is given to those active forms of oxygen that are generated as a result of decomposition of oxy- and peroxycytochrome P450 (Halpert et al., 1986; Ortiz de Montellano and Reich, 1986; Stevens and Halpert, 1988; Reed et al., 1988; Guengerich and Bocker, 1988; Ortiz de Montellano, 1989; Guengerich, 1990). According to this point of view, inactivation of cytochrome P450 occurs basically due to H O , formed at the active site   of cytochrome P450, and is the result of decomposition of peroxy complexes. H O generated as a result of dismuta  tion of superoxidative anions exerts an insigni"cant inactivation e!ect (Mengazetdinov et al., 1989). It is considered that modi"cation of the amino acid residues at the active site of cytochrome P450 by H O is a signi"cant step in   hemoprotein inactivation (Davies et al., 1987). Such insigni"cant structural changes in the enzyme under the in#uence of H O lead to functional changes in the enzyme. There  fore, cytochrome P450 is considered the universal &&enzymesuicide'' and hydrogen peroxide appears to be the main reason for its inactivation (Karuzina and Archakov, 1994). During incubation of higher plant microsomes with NADPH, decreases in cytochrome P450 hydroxylase and demethylase and time-dependent activities are observed (Reichart et al., 1984). These are the result of autocatalytic generation of the active forms of oxygen at the active site of hemoprotein or of activation of endogenous substrates. The ratio of active to inactive forms of hemoprotein depends signi"cantly on plant age. In microsomes of etiolated maize seedlings NADPH-dependent demethylase activity reaches a maximum in 7-day-old seedlings. Afterward,

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the P450:P420 ratio steadily decreases. In 14-day-old microsomes NADPH-dependent monooxygenase activity has completely disappeared. The loss of N-demethylase activity is accompanied by accumulation of peroxidase (Khatisashvili et al., 1993a, b). The substitution of NADPH-dependent monooxygenase by H O -dependent peroxidase activity is especially evident in   the case of incubation of microsomes with #avanoids (Khatisashvili et al., 1994, 1997). Seven-day-old microsomes intensely oxidize only methoxylated #avanoids (nobiletin and 5-demethyl-nobiletin) by an NADPH-dependent mechanism. In the presence of H O , this reaction does not proceed. Fourteen-day-old   microsomes oxidize only hydroxylated #avonoids (limocytrin and quercetin). After complete nobiletin oxidation, 7day-old microsomes do not react on addition of NADPH and substrate. They oxidize hydroxylated #avonoids and O-demethylated products of nobiletin via an H O   dependent pathway. Cytochrome P450 is a multifunctional enzyme catalyzing oxidative and peroxidative reactions (Archakov and Bachmanova, 1990; Ruckpaul et al., 1989; Archakov and Zhukov, 1989; Zhukov et al., 1989). The data presented indicate that P450 accepts peroxidase activity as a result of conversion. In plants, in contrast to animals instead of inactivation, transformation of monooxygenase activity into peroxidase occurs. 8. ACTION ON THE CELL STRUCTURE

8.1. The Movements of Toxicants in the Plant Cells Obviously, in this section, the question of whether the plant cell appears to be a small factory that continuously assimilates noncharacteristic cellular compounds such as xenobiotics from the environment, deeply metabolizing or accumulating them in the form of nontoxic conjugates, is answered. This is an extremely important problem as the plants prove to be the main ecological power of nature. The importance of plant ecological potential is increasing due to the increase in the amounts and varieties of di!erent pollutants, including organic toxicants. The detoxication potential of plants is determined by the ability of the cell to take up and transform di!erent concentrations of toxicants, maintaining at the same time regular metabolic properties. The data obtained during the last decade con"rm that all kinds of organic xenobiotics permeating the cell lead to the divergence of cellular ultrastructure and metabolism (Buadze, 1988; Durmishidze, 1988; Sengupta et al., 1989; Kumar and Subrash, 1990; Allnu! et al., 1991). Today, the great majority of investigations concerning how xenobiotics act on plant cell ultrastructure have been done using inorganic xenobiotics. Organic xenobiotics systematically have not been investigated.

15

FIG. 1. Fragment of maize root cell after 10 min incubation in [1-C]phenoxyacetic acid. Insigni"cant amounts of labeled sections are observed. ;50,000.

The authors have tried to "ll this gap by studying the most important representatives of alkanes, alkenes, arenes including polycyclic carcinogenic hydrocarbons, phenols, and phenoxyacetic acids. The above-mentioned preparations labeled with radioactive carbon and tritium were used. Localization and distribution of organic xenobiotics were investigated by the method of electron microscopic authoradiography. Investigations were carried out using di!erent annual and perennial plants as well as plant cell and tissue cultures. The e!ect of toxicant action was estimated as the divergence of cell ultrastructure. Penetration, transformation, and localization of toxicants in plant cells were estimated by measuring radioactive label. It was reported that in some cases even 10 min incubation was enough to bind the label in plant cells. Incubation of [1!C]Phenoxyacetic acid with cells of maize apex revealed that the label occupied more than 3% of the total cell area (Fig. 1). By that time the label was found in the nucleus, nucleolus, and vacuoles. A di!erent picture was observed in case of sun#ower cells incubated with phenoxyacetic acid. After 10 min of incubation the label covered more than 16% of the total cell area. The label concentrated mainly in the intracellular space and a smaller amount in the nucleus. Analogous to sun#ower, phenoxyacetic acid deeply penetrates pea cells. Penetration of toxicant through the cell membrane, cytoplasm, nucleus, and nucleolus is evident in Fig. 2. Maize is more resistant to penetration of 2,4-[1-C]D and [1,12-C]benzidine, whereas these toxicants more deeply penetrate pea and sun#ower cells, occupying various subcellular organelles (Figs. 3}5). As a result of 30 min incubation of the same toxicants with the same plant cells, the following picture was observed: the label penetrated maize cells the least, cells occupying less than a 5% of the

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FIG. 2. Fragment of root cell of pea after 10 min incubation in [1-C]phenoxyacetic acid. Intensive penetration of label through the cell membrane is observed. ;100,000.

total area, mainly the nucleus and vacuoles, with insigni"cant amounts of label in cytoplasm and membranes. Toxicants occupied more than 30% of the intracellular area in sun#ower and pea, including mitochondria, plastids, nucleus, and nucleolus (Buadze et al., 1985a, b, 1986). Calculation of the absorbed radioactivity revealed that the lowest percentage is characteristic of maize leaves (7%), the medium exponent has pea at 22%, and the highest was, sun#ower at 50%. Some attempts were made to elucidate at the ultrastructural level the changes in plant cells under the in#uence of di!erent toxicants. 8.2. Alkanes and Alkenes Under the in#uence of methane, in the cells of maize seedlings in the upper part of the leaf, chloroplasts with an elongated shape, containing grana, were distributed along the periphery of the cell. In some chloroplasts, there were starch accumulations. In some cases, chloroplasts contained electron-dense matrix, and grana and thylakoids were not seen. On the external chloroplast membranes, vacuolar bubbles were observed. Lamellar membranes were observed along the entire chloroplast on the long axis. Large quantities of lipid droplets and mitochondria with an electron-dense matrix and enlarged cristae were concentrated in the cytoplasm around chloroplasts. Simulatenously, the "gures reveal the complete destruction of chloroplasts without double membranes. Separate grana were smashed and located in a brightened cytoplasm matrix. In the middle part of the leaf, chloroplasts had a cresent shape and in most cases were grouped along the cell circle. Destroyed grana and chloroplasts without double membranes

FIGS. 3}5. Fragments of root cells of maize (3), sun#ower (4), and pea (5) after 10 min incubation in 2,4-[1-C]D. Penetration of the label into di!erent cell structures in clearly observed. ;50,000.

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were observed in some cells. Cytoplasm was observed here and there, and mitochondria had a bright or electron-dense matrix. In the lower part of the leaf, the chloroplasts were more circular, and most of the mitochondria had a bright matrix. No starch grains were observed. Under the action of ethane in the upper part of the leaf, quite normal chloroplasts with crescent-shaped grana were visible; a few of them were elongated. Some cell chloroplasts contained large amounts of starch grains. In the middle part of the leaf, chloroplasts acquired an elongated shape, in most cases without grana. Chloroplast matrix was barely visible. Chloroplasts were surrounded by so-called, swollen membrane. Gradually, the stages of chloroplast degradation were observed when surrounding membrane disappeared and dissociation of grana into thylakoids occurred. Mitochondria were invaginated with clearly expressed electron-dense matrix and broadened cristae. This degradation was observed. The cytoplasm was bright (Buadze et al., 1979b). Particular declinations in chloroplast shape and structure in the lower part were not observed. Chloroplasts had a crescent shape with clearly observed grana and thylakoids; mitochondria were characterized by swollen cristae and a dense matrix. The in#uence of propane on cells in the upper part of ryegrass leaf led to chloroplasts with di!erent shapes from semicircular-ellipsoid to crescent. The grana and thylakoids were well observed. Nearly all chloroplasts contained starch grains. Grana were shifted to one side of the chloroplasts. Cytoplasm was saturated by ribosomes. In the middle part of the leaf, the chloroplast matrix was bright, and the shape changed from circular to crescent; grana were visible, but their amount was limited. No starch grains were observed; mitochondria were brightened (Buadze et al., 1979b). The appearance of starch grains was marked in the lower part of chloroplasts. Grana were chaotically arranged in the membrane, and mitochondria were present in regular amounts. Among the other low-molecular-weight alkanes, the e!ect of butane on pea leaves was studied. It was found that: In the upper part of the leaf, chloroplasts are of di!erent shape, from semicircular to crescent, and grana arc present in small quantities and not always along the diagonal. Starch grains are elongated. The gradual degradation of chloroplasts and mitochondria is visible on electron micrographs. The matrix of mitochondria is thickened with gradual swelling of the cristale and, "nally, mitochondria rupture along internal crista borders. In the middle part, chloroplasts are of di!erent shape and with uncertain location of grana. Invagination of chloroplasts is observed. The gradual degradation of chloroplasts

17

FIG. 6. Fragment of maize leaf cell incubated in methane. Upper part of the leaf. Note the total destruction of chloroplasts. ;60,000.

is noticeable, with the di!erence that lamellae of grana at the end take on a swollen vacuole shape. In the lower part of the leaf, chloroplasts are of di!erent shape, mostly semicircular, and matrix is bright. Grana and starch grains are observed in small quantities. Mitochondria are bright, but contain membrane without cristae. The agranular structure of endoplasmic reticulum is characteristic. The size of chloroplasts are increases from methane to butane, indicating swelling ability. The most sensitive to the compounds studied appeared to be the cells in the upper part of the leaf (Figs. 6, 7). Densitometry of chloroplast thylakoids demonstrated that the distance between membranes indicates the swelling after 24 h of exposure in the atmosphere of the examined alkanes (Buadze et al., 1979b).

FIG. 7. Fragment of maize leaf cell incubated in methane. Upper part of the leaf. Chloroplasts maintain their ultrastructural organization. ;100,000.

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FIG. 8. Fragment of maple leaf incubated in [1,6-C]benzene. Note the change in structure of the photosynthetic apparatus and accumulation of osmiophilic insertions. ;60,000.

8.3. Compounds Containing an Aromatic Nucleus Under the in#uence of [1,6-C]benzene on cell ultrastructural organization in leaves of perennial woody plants, pathological changes in the photosynthetic apparatus were ascertained. In particular, it was expressed in disorganization of the chloroplast}lamellar}grana complex and in accumulation of osmiophilic inclusions (Fig. 8). Many years of experience allowed the authors to make a list of perennial plants with respect to their increased resistance to benzene: lime, maple, "r, poplar, ordinary spruce, nut tree, platan, cypress, ash tree, and silver spruce. o-Nitrophenol, 2,4-dinitrophenol, and nitrobenzene completely destroy cell ultrastructure in the lower and upper parts of the leaf. Benzene, phenol, o-nitrophenol, phenol and o-cresol manifest pathological e!ects only in the lower part of the leaf. Probably, the di!erent toxicity of xenobiotics is

FIG. 9. Fragment of maize root cell incubated in benzidine solution. Note the intensive invagination of nuclear membrane. ;60,000.

FIG. 10. Fragment of maize root cell incubated in benzidine solution. Note the mitochondria with bright matrix. ;60,000.

conditioned not only by the presence of de"nitive functional groups, but also by their disposition in the molecule (Meskhi et al., 1973). The "rst signs of destruction of the cell ultrastructure, under the in#uence of polycyclic hydrocarbons, are noticeable in nuclei. The nuclear membrane con"guration is signi"cantly changed (Fig. 9). The nucleus itself becomes invaginated. With the following increase in the concentration of benzidine, benz[a]anthracene, and benzo[a]pyrene, &&chromatin coagulation'' of di!erent forms and sizes is observed, which indicates the violation of DNA synthesis. Mitochondria lose their content and become bright (Fig. 10). Finally, the cell's complete destruction is clearly expressed (Fig. 11). Plastids proved to be the most resistant subcellular organelles. The small concentrations of benzo[a]pyrene do not have pathologic e!ects on cell structure, as their oxidative degradation takes place and the intermediates participate in cell

FIG. 11. Fragment of maize root cell incubated in benz[a]anthracene solution. Note the total cell destruction. ;40,000.

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metabolism with subsequent oxidation to carbonic acid (Buadze et al., 1979a). The in#uence of dinitro-o-cresol and 2,4-D on the photosynthetic apparatus of vine leaf cells depends directly on herbicide concentration in the cell. An increase in concentration from 0.01 to 1% enhances the degree of damage to the lower part of the leaf. Degradation of cell ultrastructural organization and particularly of chloroplasts, in cells of the upper part of the leaf, becomes noticeable when the herbicide concentration is 0.1%. When the concentration of DNOC is equal to 1%, complete destruction of the cell is clearly expressed DNOC causes the damage to the photosynthetic apparatus as well as to the entire cell structural organization. Under the in#uence of DNOC, considerable inhibition of mitochondrial oxidative phosphorylation is observed (Stupnikow, 1975). According to the author's data, with an increase in plant incubation time from 24 to 72 h in a solution of 2,4-D (0.003 mg/ml), increased destruction of cell structure is observed. The surface of maize leaf cells partially loses its characteristic structural roughness, and stomata are submerged into the epidermis. Cells of lemon plant leaf are characterized by a high twisting of cristae with spans; stomata become more rounded because of #attening of surrounding epidermal cells. It was established that under the in#uence of herbicide, vine leaf surface structure loses its normal outline. The cristae become smooth, lose their roundness, and the stomata decrease in size. These peculiarities of the leaf epidermis structure promote the loss of their elasticity (Deverall, 1980; Buadze, 1991). Hence, on the basis of the above-indicated data it can be concluded that plants signi"cantly di!er in the ability to assimilate organic toxicants, which, on penetration into cytoplasm, are incorporated into subcellular structures with di!erent intensities. Evidently for the "rst 30 min toxicants penetrate into and are accumulated in subcellular organelles. Simultaneously, speci"c enzymes participating in the subsequent oxidative transformations of toxicants are induced (Ugrekhelidze, 1976; Khatishashvili et al., 1997). All of the toxicants examined caused changes to di!erent degrees in plant cell structure. Though the general cytological picture remains almost unchanged, it should be taken into consideration that the action of low metabolic concentrations of chemical toxicants on plants causes some diversion in ultrastructure (enhancement of periplasmic space, decrease in total disappearance of plasmodemos, enhancement of the volume of endoplasmic reticulum, etc.). The in#uence of toxicants and their intermediates is "rst of all directed to the most sensitive photosynthetic apparatus of the plant cell (Sharma et al., 1989; Furikawa, 1991). Simultaneously, enzymatic systems of the Krebs cycle and of oxidative phosporphylation are inhibited.

Biosynthesis of ATP and other energetically important di- and trinucleotides is substantially decreased (Bataynen et al., 1986). The violation of plant cell structure as a result of the action of penetrating organic toxicants reveals the mechanism behind the development of cell pathology, and allows estimation of maximally permissible doses of toxicants for each plant and their ecological potential. 9. CONCLUSIONS

The intensity of organic xenobiotic absorption by plants through roots and leaves is determined by such factors as the chemical structure of molecules (hydrophilicity, molecular mass, dissociation) and penetration conditions (temperature, pH, xenobiotic concentration, etc.). Organic xenobiotics penetrate leaf cells through stomata as well as through cuticle. The following main pathways of xenobiotic detoxication exist in higher plants: conjugation with endogenous compounds*peptides, sugars, amino acids, and organic acids; oxidative degradation from initial hydroxylation of alkyl and aryl groups to deep consequent oxidation of the entire xenobiotic molecule. Enzymes catalyzing the oxidative degradation of xenobiotics are localized in cytoplasm and in separate cell organelles. Such an intracellular distribution of enzymes considerably increases plant detoxication ability. For the deep oxidative degradation of xenobiotics, increased polarity is needed, and is reached by their initial monooxygenation. All components of a monooxygenase system are identi"ed in plants and their inductive nature is determined. Transformation of a monooxygenase enzyme system into peroxidase takes place with aging of plants. Once into a plant cell, toxicants are incorporated into cell organelles with di!erent intensities. Depending on the concentration and respective toxic e!ect, three levels of action of xenobiotics on plants can be determined: (1) having no noticeable deviations in cell ultrastructure and all biological processes; (2) having clearly expressed declinations in cell ultrastructure and main cellular processes; (3) leading to complete cell destruction and plant death. REFERENCES Allnu!, F. C. T., Ewy, R., Renganathan, M., Pan, R. S., and Dilley, K. A. (1991). Nigericin and hexylamine e!ects on localized proton gradients in thylacoids: Biochem. Biophys. Acta Bioenerg. 1059, 28}36. Andreopoulos-Renaud, U., Glas, J., Falgoux, D., and Schiedecker, D. (1975). Absorption D'un polyethyleneglycol par de jeunes plantes de haricot et de cotonnier. Dosage par chromatographie on phase gaze use sur exudat de tige. Cr. Acad. Sci. D 280, 2333}2340. Archakov, A. I. (1975). In ¹he Microsomal Oxidation, pp. 53}82. Nauka, Moscow.

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Archakov, A. I. (1983). Oxygenases of Biological membranes. In XXX
Buadze, O. A. (1991). Structural}Functional Basis of Xenobiotics Action on Plant Cells Organisms. Author's abstract of doctoral dissertation, Tbilisi. [In Russian.] Buadze, O. A., Durmishidze, S. V., Kakhaya, M. D., and Zaalishvili, G. V. (1979a). The study of e!ect of di!erent concentrations of benzidine, benzathracene and 3,4-benzpyrene on root ultra-structure of maize. In Metabolism of Chemical Pollution of Biosphere in Plants (S. V. Durmishidze, Ed.), pp. 179}195. Metsniereba, Tbilisi. [In Russian with English abstract.] Buadze, O. A., Kakhaya, M. D., and Zaalishvili, G. V. (1979b). The in#uence of the lowest alkanes on chloroplast ultrastructure of some plants. In =orks of Session on Defence of Environment, pp. 15}16. Tbilisi. [In Russian.] Buadze, O. A., Durmishidze, S. V., and Apakidze, A. V. (1985a). The ultrastructural aspects of 2,4-D herbicide penetration and localization in plant cell. Proc. Georg. Acad. Sci. 119, 409}413. [In Russian with English abstract.] Buadze, O. A., Durmishidze, S. V., Kakhaya, M. D., Katsitadze, K. P., and Apakidze, A. V. (1985b). The electronmicroscopic study of some questions of phenoxyacetic acid movement, localization and utilization in some plants. Proc. Georg. Acad. Sci. Biol. Ser. 11, 311}318. [In Russian with English abstract.] Buadze, O. A., Lomidze, E. P., Kakhaya, M. D., and Gagnidze, L. P. (1986). The uptake and distribution of radioactive label of 1-12-C-benzidine in plant cell. In =orks of Conference of ;zbekistan Biochemists, pp. 181}182. Tashkent. [In Russian.] Bukovac, M. T., Petracek, P. D., Fader, R. G., and Morse, R. D. (1990). Sorption of organic compounds by plant cuticles. =eed Sci. 38, 289}298. Burrows, W. J., and Leworthy, D. P. (1976). Metabolism of N,Ndiphenylurea by cytokinin-dependent tobacco callus: Identi"cation of the glucoside. Biochem. Biophys. Res. Commun. 70, 1109}1117. Burt, M. E., and Corbin, F. T. (1978). Uptake, translocation and metabolism of propham by wheat (¹riticum aestivum), sugarbeet (Beta vulgaris), and alfalfa (Medicago sativa). =eed Sci. 26, 296}302. Cabanne, F., Giallardon, P., and Scalla, R. (1985). Phytotoxicity and metabolism of chloroturon in two wheat varieties. Pestic. Biochem. Physiol. 23, 212}220. Carringer, R. D., Rieck, C. E., and Bush, L. P. (1978). Metabolism of EPTC in corn (Zea mays). =eed Sci. 26, 157}163. Cassagne, C., and Lessire, R. (1975). Studies on alkane biosynthesis in epidermis of Allium porrum L. leaves. 4. Wax movement into and out of the epidermal cells. Plant Sci. ¸ett. S.5, 261}266. Chamberlain, K., Butcher, D. N., and White, J. C. (1986). Relationship between chemical structure and phloem mobility in Ricinus communis var. Gibsonii with reference to a series of W-/l-naphthoxy/-alcanoic acids. Pestic. Sci. 17, 48}52. Chandler, J. M., Basler, E., and Santelman, P. W. (1974). Uptake and translocation of alachlor in soybean and wheat. =eed Sci. 22, 253}259. Chang, F. Y., and Vanden Born, W. H. (1971). Dicamba uptake, translocation, metabolism and selectivity. =eed Sci. 19, 113}122. Chkanikov, D. I. (1985). Metabolism of 2,4-D in plants. ;sp. Sov. Biol. 99, 212}225. [In Russian.] Chkanikov, D. I., Makeev, A. M., Pavlova, N. N., Artemenko, E. N., and Dubovoi, V. P. (1976). The role of 2,4-D metabolism in plant resistance to this herbicide: Agrokhimiya 2, 127}132. [In Russian.] Chkanikov, D. I., Makeev, A. M., Pavlova, N. N., and Nazarova, T. A. (1982). 2,4-D metabolism in cultural cereals. Physiol. Rastenii 29, 542}547. [In Russian with English abstract.] Chrikishvili, D. I., Ugrekhelidze, D. Sh., and Mithaishvili, T. I. (1977). Products of phenol conjugation in maize. Bull. Georg. Acad. Sci. 88, 173}176. [In Russian with English abstract.]

REVIEW Cole, D. J., and Owen, W. J. (1987). Metabolism of metalaxyl in cell suspension cultures of ¸actuca satiira, L. and
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Durmishidze, S. V., and Ugrekhelidze, D. Sh. (1968b). Absorption and conversion of butane by higher plants. Dokl. Akad. Nauk SSSR 182, 214}216. [In Russian with English abstract.] Durmishidze, S. V., and Ugrekhelidze, D. Sh. (1975). Absorption and transformation of methane by plants. Physiol. Rastenii 22, 70}73. [In Russian with English abstract.] Durmishidze, S. V., Ugrekhelidze, D. Sh., Djikia, A. N., and Tsevelidze, D. Sh. (1969). The intermediate products of enzymatic oxidation of benzene and phenol. Dokl. Akad. Nauk SSSR 184, 466}469. Durmishidze, S. V., Ugrekhelidze, D. Sh., and Djikiya, A. N. (1974a). Absorption and transformation of benzene by higher plants. Physiol. Biochim. Kult. Rastenii 6, 217}221. [In Russian with English abstract.] Durmishidze, S. V., Ugrekhelidze, D. Sh., and Djikiya, A. N. (1974b). Absorption and transformation of toluene by higher plants. Prikl. Biokhim. Microbiol. 10, 673}676. [In Russian with English abstract.] Durmishidze, S. V., Ugrekhelidze, D. Sh., and Djikya, A. N. (1974c). Uptake of benzene by fruits from atmosphere. Prikl. Biokhim. Microbiol. 10, 472}476. [In Russian with English abstract.] Durmishidze, S. V., Ugrekhelidze, D. Sh., and Djikya, A. N. (1974d). Uptake and transformation of benzene by higher plants. Physiol. Biochem. Kult. Rastenii 6, 217}220. [In Russian with English abstract.] Durmishidze, S. V., Djikya, A. H., and Lomidze, E. P. (1979). Uptake and transformation of benzidine by plants in sterile conditions. Dokl. Akad. Nauk SSSR 247, 244}247. [In Russian with English abstract.] Durmishidze, S. V., Ugrekhelidze, D. Sh., and Kakhniashvili, Ch. A. (1982). Metabolism of phenoxyacetic acids in plants: Conjugation products of phenoxyacetic and 2,4-dichlorophenoxyacetic acids with peptides. In ¹he Fifth International Congress of Pesticide Chemistry (J;PAC), Kyoto, Japan, 1982, Abstract Va-2. Durst, F. (1991). Biochemistry and physiology of plant cytochrome P-450. In Frontiers in Biotransformation, Vol. 4, pp. 191}232. Academie-Verlag, Berlin. Eastin, E. F. (1971). Fate of #uorodifen in resistant peanut seedlings. =eed Sci. 19, 261}267. Edwards, R., and Owen, W. J. (1989). The comparative metabolism of the s-triazineherbicides atrazine and terbutryne in suspension cultures of potato and wheat. Pestic. Biochem. Physiol. 34, 246}254. Epstein, E., and Lavee, Sh. (1977). Uptake, translocation, and metabolism of IAA in the olive (Olea europea): Uptake and translocation of [1C]IAA in detatched Manzanilla olive leaves. J. Exp. Bot. 28, 619}625. Eynard, I. (1974). Determination on foliage of surfactant solution by a radioisotope technique. Allionia 17, 131}135. Feung, Ch., Hamilton, R. H., and Mumma, R. O. (1975). Metabolism of 2,4-dichlorophenoxyacetic acid: 10. Identi"cation of metabolites in rice root callus tissue cultures. J. Agr. Food Chem. 24, 1013}1019. Fleeker, J., and Steen, R. (1971). Hydroxylation of 2,4-D in several weed species. =eed Sci. 19, 507}513. Fonne-P"ster, R., Gaudin, J., Kreuz, K., Ransteiner, K., and Eber, E. (1990). Hydroxylation of primisulfuron by an inducible cytochrome P-450-dependent monooxygenase system from maize. Pestic. Biochem. Physiol. 37, 165}175. Franke, W. (1975). Sto!aufnahme duK rch das Blatt under besondere Berucksichtigung der Ektodermen. Bodenkultur 26, 331}340. Frear, D. S., and Swanson, H. R. (1972). New metabolites of monuron in excised cotton leaves. Phytochemistry 11, 1919}1923. Frear, D. S., Swanson, H. R., Mansager, E. R., and Wien, R. G. (1978). Chloramben metabolism in plants: Isolation and identi"cation of glucose ester. J. Agr. Food Chem. 26, 1347}1354. Frear, D. S., Swanson, H. R., and Mansager, E. R. (1983a). Aci#uorfen metabolism in soybean: Diphenylether bond cleavage and the formation

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of homoglutathione, cysteine, and glucose conjugates. Pestic. Biochem. Physiol. 20, 299}310. Frear, D. S., Swanson, H. R., Mansager, E. R., and Tanaka, F. S. (1983b). Metribuzin metabolism in tomato: Isolation and identi"cation of Nglucoside conjugates. Pestic. Biochem. Physiol. 19, 270}281. Frear, D. S., Swanson, H. R., and Mansager, E. R. (1985). Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates. Pestic. Biochem. Physiol. 23, 56}65. Furikawa, A. (1991). Inhibition of photosynthesis of Populus euramericana and Helianthus annus by SO , and NO and O . Ecol. Res. 6, 79}86.    Garner, W. Y., and Menzer, R. E. (1986). Metabolism of N-hydroxymethyl dimethionate in bean plants. Pestic. Biochem. Physiol. 25, 218} 232. Gestetner, B., and Coon, E. E. (1974). The 2-hydroxylation of transcinnamic chloroplasts from Melilotus alba Ders. Arch. Biochem. Biophys. 163, 671}623. Gordeziani, M., Durmishidze, S., and Kurashvili, L. (1979). N-demethylated and p-hydroxylated activities of soybean and maize cotyledons. Proc. Georg. Acad. Sci. 96, 717}720. [In Russian with English abstract.] Gordeziani, M., Durmishidze, S., Bobokhidze, E., and Lomidze, E. (1986). Microsomal diethylation of diethylanilin and p-nitroanisole in mixed system plant microsomes}mitochondria. Proc. Georg. Acad. Sci. 12, 42}46. [In Russian with English abstract.] Gordeziani, M., Durmishidze, S., Khatisashvili, G., Adamia, G., and Lomidze, E. (1987). The investigation of biosynthetic and detoxication ability of plant cytochrome P-450. Dokl. Akad. Nauk SSSR 295, 1491}1493. [In Russian with English abstract.]

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