Glutathione S-transferase activity in spruce needles

PESTICIDE

BIOCHEMISTRY

AND

Glutathione

PHYSIOLOGY

37, 211-218 (1990)

S-Transferase

Activity in Spruce Needles

PETER SCHR~DER,***GERALD L. LAMOUREUX,~ DONALD G.Rus~~ss,t HEINZRENNENBERG* *Fraunhofer Institute for Atmospheric Environmental Garmisch-Partenkirchen, Federal Republic of Germany, University Station, P.O. Box 5674,

Research, Kreuzeckbahnstr. and tBiosciences Research Fargo, North Dakota 58105

AND

19, D-8100 Laboratory, State

Received November 22, 1989; accepted March 30, 1990 Glutathione S-transferase activity was present in extracts from needles of two different spruce species (Picea abies and Picea glauca). In vitro conjugation studies were conducted with three 14C herbicides and one r4C fungicide: atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), fluorodifen (2,4’-dinitro-4-trifluoromethyl diphenyl-ether), propachlor (2-chloro-N-isopropylacetanilide), and pentachloronitrobenzene (PCNB). The enzymes from both P. abies and P. glauca showed the highest rates of enzymatic conjugation for fluorodifen as the substrate while intermediate to low rates of enzymatic conjugation were observed with PCNB and propachlor. Atrazine was not an appreciable substrate for the enzymes of either species. The water-soluble “‘C conjugation products of the enzymatic reactions were assayed by liquid scintillation spectrometry. The [r4C]glutathione conjugates from fluorodifen and PCNB were identified by a combination of thinlayer chromatography (TLC), high-performance liquid chromatography (HPLC), and fast atom bombardment mass spectrometry and the [‘4C]glutathione conjugate of propachlor was identified by TLC and HPLC comparison to an authentic standard. The catalytic properties of ghrtathione S-transferase from P. abies were analyzed with CDNB as substrate. The apparent K,,, values were 0.14 &for GSH and 0.67 mi%ffor CDNB, respectively, the pH optimum was between 7.6 and 8.0, and the temperature optimum was 4045°C. The activation energy was calculated to be 32.4 k.l B 1!390 Academic Press, Inc. mol-‘.

chlorinated nitrobenzenes, and related compounds with the sulfur containing Many plant and animal species are capa- tripeptide glutathione (1, 2). In some plant ble of detoxifying halogenated xenobiotics species, GST activity can be stimulated by by conjugation to carbohydrates or to glu- antidotes (2,3), leading to an increase in the tathione (GSH).2 Although the latter reac- resistance to xenobiotics. tion can proceed spontaneously at low The occurrence of halogenated hydrocarrates, it is predominantly mediated by glu- bons and herbicides in the atmosphere is tathione S-transferase enzymes (GSTs, EC not restricted to highly industrialized or ag2.5.1.18). The GSTs are known to catalyze ricultural areas, but is also evident in reconjugation of chlorotriazines, chloroacet- mote areas, e.g., the alpine mountains in amides, diphenyl ethers, thiocarbamates, Germany (4-6). These compounds appear to be transported in the atmosphere over r To whom correspondence should be addressed. long distances and to be deposited in signif’ Abbreviations used: Atrazine, 2-chloroicant amounts in the alpine forests. Re4-ethylamino-6-isopropylamino-s-triazine; BuOH, ncently, evidence has been presented for the butanol; CDNB, chloro-2,4-dinitrobenzene; DCNB, presence of several nitrated and chlorinated 1,2-di-chloro-4nitrobenzene; DNP, 2,4-dinitrophenyl moiety; FAB-MS, fast atom bombardment mass spec- phenolic compounds in cuticles of spruce trometry; fluorodifen, 2,4’-dinitro-4-Wluoromethyl (7) and pine (8) needles with concentrations diphenylether; GST, glutathione S-transferase; HAc, ranging from 7 to 30 rig/g DW. In a third glacial acetic acid; PCNB, pentachloronitrobenzene; study, experiments were conducted comp-NBC, p-nitrobenzoyl chloride; propachlor, 2paring deposition of hexachlorobenzene, chloro-N-isopropylacetanilide; PVPP, polyvinylpolypyrrohidone; RAM, radioactivity monitor. 2,4-dinitrophenol, 2,4-dinitro-o-cresol, and INTRODUCTION

211 0048-3575/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

212

SCHRiiDER

4nitrophenol in trees from Bayreuth, the Fichtelgebirge, and locations close to a highway (9). Although these substancesare known to be photolytically degraded and to undergo rapid metabolism, increasing concentrations in the needles were found with time, indicating extremely high doses of the original compound being deposited on the trees. Similar results were reported for the enrichment of Ci- and C,-chlorocarbons in pine needles (10). All four studies indicated the possibility of damage to trees exposed to airborne organic pollutants that possessedsimilar properties to uncouplers and herbicides. The presence of GSTs in forest trees may, therefore, serve the beneficial function of detoxifying atmospheric xenobiotics. GSTs from crops and weeds have been intensively studied (2,22), but data on these enzymes in forest trees are lacking. In the present study, GST activity was analyzed in needles from two different species of spruce, a major forest tree in remote mountain areas of the northern hemisphere. MATERIALS

AND

METHODS

Plant material. The experiments were performed with present year (1988) needles from Norway spruce (Picea abies L. Karst., sites close to Garmisch-Partenkirchen, FRG) and white spruce (P. glauca L. Moench, sites at Fargo, ND). The needles were frozen and stored in liquid nitrogen immediately after harvesting. Chemicals. Equine liver GST (Sigma Equine Liver Affinity Enzyme G6511), CDNB, GSH, and PVPP were from Sigma Chemical Co. (St. Louis, MO); DCNB and p-NBC were from Kodak (Rochester, NY). All other chemicals used were research grade commercial materials. [14C-ringULJ-Atrazine (2-chloro-4-ethylamino-6isopropylamino-s-triazine, sp act 21.3 lXi/lJ,mol, radiochemical purity >98%) was from Dow Chemical Co. (Midland, MI). The specific activity of the [14Clatrazine was diluted to 2.2 ~Ci/~mol. [‘4C-CF,]Fluorodifen (2,4’-dinitro-4-trifuoromethyl

ET AL.

diphenylether, sp act 2.95 @i/kmol, radiochemical purity >95%) was from Ciba Geigy (Greensboro, NC). [i4C]PCNB ([‘4C-ring-ULlpentachloronitrobenzene, 1.77 &i/kmol) was synthesized by the method of Kadunce and Lamoureux (11) and [‘4C-carbonyl]propachlor (2-chloroN-isopropylacetanilide, sp act 2.73 pCi/p,mol, radiochemical purity >95%) was synthesized by the method of Lamoureux et al. (12). Glutathione conjugate standards of fluorodifen [S-(4-trifluoromethyl-2nitrophenyl)glutathione], propachlor [S-2-(N-isopropylacetanilide)glutathione], and CDNB [S-(2,4-dinitrophenyl)glutathione] were synthesized by standard methods and the structures of these compounds were verified by FAB-MS. In vim-studies. Freshly harvested P. abies and P. glauca needles were cut longitudinally and then in 2-mm cross sections. The cut needles (0.5 g) were vacuum infiltrated with 500 ~1 of potassium phosphate buffer (0.1 M, pH 7.8) containing 61.5 nmol of [‘4C]fluorodifen and incubated at 30°C in a glass tube. After 20 hr the incubation was stopped by withdrawing the liquid phase with a glass syringe. The tissue was washed twice with 2 ml of acetone. The incubation liquid and washing solutions were combined and saved for later analysis. The needles were powdered by a tissuemizer (Technical Products Int., St. Louis, MO) under liquid nitrogen in a stainless-steel tube. Five volumes of 70% acetone was added, and, after centrifugation, the supernatant was partitioned with methylene chloride:l% acetic acid (8:3, v/v). A microfuge was used to facilitate phase separation. A 50-~1 aliquot of the aqueous phase, which contained the water-soluble conjugates, was added to 5 ml of Beckman Readi-gel and monitored for radioactivity in a LS 6800liquid scintillation counter (Beckman, Fullerton, CA). Aliquots of both, the extract and the incubation medium from P. gfauca incubations, were chromatographed on HF 250 Anasil thin-layer plates (Analabs, Kansas City, MO) and developed to

GST

IN

SPRUCE

NEEDLES

213

The reaction was started by the addition of varying amounts of enzyme. Controls lacking GST or GSH were measured. Commercially available GST from equine liver was used as a reference. Assays using DCNB = 8.5) andp-NBC (e3i0 (E345nmr&f-‘cm-i mM-‘cm-’ = 1.9) as substrates were Krformed under the conditions described above, with the modification that in the caseof DCNB the enzyme was incubated in 100 mM, pH 7.5, potassium phosphate buffer. Protein in the enzyme extracts was Preparation of enzyme extracts. P. abies needles were pulverized in liquid nitrogen measured by the method of Bradford (14) and extracted at 4°C in a Model CB6 War- using bovine serum albumin as a standard. ing blender (New Hartford, CT) with 1.5 Activation energy was calculated by the times their weight of PVPP in 10 vol (w/v) method of Dixon and Webb (15). Enzyme assays with radiolabeled xenobiof potassium phosphate buffer (0.1 M, pH 7.8) that contained 0.5% Triton X-100. The otics. The radiolabeled substrates were crude extract was centrifuged twice at added to enzyme extracts from P. abies and 20,OOOg.Proteins in the supematant were P. glauca or to commercial equine liver precipitated by stepwise addition of solid GST and incubated for several hours at ammonium sulfate to 40, 65, and 80% satu- 30°C. The assay mixtures (total vol 1 ml) ration. During this procedure, the pH was consisted of 100 mM potassium phosphate maintained at 7.8 by the dropwise addition buffer (pH 7.8), 1 mM GSH, and 60 WM 14C of 10N NaOH. After each step the extracts substrate dissolved in acetone. The reacwere centrifuged at 20,OOOgand the pellets tions were initiated by addition of enzyme. were resuspended in 2 ml 20 r& potassium Fifty-microliter aliquots were withdrawn phosphate buffer, pH 7.8. Samples were from the reaction mixture and partitioned then dialyzed for 24 hr against 300 vol of against 550 ~1 of methylene chloride:l% potassium phosphate buffer (10 mM, pH acetic acid (8:3,v/v). After partitioning (a 7.8) and centrifuged at 5OOOg.The superna- microfuge was used for phase separation), tants were stored directly or after ly- the aqueous phases were analyzed for waophilization in a freeze drier. These samples ter-soluble conjugates by liquid scintillation were purified and desalted by using a Sep spectrometry in 5 ml of Read&gel (BeckPak Cl8 column (Waters, Milford, MA) mann Instruments). Reaction mixtures depreconditioned with potassium phosphate void of enzyme or GSH were used as conbuffer. Enzyme extracts of white spruce trols. needles were prepared in the same way. When radiolabeled PCNB was used as a Spectrophotometric enzyme assay and substrate, the reaction mixtures were diprotein determination. GST activity was luted with 3 vol of 1% HAc at the end of the determined at 25°C and 340 nm as described experiments and applied to Sep Pak Cl8 by Habig et al. (13) using a single beam columns and fractioned with increasing Spectronic 1220 photometer (Milton-Roy, concentrations (from 0 to 100%) of CH,CN Unterfohring, FRG) or a dual beam pho- in 1% HAc. Fractions were collected and tometer DU7 (Beckman, Fullerton, CA). checked for radiolabel. The reaction mixThe assay mixture (total ~013 ml) consisted tures (500 l.~laliquots) were also analyzed of 100mM potassium phosphate buffer, pH by TLC as described above. 6.4; 1.0 mM CDNB (e340nmm&f-‘cm-i = Purification of conjugates. Water9.6), dissolved in EtOH; and 1 mM GSH. soluble conjugates were collected and con15 cm in a mixture of BuOH:HAc:water (12:3:5) in paper-lined tanks. S-(2,4dinitrophenyl)glutathione was used as an internal standard. The chromatograms were scanned for radioactivity in a Packard 7220 Radiochromatogram-Scanner (Packard Instrument Co., Downers Grove, IL) and for uv-absorbing compounds. Radioactive zones were removed, suspended in Beckman Readi-Gel:H,O (10:3), and quantitled by scintillation spectrometry .

214

SCHRdDER

centrated to dryness in a rotary vacuum evaporator. After resuspension in 15% CH,CN/l% HAc, the samples were analyzed by reversed-phase HPLC. Separation was on a 3.9 mm x 30 cm+Bondapak C,s column (Waters Assoc., Milford, MA) using different organic gradients in 1% HAc at a flow rate of 1.5 ml/min. System A: isocratic elution with 20% acetonitrile; system B: linear 40-min gradient from 20 to 60% acetonitrile; system C: linear gradients from 20 to 60% acetonitrile in 40 min and from 60 to 100% acetonitrile in 10 min; system D: linear 30-min gradient from 25 to 55% acetonitrile. Separated compounds were detected simultaneously by a radioactive monitor (LB 503 RAM, Berthold, Wildbad, FRG) and an uv-monitor (Chromatronix, Berkeley, CA) at 254 nm. The peaks were collected and concentrated to dryness in a rotary evaporator. After resuspension in 15% aqueous CH,CN, the samples were separated again by HPLC using system A with the RAM out of line. The purified peaks were collected, transferred to l-ml microflex glass conical tubes with 600 ~1 70% acetone in glass distilled water and concentrated to dryness under a stream of NZ. Samples were stored at - 18°C for further use. Mass spectral analyses. Fast atom bombardment mass spectrometry was performed with a MatlVarian CHS-DF equipped with an Ion-Tek Saddlefield gun (Varian, Sunnyvale CA). The samples were dissolved in a matrix of 0.2 ~1 of glycerol, 0.1 ~1 methanol, and 10 pg oxalic acid and placed on a copper probe tip. Ionization followed FAB using xenon as the primary beam. RESULTS

In vivo conjugation of jkorodifen. Excised needle sections of P. glauca and P. abies, vacuum infiltrated with [14C]fluorodifen and incubated for 20 hr, partially metabolized the fluorodifen to water-soluble 14C products. The products from the P. glauca reaction were characterized by

ET

AL.

HPLC system A and by TLC. The incubation medium contained 4.2 and 2.1% glutathione conjugate and the needle pieces contained 9 and 9.2% glutathione conjugate in the P. glauca and P. abies experiments, respectively. Approximately, 90% of the total applied radioactivity was recovered. A conversion of 730 and 620 pmol g fresh wt-’ hr-’ for P. glauca and P. abies incubations, respectively, was calculated from these in vivo studies. Although these rates of conversion of fluorodifen are much lower than that observed in pea (l), these findings indicate that needles of two different Picea species are capable of metabolizing fluorodifen by glutathione conjugation. In vitro conjugation of PCNB, atrazine, j7uorodifen, and propachlor. A significant

rate of formation of water-soluble 14C products was observed upon incubation of [‘4C]fluorodifen, propachlor, or PCNB with enzyme extracts from P. glauca or P. abies (Fig. 1). [‘4C]Atrazine, however, was not converted to 14C water-soluble products at a significant rate under these conditions. The Rf values of the original substrates and the products obtained from these reactions are shown in Table 1. [14C]PCNB yielded two radioactive products that were detected by TLC (Rf0.20, 19.1%, and Rf0.45, 46.8%) and by HPLC on system B (peak 1, 9.6 min; peak 2, 20 min). The metabolite present in peak 1 was analyzed by positive and negative ion FAB-MS. The isotope abundance ratio of the molecular ion cluster was consistent for a compound with a molecular weight of 553 that contained five chlorines. This metabolite was concluded to be S-(pentachlorophenyl)glutathione. Mass spectral analysis of the metabolite present in peak 2 was not successful, but it was probably a diglutathione conjugate or a product produced from a glutathione conjugate by oxidative or hydrolytic reactions (16). Fluorodifen was also converted to a water-soluble product upon incubation with enzyme extracts from P. glauca. This metabolite was indistinguishable from a glutathione conjugate standard of fluorodifen by

21.5

GST IN SPRUCE NEEDLES TABLE Rr Values

1

of Different GSH Conjugates HF 250 &TLC Plates

Product Rr

Substrate R,

Atrazine Fluorodifen Propachlor PCNB Peak 1

0.45 (90.2%) 0.37 (86.2%)

0.80 (-100%) 0.87 (8.7%) 0.80 (7.2%~)

Peak 2 DNP-SG conjugate (int. standard)

0.45 (46.8%)

-___

Substrate

on Anasil

--

0.20 (19.1%) 0.87 (2.7%)

_-_ -_ _ p- ,: ? P 60. 6 '0 60. m

- - ',-

- - - -a- - ,

c r-

1

4 19

FIG. 1. Time study of conjugation of different xenobiotics. Sixty nanomoles of each substrate was incubated at pH 7.8 with I mmol of GSH and 0.13 and 0.27 mg of proteinlml in the Norway and white spruce reactions, respectively. (a) P. glauca; (b) P. abies; and (c) nonenzymatic controls. x-x, fluorodifen reaction; A--A, propachlor reaction; U-0, PCNB reaction; l ...*., atrazine reaction.

both TLC and HPLC analysis. After purification by HPLC in a manner similar to that described for S-(pentachlorophenyl)glutathione, this product was identified by positive ion FAB-MS as S(4-trifluoromethyl-2-nitrophenyl)glutathione (Fig. 2). The FAB-MS was characterized by an intense molecular ion cluster at MHf at 497 (77%), MNaf (20%), MK+ (12%), and ion fragments at m/z 422 (10%) and 368 (22%). The ion fragments correspond to the loss of the glycine and glutamyl residues, respectively. The general nature of the spectrum was very similar to the positive ion FABMS spectrum of the homoglutathione conjugate of propachlor reported recently (17). An identical FAB

0.45

Note. Development (15 cm, room temperature) in paper-lined tanks with BuOH:HAc:H,O (12:3:5). Percentage of total radiolabel in parentheses. PCNB recovery is low because of high volatility of parent and products.

mass spectrum was obtained from a synthetic standard glutathione conjugate of fluorodifen prepared by a previously described method (18). Standard glutathione conjugates of propachlor and atrazine were prepared and identified by FAB-MS (19). These glutathione conjugates were used as chromatographic standards in the TLC and HPLC analysis of the enzymatic reactions of propachlor and atrazine. The major metabolite of propachlor also appeared to be a glutathione conjugate (19). No appreciable products were detected by HPLC and TLC analysis with atrazine as the substrate. After 19 hr in the presence of enzyme extracts from P. glauca and P. abies, atrazine was not metabolized at rates significantly above the control rate with either enzyme preparation (Figs. la-lc; Table 2), whereas 100% of the fluorodifen and the propachlor and 20% of the PCNB had been converted to glutathione conjugates by P. abies (Fig. lb). These conjugates were formed at rates significantly above the control rates (Fig. lc, Table 2). Fluorodifen and PCNB were also metabolized by enzyme extracts from P. glauca at appreciable rates, 85 and 47%, respectively (Fig. la). The crude enzyme preparations from P. glauca and P. abies were similar in their

216

SCHRdDER

ET AL. +2H=368

100

80 8 5 ?1 5 60 : 40 -d 2d m a20

2

i 100

0

FIG. 2. Fast atom bombardment (FAB) mass spectrum of the spruce GST-derived GSH conjugate ofjluorodifen. Peaks marked with an asterisk (*) are due to the glycerol matrix and are also observed in a blank spectrum of the glycerol/ oxalic acid matrix. For detailed discussion see text.

specific activities toward fluorodifen (Table 2). With PCNB as a substrate, the GST from P. gluuca was more active, but in incubations at pH 7.8 with propachlor as the substrate, only GST from P. abies appeared to be active. Propachlor incubations with P. glauca extracts at pH 6.4, however, resulted in a specific activity of 0.6 nmol conjugate formation per hour (data not shown). Because of the high nonenzymatic rate of GSH with propachlor, some uncertainty exists regarding the relative activity TABLE 2 Specific Activities of the Spruce Needle GST from P. abies and P. glauca for the Conversion of Different Xenobiotics of GSH Co&gates at pH 7.8 Substrate

P. abies

P. glauca

nmol product hr- ‘ mg protein - ’ Atrazine

Fluorodifen PCNB Propachlor

0.08 6.80


0.86

1.47

0.91

co.01

Note. Aliquots of enzyme extracts were incubated with 60 nmol of the respective substrate for 3 hr and assayed as described under Materials and Methods. Values are corrected for nonenzymatic rates.

of propachlor as a substrate for GST from P. glauca and P. abies. Catalytic properties of GST from P. abies. CDNB, DCNB, and p-NBC are

model substrates for GST activity that can be assayed by spectrophotometric methods. They were evaluated as substrates for GST for P. abies. Based on initial rate data, CDNB was the most effective substrate (Table 3). The apparent KM values, as calculated from Lineweaver-Burk plots, were TABLE 3 Properties of the P. abies Needle GST in the 40 to 65% Ammonium Sulfate Cut Using DifSerent Substrates Substrate 13.8 f

Specific activity (nmol tin-’ mg-‘)

(GSHIDCNB) (GSH/p-NBC) (GSHKDNB)

pH optimum

(GSWCDNB)

Temperature optimum (“a Activation energy &.l mol-‘) K,,, values (mMl GSH CDNB V,, (nA4 min-‘) Note. For extraction

11.5 221.0

(GSWCDNB)

40.045.0

(GSWCDNB)

32.4 0.14 -’ 0.67 f

(GSHKDNB) see Materials

356.7

and Methods.

1.4

jr 4.2 k 63.0 7.6-8.0

0.05 0.1

2 129.9

GST

IN SPRUCE

0.14 -t 0.05 r&f for GSH and 0.67 ? 0.1 mA4 for CDNB. The V,, was 10.7 + 3.9 nmol/min. The enzyme had a broad temperature optimum of 40-45”C at pH 7.8 in 100 n&f potassium phosphate buffer. The activation energy of the enzyme in the temperature regime between 15 and 40°C as calculated from an Arrhenius plot by the method of Dixon and Webb (15), was 32.4 kJlmo1. DISCUSSION

Glutathione S-transferase activity capable of the detoxificaion of xenobiotics appears to be constitutive in the needles of P. glauca and P. abies. As evidenced by in vivo studies with tluorodifen in needles of both species,this detoxification mechanism appears to operate in vivo. Fluorodifen does not undergo significant nonenzymatic glutathione conjugation in vitro and, therefore, it can be concluded that this process of conjugate formation is enzymatic. As shown in earlier studies by two of the authors (2, 20), processing via y-glutamylcysteine and cysteine conjugates is possible in vivo as well as in crude extracts. In the present study, however, a further processing of the primary SG conjugate was not observed. The GSTs from P. glauca and P. abies appear to have slightly different specific activities toward the xenobiotics tested. Whereas fluorodifen was conjugated at equal rates in both species, P. glauca GST metabolized PCNB twice as fast as P. abies GST did. Propachlor, which was metabolized by P. abies at pH 7.8, did not show significant enzymatic conjugation rates with P. glauca at pH 7.8, but at pH 6.4. Such differences could be attributed to differences in the GST isozymes present in these species (2, 3, 11). Neither species has appreciable GST activity with atrazine as a substrate; however, GST activity for atrazinc appears to be limited to a few species such as Zea, Sorghum, and Saccharum (2, 21). Some GST isozymes can be induced in Zea and Sorghum upon exposure to certain

217

NEEDLES

xenobiotics (21). Whether an induction like this can also be obtained in spruce requires further studies. The catalytic properties of GST from P. abies appear to be similar to those observed with GST from other species of higher plants (2, 11). For the P. abies enzyme, CDNB, DCNB, and p-NBC were found to be substrates. Conjugation rates for CDNB, however, are about 20 times higher than with DCNB and p-NBC. This is in good accordance with studies in rats, which also showed highest conjugation rates for CDNB, whereas other compounds were only poor substrates (13). Further studies are needed to determine (i) the substrate specificity of spruce GST, (ii) the mechanisms by which the resulting glutathione conjugates are further metabolized, and (iii) the ecological significance of trees exposed to elevated concentrations of airborne xenobiotics. ACKNOWLEDGMENTS The authorsthank VeronikaMeischner and Stephan Pflugmacher for their assistance in preparing Norway spruce extracts, Hans Papen for helpful discussions, Carole Jean Lamoureux for the excellent FAB-MS analyses, and Dick Zaylskie for preparation of the computer graph of the fluorodifen conjugate (Fig. 2). REFERENCES

1. D. S. Frear and H. R. Swanson, Metabolism of substituted diphenylether herbicides in plants. I. Enzymatic cleavage of fluorodifen in peas (Pisum sativum L), Pestic. Biochem. Physiol. 3, 473 (1973).

G. L. Lamoureux and D. G. Rusness, The role of glutathione and glutathione-S-transferases in pesticide metabolism, selectivity, and mode of action in plants and insects, in “Glutathione: Chemical, Biochemical and Medical Aspects” (D. Dolphin, R. Poulson, 0. Avramovic, Eds.) Vol. BIB, pp. 153-l%, Wiley, New York, 1989. 3. T. J. Mozer, D. C. Tiemeier, and E. G. Jaworski, Purification and characterization of corn glutathione S-transferase, Biochemistry 22, 1068 2.

(1983).

4. W. Elling, S. J. Huber, B. Bankstahl, and B. Hock, Atmospheric transport of atrazine: A simple device for its detection, Environ Pollut. 48, 77 (1988).

5. M. Koval, H. Giehl, P. Matuska, D. ScharBe, H. E. Scheel, K. Schlitt, W. Seiler, and J. Wer-

218

6.

7.

8. 9.

10.

11. 12.

13.

14.

SCHRtiDER hahn, Bestimmung der horizontalen und verikalen Verteilung verschiedener Spurengase im Rahmen des Tulla-Projekts, AbschIuBbericht ISPRA, 1986. J. Milller, Aromatic and chlorinated hydrocarbons in forest areas, in “Mechanisms and Effects of Pollutant-Transfer into Forests” (H. W. Georgii, Ed.), Kluwer pp. 133-139, Academic Pub., 1989. A. Reischl, M. Reissinger, and 0. Hutzinger, Accumulation of organic air constituents by plant surfaces. III. Occurrence and distribution of atmospheric organic micropollutants in conifer needles, Chemosphere, 16, 2647 (1987). C. Gaggi and E. Bacci, Accumulation of chlorinated hydrocarbon vapour in pine needles, Chemosphere 14(5), 451 (1985). M. Hinkel, A. Reischl, K.-W. Schramm, F. Trautner, M. Reissinger, and 0. Hutzinger, Concentration levels of nitrated phenols in conifer needles, Chemosphere, in press. K. Bachmann and J. Polzer, The determination of phosgene in the lower troposphere, in “Mechanisms and Effects of Pollutant-Transfer into Forests (H. W. Georgii, Ed.), pp. 125-132, Kluwer Academic Pub. 1989. R. E. Kadunce and G. L. Lamoureux, Synthesis of Pentachloronitrobenzene-‘4C,, J. Lab. Comp. Radiopharm. 12, 459 (1976). G. L. Lamoureux, L. E. Stafford, and F. S. Tanaka, Metabolism of 2-chloro-N-isopropylacetamide (Propachlor) in the leaves of corn, sorghum, sugarcane and barley, J. Agric. Food Gem. 19, 346 (1971). W. H. Habig, M. J. Pabst, and W. B. Jacoby, Glutathione S-transferases: The first step in mercapturic acid formation, J. Biol. Chem. 22, 7130 (1974). M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of pro-

ET AL.

15. 16.

17.

18.

19.

20.

21.

22.

tein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248 (1976). M. Dixon and E. C. Webb, “Enzymes,” pp. 145166 Academic Press, New York, 1964. G. L. Lamoureux and D. G. Rusness, Pentachloronitrobenzene metabolism in peanut. 1. Mass spectral characterization of seven glutathionerelated conjugates produced in vivo or in vitro, J. Agric. Food Chem. 28, 1057 (1980). G. L. Lamoureux and D. G. Rusness, Propachlor metabolism in soybean plants, excised soybean tissues, and soil, Pestic. Biochem. Physiol. 34, 187 (1989). R. H. Shimabukuro, G. L. Lamoureux, H. R. Swanson, W. C. Walsh, L. E. Stafford, and D. S. Frear, Metabolism of substituted diphenylether herbicides in plants. II. Identification of a new fluorodifen metabolite, S-(2-nitro4-trifluoromethylphenyl)-glutathione in peanut, Pesric. Biochem. Physiol. 3, 483 (1973). G. L. Larsen and R. Ryhage, Fast atom bombardment mass spectra of mercapturic acid-pathway metabolites of propachlor (2-chloro-N-isopropylacetanilide), Xenobiotica 12, 855 (1982). G. L. Lamoureux and D. G. Rusness, Malonylcysteine conjugates as end-products of glutathione conjugate metabolism in plants, in “IUPAC Pesticide Chemistry, Human Welfare and the Environment” (J. Miyamato et al., Eds.), p. 295, Pergamon , New York, 1983. G. L. Lamoureux, H. R. Shimabukuro, H. R. Swanson, and D. S. Frear, Metabolism of 2chloro-4-ethylamino-6-isopropylaminos-triazine (atrazine) in excised Sorghum leaf sections, J. Agric. Food Chem. 18, 81 (1970). K. P. Timmerman, Molecular characterization of corn glutathione S-transferase isoenzymes involved in herbicide detoxication, Physiol. Plant. 77, 465 (1989).