Cytochrome P450 monooxygenase and glutathione S-transferase activity of two Australian termites: Mastotermes darwiniensis and Coptotermes acinaciformis

Pergamon

0965-1748(94)E0016-A

Insect Biochem. Molec. Biol. Vol. 24, No. 9, pp. 929-935, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00

Cytochrome P450 Monooxygenase and Glutathione S-Transferase Activity of Two Australian Termites: Mastotermes darwiniensis and Coptotermes acinaciformis V. S. HARITOS,t~ J. R. J. FRENCH,§ J. T. A H O K A S t Received 22 September 1993; revised and accepted 16 February 1994

The major detoxication enzymes have been characterized in preparations of two economically important termite species. Mastotermes darwiniensis microsomes contained a similar quantity of total cytochrome P450 as Coptotermes acinaciformis but the activities of aldrin epoxidase (AE), 7-etboxyresorufin O-deethylase (EROD) and 7-ethoxyeoumarin O-deethylase (ECOD) were 4.5, 5.8 and 17 times higher, respectively. Compared with other insects, AE activity in these two termite species is low, EROD activity moderate and ECOD activities are high, especially in M. darwiniensis. The cytosolic GST activity of C. acinaciformis toward ehloro-3,5-dinitrobenzene (CDNB) was 3.13/~ mol min-t mg-t, > 3 fold higher activity than M. darwiniensis cytosol. Apparent Km values of 1.87 mM for CDNB and 0.84 mM for glutathione were determined in C. acinaciformis cytosol. 7,8-Benzoflavone dramatically reduced EROD activity in microsomes of both termite species but had significantly lower inhibitory effect on ECOD activity. The addition of SKF 525A to termite microsomes inhibited both EROD and ECOD activities. Multiple isoenzymes of cytochrome P450 in termites are indicated by these findings. The distinctly different substrate specificities and 2maxof the CO difference spectra from M. darwiniensis and C. acinaciformis microsomes may arise from differences in the cytochrome P450 isoenzymes between the two species. Cytochrome P450 monooxygenases Glutathione S-transferases Termites Isoptera

formation of hydroxides, epoxides, N- and S-oxides and other products. GSTs catalyse the conjugation of Cytochrome P 450 monooxygenases (E. C. 1.14.14.1) and organic molecules possessing a reactive electrophilic glutathione S-transferases (GSTs, E.C. 2.5.1.18) are two centre with the thiol group of the tripeptide glutathione. important multicomponent enzyme systems involved The effect of both of the transformations is to convert in the metabolism of a broad range of foreign and lipophilic chemicals into water soluble products that can endogenous chemicals. In mammals and some insects be excreted. Products of cytochrome P450 monocytochrome P450 monooxygenases and GSTs have been oxygenase activity may be further metabolized by conjushown to exist as a number of isoenzymes with varying gation with endogenous molecules such as glutathione, substrate specificities (Jakoby and Habig, 1980; Clark, sulphate and glucuronic acid to aid in their excretion 1989; Soderlund and Bloomquist, 1990). (Jakoby and Habig, 1980). Cytochrome P450 monooxygenases catalyse a variety Monooxygenases play an important role in the of oxidative reactions which incorporate an atom of synthesis and degradation of insect hormones such as molecular oxygen into the substrate resulting in the juvenile hormone and ecdysones and in the oxidative metabolism of fatty acids (Hodgson, 1985; Ronis and Hodgson, 1989). The interaction of phytotKey Centre for Applied and Nutritional Toxicology, RMIT- phagous insects with different host plants can lead to an University,GPO Box 2476V, Melbourne,Australia and Divisionof induction of detoxication enzymes as a response to Forest Products, CSIRO, Private Bag I0, Clayton, Australia. changing allelochemical (deterrence chemicals) content of SAuthor for correspondence. §Division of Forest Products, CSIRO, Private Bag 10, Clayton, the plants (Brattsten et al., 1977; Terriere, 1984; Berenbaum, 1991). Australia. INTRODUCTION

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V.S. HARITOS et al.

One of the most important aspects of insect biotransformation is the metabolism of pesticides and biochemical mechanisms of resistance. The role of monooxygenases is of primary and critical importance in these respects due to their lack of substrate specificity, the wide range of biotransformations carried out and the ability of the cytochrome P450 monooxygenase system to be induced by exposure to many chemicals (Kulkani and Hodgson, 1984; Hodgson, 1985). The protection afforded by GSTs in insects is evident by the induction of these enzymes after exposure to sublethal concentrations of insecticides and high constitutive levels in certain insecticide resistant strains (Motoyama and Dauterman, 1977; Chiang and Sun, 1993; Lagadic et al., 1993). The consumption of plants containing allelochemicals by phytophagous insects can also lead to rapid changes in GST activity (Moldenke et al., 1992). Cytochrome P450 monooxygenases and GSTs are the most intensively studied detoxication enzymes. They have been detected in animal tissues, plants and prokaryotes but by far the most is known about mammalian enzymes. The detoxication enzymes of insects such as the housefly Musca domestica, Drosophila melanogaster and Spodoptera spp. have been given some attention but by comparison, nothing is known of the detoxication enzymes of termites (order: Isoptera). Yet termites are highly important insects both ecologically and economically. Termites play a vital role in the global cycling of carbon and trace elements as they digest cellulose from wood, grass and humus for energy with the aid of intestinal microorganisms (Waller and LaFage, 1987). Termites are also major structural and crop pests in many continents including Australia, Africa, Asia and the Americas. They impact on many economic activities but most significantly damage building timbers and forest, fruit and ornamental trees (French, 1986). The North Australian termite, M. darwiniensis and C. acinaciformis are Australia's most destructive termites and are distinguished by their ability to consume most wood types. For termites, an effective detoxication system could aid the consumption of different woods containing a variety of allelochemicals. Recently we reported the metabolism of a chlorinated environmental pollutant by M. darwiniensis and C. acinac(formis (Haritos et al., 1993). 4,4'-Dichlorobiphenyl incorporated in their food was metabolized to the hydroxylated derivative and excreted by the termites in the faeces. Polychlorinated biphenyls are metabolized by mammalian cytochrome P450 monooxygenases to hydroxylated derivatives (Safe et al., 1975) and the excretion of a similar metabolite from exposed termites suggested there was effective monooxygenase activity in termites. We have investigated the detoxication activity of microsomal and cytosolic enzymes of M. darwiniensis and C. acinaciformis using standard biochemical substrates. Comparisons of activity within the two termite species and between termites and other insects are then made.

MATERIALS AND M E T H O D S

Chemicals All reagents were of analytical grade, HPLC grade or equivalent. 7-Ethoxyresorufin, 7-ethoxycoumarin, chloro-3,5-dinitrobenzene (CDNB), 7-hydroxycoumarin, dithiothreitol, ethylenediamine tetraacetic acid (EDTA), glutathione (GSH) and 7,8-benzoflavone were purchased from Sigma, St. Louis, U.S.A. Glucose-6phosphate, nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphate dehydrogenase were obtained from Boehringer Mannheim, Germany. Resorufin was purchased from Eastman Kodak, U.S.A. Aldrin and dieldrin were obtained from ChemService, U.S.A. SKF 525A was a gift of Smith Kline and Beecham, U.K. Insects M. darwiniensis Froggatt (Isoptera:Mastotermitidae) and C. acinaciformis Froggatt (Isoptera: Rhinotermiti dae) were collected from field colonies in Kakadu National Park, Northern Territory, Australia. M. darwiniensis colonies were kept for a short time at 3TC and 75% relative humidity and C. acinaciformis were kept at 27°C and 75% relative humidity. The worker termites of M. darwiniensis only and mixtures of soldiers and worker termites of C. acinaciformis were used for the study. Preparation of microsomes Termites (8-16g) were separated from mound materials and killed by freezing at -20°C for 15 min. The whole bodies were homogenized in 5vol of cold 0.1 M sodium phosphate buffer, pH 7.4, containing 1 mM dithiothreitol and 1 mM EDTA using an UltraTurrax tissue homogenizer (Janke and Kunkel, Germany) in pulses, until a smooth consistency was obtained (ca. 40s). The homogenate was strained through a double layer of muslin cloth prior to centrifugation at 10,000g for 20min at 4°C. The lipid was removed and the microsomal pellet was obtained from the supernatant after centrifugation at 100,000g for 1 h at 4°C in a Beckman LM-80M Ultracentrifuge equipped with a 55Ti rotor. The pellet was resuspended in 0.1 M sodium phosphate buffer containing 20% glycerol (v/v) and stored with the cytosolic fraction at -80°C. Enzyme assays The content of cytochrome P450 in the microsomal samples were determined by the CO difference method of Omura and Sata (1964) and Rutten et al. (1987) using a Shimadzu UV-3000 spectrophotometer. Briefly, termite microsomes were diluted with 10vol of chilled 0.1 M sodium phosphate buffer, pH 7.4, containing 20% glycerol (v/v). Half the sample was bubbled with CO for 30 s and both sample and reference cu,vettes reduced with 1.14M sodium dithionite (10/~l). The difference spectrum was measured between 500~400nm after

DETOXICATION ENZYMES OF TERMITES 5-7 min. An extinction coefficient of 91 mM ~cm ~ was used for quantification. Aldrin epoxidase (AE) activity of termite microsomes was measured by the method of Wolffet al. (1980). Final assay volume was 1 ml which contained 0.1 M sodium phosphate buffer, pH 7.4, 2.5 mM MgC12, 50 mM KCI, a N A D P H regenerating system containing 0.3raM NADP, 1.5 mM glucose 6-phosphate and 2.5 units glucose 6-phosphate dehydrogenase and ca. 0.8 mg microsomal protein. The assay was initiated by the addition of aldrin (0.2 mM final concentration) after a 2 min preincubation of the mixture and carried out with shaking for 15 min at 28°C. The activity was terminated by the addition of n-hexane (2 ml) and the mixture vortexed for 3 min. The layers were separated by centrifugation at 2000g and an aliquot was removed for analysis of dieldrin concentration using Shimadzu GC14A gas chromatograph and 63Ni electron capture detection. Split injections of 1/~1 were made onto a 25 m x 0.22 mm DB-5 capillary column (SGE, Australia) at 200°C programmed to 250°C at 10°C per min. The injection port and detector temperature were held at 250°C and 270°C respectively. The concentration of dieldrin was measured from a calibration curve constructed from dieldrin standards in the range 100 to 1000 ng/ml. Microsomal ethoxyresorufin-O-deethylase (EROD) activity was measured using a modified Pohl and Fouts (1980) method. The assay mixture consisted of 0.1 M Tris buffer pH 7.6, 2.5 mM MgC12, 50 mM KC1, 1.2 mg BSA, N A D P H regenerating system (as above) and ca. 0.8 mg termite microsomal protein. The assay was initiated by the addition of 7-ethoxyresorufin (final concentration 1.2 #M) at 28°C after a 2 min incubation. Ethoxycoumarin-O-deethylase (ECOD) activity of termite microsomes was measured using the method of Ullrich and Weber (1972) modified as follows. The addition of 7-ethoxycoumarin (final concentration 0.5 raM) initiated the assay which contained 0.1 M Tris buffer, pH 7.4, 0 . 5 m M MgC12, 10mM KC1, BSA 1.2rag, N A D P H regenerating system (as above) and ca. 0.8 mg termite microsomal protein in a 2 ml total volume. The assay was carried out at 30°C after a 2 rain incubation. E R O D assays were terminated with methanol (2 ml) and ECOD assays with 5% zinc sulphate and saturated barium hydroxide solution (1 ml each). The concentration of products from the assays, resorufin and 7-hydroxycoumarin respectively, were measured using a Hitachi F-4500 fluorimeter at excitation/emission wavelengths of 530/585nm for resorufin and 380/452nm for 7-hydroxycoumarin. Quantity of product formed was determined from standard curves in the ranges of 0.25-2nmol/ml and 0.13-1 nmol/ml, respectively. Prior experiments using M. darwiniensis microsomes incubated between 20-36°C at 4°C intervals determined 28°C and 30°C to be the temperatures of maximal E R O D and ECOD activity. E R O D activity was linear over the time range of 5-20 min; a 15 min incubation time was selected as providing adequate sensitivity.

931

Monooxygenase inhibitors, 7,8-benzoflavone and SKF 525A were added to E R O D and ECOD assay mixtures prior to addition of the substrates. 7,8-Benzoflavone was added in 20/~1 of methanol giving final assay concentrations of 10 and 100 #M. SKF 525A was added in 0.1 ml Mili-Q water to produce final concentrations of 125 and 250 #M. GST activity of termite cytosol was measured by the method of Habig et al. (1974) monitoring the formation of C D N B - G S H conjugate at 340nm (E = 9 . 6 m M ~cm ~) over 1.5min using a Shimadzu UV-2101 spectrophotometer equipped with a thermostatted cell block. The assay was carried out at 37°C in 0.1 M sodium phosphate buffer at pH 6.5 and was corrected for non-enzymatic reaction rate. The optimal assay temperature for CDNB activity of termite cytosol was investigated between 25-46°C at 3°C intervals and found 37-40°C to produce maximal activity. The standard assay mixture for the measurement of activity contained 2.5 mM CDNB and 6 mM GSH. The apparent kinetic parameters Km and Vmaxwere determined for CDNB and GSH in C. acinaciformis cytosol. The final concentration of CDNB was varied over 5 values between 0.25-5 mM (GSH assay concentration 6 mM) and GSH over 5 values between 0.25-6 mM (CDNB assay concentration 2.5 raM). Each experimental point was measured in quintuplicate and the mean value and standard deviation determined. Estimates of Km and Vmax were calculated with a least squares non-linear fitting procedure of velocity against substrate concentration (Cleland, 1979) utilizing SYSTAT 5.1 data handling computer package. Protein concentrations were measured by the method of Lowry et al. (1951) using BSA as standard protein.

RESULTS Cytochrome P450 determinations The dithionite reduced, CO difference spectra of termite microsomes show an absorbance maxima near 450 nm with only a small amount of the degradation product, cytochrome P420 ( < 5 % of total) produced (Fig. 1). M. darwiniensis microsomes show a hypochromic shift in wavelength of maximum absorbance to 448 nm in the CO difference spectrum whereas C. acinaciformis microsomes appeared at 450 nm consistently. Although termites were processed from different field collections there was only small variation in the measured cytochrome P450 content of the termite microsomes (Table 1). M. darwiniensis and C. acinaciformis microsomes have similar cytochrome P450 content per mg of microsomal protein from whole body preparations. Microsomal monooxygenase activities The catalytic activities of AE, E R O D and ECOD from termite microsomes are given in Table 1. C.

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V . S . H A R I T O S et al.

0.03-

T A B L E 1. Monooxygenase characteristics and activities of M. darwiniensis and C. acinacformis microsomes prepared from whole body homogenates Monooxygenase characteristic

A

0'02--

O.O1Z 00 ,n,,

o

(,rj ¢1

0

,400 /

450 WAVELENGTH

500

(NM)

F I G U R E 1. CO difference spectra of dithionite reduced microsomes of C, acinaciformis (A, 2m,x 450 nm) and M. darwiniensis (B, "~max 448 nm). Whole body homogenate yielded similar quantities of cytochrome P450 per mg protein in both termite species. Minor a m o u n t s of cytochrome P420, the degradation product ofcytochrome P450, are formed during processing.

acinaciformis microsomes had considerably lower activity toward all substrates used. The AE activity of M. darwiniensis is 6 times that of C. acinaciformis, the EROD activity 8 times higher and the ECOD activity 21 times higher per mg of microsomal protein. When the monooxygenase activities are expressed per nmol of cytochrome P450 in the microsomes, AE of M. darwiniensis is 4.5 times higher than that of C. acinaciformis, the EROD activity 5.8 times higher and ECOD activity 17 times higher. Monooxygenase inhibition

activity

assay

requirements

and

N A D P H was a major requirement for E R O D and ECOD activity in both termite species. Metabolism of 7-ethoxyresorufin in M. darwiniensis and C. acinaciformis microsomes was reduced to l and 16%, respectively, of normal and 7-ethoxycoumarin reduced to 2 and 1% of normal, in the absence of an added source of NADPH. The degree of inhibition of monooxygenase activity by 7,8-benzoflavone and SKF 525A was dependent on the concentration of inhibitor (Fig. 2). 7,8-Benzoflavone dramatically reduced E R O D activity in both M. darwiniensis and C. acinaciformis but had a significantly lower inhibitory effect on ECOD activity. SKF 525A inhibited both E R O D and ECOD activities of M. darwiniensis and C. acinaciformis but affected EROD activity more than ECOD. In general, M. darwiniensis monooxygenase activities were more susceptible to inhibition by SKF 525A than C. acinaciformis.

Cytochrome P 4 5 0 * t ( n m o l m g t protein) Cytochrome P450" (AA mg t protein) 2m,X (50(P400 rim) AE;~ (nmol min i m g - i ) EROD§ (nmol m i n - t mg i ) ECOD¶ (nmol m i n - i m g I ) AE ( n m o l m i n i nmol P450 i) EROD (nmol min Lnmol P450 i) ECOD (nmol min i nmol P450 l)

M. darwiniensis

C. acinaciJormis

0.142 ! 0.005

0.128 + 0.009

0.00636 _+ 0.0003

0.00563 _+ 0.0004

448 0.078 _+ 0.005

450 0.013 _+ 0.001

0.015 +_ 0.001

0.002 _+ 0.000

0.259 _+ 0.021

0.012 + 0.001

0.551 _+0.031

0.121 _+ 0.001

0.105 ± 0.008

0.018 + 0.003

1.830 _+ 0.145

0.108 _+ 0.012

Data represent the means + SE of two separate experiments each with three or more determinations except * where data represent the mean ___SE of at least 6 separate determinations. t A s s u m e s an extinction coefficient of 91 m M i cm ~. :~AE aldrin epoxidase. §EROD 7-ethoxyresorufin O-deethylase. ¶ E C O D 7-ethoxycoumarin O-deethylase.

Glutathione S-transferase activity The cytosolic fraction of termites has moderate to high conjugating activity toward CDNB. Mean activities for M. darwiniensis and C. acinaciformis were 0.877 _+ 0.028 and 3.131 _+ 0.083/~mol min 1mg ~. Apparent Km and Vm,x values and standard errors for CDNB were calculated as 1.87_+0.19mM and 5.01 _+0.22/~molmin ~mg ~ protein. The same parameters for GSH were calculated as 0.91 _+ 0.08 mM and 3.65 ± 0.10 pmol min ~mg -~. The curves obtained from the plots of substrate concentration against velocity are shown in Fig. 3. Specific activity of termite cytosol increased linearly with increasing assay protein (r2= 0.996) in the range 44-176 pg. DISCUSSION The role of detoxication enzymes of insects in metabolizing insecticides, hormones, allelochemicals and other xenobiotics is well established. The major detoxication enzymes of many insects have been characterized and reported, however, we believe this to be the first report of these enzymes and activities in the order Isoptera. There are a number of reasons why understanding of this system in termites is of great interest. Firstly, on a global scale, termites consume around 7 billion tonnes of biomass each year and release between 4 and 40% of the total yearly emission of methane (Zimmerman et al., 1982; Khalil et al., 1990). Termites therefore are highly successful insects and an integral component of the biosphere. Also, knowledge of the ability of termites to detoxify pesticides, especially the organochlorines used

DETOXICATION ENZYMES OF TERMITES lOO~

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20

40

60

7,8-benzoflavone

j

80

0

100

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100

2o

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o'o

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acinaciformis ----O.-- EROD

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>,

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FIGURE 2. The differential inhibition of monooxygenase activity by 7,8-benzoflavone and SKF 525A in microsomal preparations from M. darwiniensis and C. acinaciformis. The effects of inhibitors on EROD (C)) and ECOD (m) activity were determined.

for their control, may in future provide starting points in the development of termiticides. The involvement of termite detoxication enzymes with wood allelochemicals may provide a basis for the existence of polyphagy in some termites but not in others. The cytochrome P450 content of microsomes prepared from whole bodies of M. darwiniensis and C. acinaciformis are similar to that of other insects prepared in this manner such as Drosophila melanogaster reported to contain 0.14 nmol mg 1protein (Ronis and Hodgson, 1989). However, individual tissues of insects can contain high levels of cytochrome P450; up to 1.52 nmol mg-J as measured in housefly fatbody (Lee and Scott, 1992). Model substrates such as aldrin, 7-ethoxyresorufin and 7-ethoxycoumarin can be used to compare the monooxygenase activities of M. darwiniensis and C. acinaciformis with other insects although AE, EROD and ECOD activities vary greatly between different insects. Variation in detoxication enzyme activity can arise through induction of activity by exposure of phytophagous insects to plant allelochemicals or from the constitutively high activities found in some pesticide resistant insect strains (Hodgson, 1985; Ronis and Hodgson, 1989). Compared with other insects AE activity in these two termite species is low. The reported AE activity of housefly (Lee and Scott, 1992) is 20-fold higher than M. darwiniensis and fall armyworm AE activity (Yu, 1991) is almost 50-fold that of C. acinaciformis. EROD activity is similar to many insects but ECOD activities are high, especially in M. darwiniensis. The termites used in this study have had no known IB 24/9

D

exposure to pesticides yet they have ECOD activities similar to pesticide resistant strains of insects. M. darwiniensis have 15-fold higher ECOD activity compared to housefly and 1.4-fold higher activity than a pesticide resistant strain of housefly (Lee and Scott, 1992). It is possible that termite detoxication activity can be induced on exposure to dietary toxicants (e.g. allelochemicals) resulting in high measured EROD and ECOD activities. The hypochromic shift to 448 nm of the CO difference spectrum of cytochrome P450, similar to that observed in M. darwiniensis microsomes, is normally associated with the development of insecticide resistance in insects (Hodgson, 1985). However, termites have never been reported to exhibit insecticide resistance, merely avoidance behaviour when exposed to certain pesticides (Lockwood et aL, 1984). Certain inhibitors of monooxygenases have been used to differentiate between isoenzymes of cytochrome P450 in mammals. 7,8-Benzoflavone is a direct acting competitive inhibitor of cytochrome P450 that is highly specific for the mammalian cytochrome P4501A group (which in the rat also exhibit a hypochromic shift to 448 nm on induction with polycyclic aromatic hydrocarbons) while SKF 525A is a specific inhibitor of the constitutive or phenobarbitone-inducible form of cytochrome P450 (Testa and Jenner, 1981). The dramatic inhibitory effect of 7,8-benzoflavone on EROD activity but not ECOD activity in M. darwiniensis and C. acinaciformis suggests that this assay is measuring the equivalent of the mammalian cytochrome P4501A group in termites. The existence of an aromatic hydrocarbon-inducible form of

934

V.S. HARITOS et al.

pigments or proteolytic enzymes. It should be noted that termites do not have eyes (except the reproductive caste) and are primarily cellulose digesters. However the exist3' ence of other inhibitory substances in termites cannot be E ruled out. The lack of cytochrome P450 degradation eproduct, cytochrome P420, in termite microsomes °~ .*Z_E 2. suggests that this enzyme is little affected by proteolytic O O enzymes during homogenization of the whole bodies. However, any effect on enzymes associated with monooxygenases such as N A D P H - c y t o c h r o m e P450 reductase is unknown at this stage. The G S T activity of termite cytosol toward C D N B is high by comparison with other insects. Insect G S T 1 2 3 4 5 activities can vary from 8 and 6 #tool min ~mg t pro[CDNB] mM tein for aphid and german cockroach respectively (Stenersen et al., 1987) to 0.1/~mol min -~ mg -1 protein for S. littoralis (Lagadic et al., 1993). M. darwiniensis specific activity is substantially lower than that of C. acinaciformis which suggests there is a qualitative difference in G S T isoenzymes or a quantitative difference in a particular isoenzyme which has good activity toward C D N B between the two species. Also, as in the case of inhibition or degradation of monooxygenases derived from whole body preparations of insects, endogenous B > o :=L and foreign substances have been found to inhibit 1 C D N B conjugation in some insects (Clark, 1989). We are undertaking the purification of termite GST isoenzymes in order to address these questions. The apparent Michaelis constant for G S H as determined in termite 1 2 3 4 5 6 cytosol falls within the common range of K m values [GSH] mM derived from invertebrate GSTs (Clark, 1989). The FIGURE 3. Plots of mean velocity (and standard deviation) vs termite apparent Km for C D N B is higher than mamsubstrate concentration for the determination of kinetic parameters Km malian values but is comparable to that of other insects and Vm~x in C. acinaciformis cytosol. Curves were obtained with varying concentrations of CDNB (plot A) and GSH (plot B) with (Lagadic et al., 1993). The termite gut contains an array of symbiotic microconcentrations of cosubstrate of 6 mM GSH and 2.5 mM CDNB. Each data point represents the mean of five measurementsand was fitted to organisms, protozoa and bacteria in the lower termites a least squares non-linear regression equation. and bacteria in the higher termites (Waller and LaFage, 1987). Obligate anaerobic microorganisms in the termite cytochrome P450 in insects, such as the mammalian gut are unlikely to be involved in cytochrome P450 cytochrome P4501A, has not been well defined. monooxygenase metabolism, however xenobiotic metabA notable finding is the much lower monooxygenase olism by other gut microorganisms cannot be ruled out. activities toward all three model substrates in C. acinaci- Where such a contribution exists whole termite body formis despite the two termite species having similar homogenate would more accurately reflect xenobiotic content of cytochrome P450 per mg of microsomal metabolism of the whole organism than processed indiprotein. There can be several reasons for this finding. vidual tissues. Different isoenzymes of cytochrome P450 may be preThe cytochrome P450 monooxygenase and G S T acsent in the microsomes from the two termite species and tivities in two of Australia's most economically importsupporting this proposal is the distinctly different sub- ant termites have been determined. This provides new strate specificities of M. darwiniensis monooxygenases information of the detoxication capability of an order and C. acinaciformis and the different wavelengths of of insects that have important global and economic absorbance maxima of the CO difference spectra. significance. Other possible reasons for the finding are that the monooxygenases of one or both termite species may be REFERENCES inhibited or degraded as a result of processing. It has Berenbaum M. (1991) Comparative processing of allelochemicals in been previously described that homogenization of whole the Papilionidae (Lepidoptera). Archs Insect Biochem. Physiol. 17, insects can release and mix endogenous substances that 213~21. are inhibitory to monooxygenases (Hodgson, 1985). Brattsten L., Wilkinson C. and Eisner T, (1977) Herbivore plant Most of the examples of endogenous inhibitors affecting interactions: mixed function oxidases and secondary plant submonooxygenases in insect preparations have been visual stances. Science 196, 1349 1352. |

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DETOX1CATION ENZYMES OF TERMITES Chiang F.-M. and Sun C.-N. (1993) Glutathione transferase isozymes of Diamondback moth larvae and their role in the degradation of some organophosphorus insecticides. Pestie. Biochem. Physiol. 45, 7-14. Clark A. G. (1989) The comparative enzymology of the glutathione S-transferascs from non-vertebrate organisms. Comp. Biochem. Physiol. 92B, 419M46. Cleland W. W. (1979) In Methods in Enzymology (Edited by Purich D. L.), Vol. 63, pp. 103 138. Academic Press, New York. French J. (1986) Termites and their economic importance in Australia. In Economic Impact and Control of Social Insects (Edited by Vinson S.), pp. 103-129. Praeger, New York. Habig W., Pabst M. and Jakoby W. (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. biol. Chem. 249, 7130 7139. Haritos V., French J. and Ahokas J. (1993) The metabolism and comparative elimination of polychlorinated biphenyl congeners in termites. Chemosphere 26, 1291-1299. Hodgson E. (1985) Microsomal mono-oxygenases. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 11, pp. 225-321. Pergamon Press, Oxford. Jakoby W. B. and Habig W. H. (1980) Glutathione transferase. In Enzymatic Basis of Detoxication (Edited by Jakoby W. B.), Vol. II, pp. 63 94. Academic Press, New York. Khalil M., Rasmussen R., French R. and Holt J. (1990) The influence of termites on atmospheric trace gases: CH4, CO2, CHCI 3, N20, CO, H z and light hydrocarbons. J. Geophys. Res. 95, 3619 3634. Kulkani A. and Hodgson E. (1984)The metabolism of insecticides: the role of monooxygenase enzymes. A. Rev. Pharmac. toxic. 24, 19-42. Lagadic L., Cuany A., Berge J. and Echaubard M. (1993) Purification and partial characterization of glutathione S-transferases from insecticide-resistant and lindane-induced susceptible Spodoptera littoralis (Boisd) larvae. Insect Biochem. Molec. Biol. 23, 467-474. Lee S. and Scott J. (1992) Tissue distribution of microsomal P450 monooxygenases and their inducibility by phenobarbital in House fly, Musca domestica L. Insect Biochem. Molec. Biol. 22, 699-712. Lockwood J., Sparks T. and Story R. (1984) Evolution of insecticide resistance to insecticides: a reevaluation of the roles of physiology and behavior. Bull. Ent. Soc. Amer. 30, 41-51. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. Moldenke A., Berry R., Miller J., Kelsey R., Wernz J. and Venkateswaran S. (1992) Carbaryl susceptibility and detoxication enzymes in Gypsy moth (Lepidoptera:Lymantriidae): influence of host plant. J. econ. Ent. 85, 1628-1635. Motoyama N. and Dauterman W. C. (1977) Purification and properties of housefly glutathione S-transferase. Insect Biochem. 7, 361 369.

935

Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. biol. Chem. 239, 2370-2378. Pohl R. J. and Fouts J. R. (1980) A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Analyt. Biochem. 107, 150-155. Ronis M. and Hodgson E. (1989) Cytochrome P450 monooxygenases in insects. Xenobiotica 19, 1077 1092. Rutten A. A., Falk H. E., Catsburg J. F., Topp R., Blaauboer B. J., von Holsteijn I., Doorn L. and van Leeuwen F. X. (1987) Interlaboratory comparison of total cytochrome P450 and protein determinations in rat liver microsomes. Archs Toxic. 61, 27 33. Safe S., Hutzinger O. and Jones D. (1975) The mechanism of chlorobiphenyl metabolism. J. agric..food Chem. 23, 851-853. Soderlund D. and Bloomquist J. (1990) Molecular mechanisms of insecticide resistance. In Pesticide Resistance in Arthropods (Edited by Roush R. and Tasnik B.), pp. 5846. Chapman and Hall, New York. Stenersen J., Kobro S., Bjerke M. and Arend U. (1987) Glutathione transferases in aquatic and terrestrial animals from nine phyla. Comp. biochem. Physiol. 86C, 73-82. Terriere L. C. (1984) Induction of detoxication enzymes in insects. A. rev. Ent. 29, 71 88. Testa B. and Jenner P. (1981) Inhibitors of cytochrome P450's and their mechanisms of action. Drug metab. Rev. 12, 1-117. Ullrich V. and Weber P. (1972) The O-dealkylation of 7-ethoxycoumarin by liver microsomes. A direct fluorometric assay. HoppeSeyler's Z. Physiol. Chem. 353, 1171 1177. Waller D. A. and La Fage J. P. (1987) Nutritional ecology of termites. In Nutritional Ecology of Insects, Mites, and Spiders (Edited by Slansky Jr F. and Rodriguez J. G.), pp. 487 532. Wiley, New York. Wolff T., Greim H., Huang M.-T., Miwa G. and Lu A. (1980) Aldrin epoxidation catalyzed by purified rat-liver cytochrome P450 and P448. High selectivity for cytochrome P450. Eur. J. Biochem. 111, 545-551. Yu S. J. (1991) Insecticide resistance in the Fall Armyworm, Spodoptera frugiperda (J. E. Smith). Pestic. Biochem. Physiol. 39, 84 91. Zimmerman P., Greenberg J., Wandiga S. and Crutzen P. (1982) Termites: a potentially large source of atmospheric methane, carbon dioxide and molecular hydrogen. Science 218, 563 565.

Acknowledgements--The technical assistance of Ms J. Butty is gratefully acknowledged and for the field collection of termites we would like to thank Messrs B. Ahmed, J. Creffield and Ms N. Chew. This work was supported by the Australian Research Council.