Comparative Biochemistry and Physiology Part C 134 (2003) 501512

Activity of carboxylesterase and glutathione S-transferase in different life-stages of carabid beetle (Poecilus cupreus) exposed to toxic metal concentrations
Grazyna Wilczeka, Paulina Kramarzb,*, Agnieszka Babczynskaa
Department of Physiology and Ecotoxicology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9, Katowice 40-007, Poland b Department of Ecotoxicology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 3, Krakow 30-387, Poland Received 7 August 2002; received in revised form 10 February 2003; accepted 10 February 2003
a

Abstract Among the cytoplasmatic enzymes responsible for neutralization of organic xenobiotics, carboxylesterases (CarE) and glutathione S-transferases (GST) play important roles. Our study tested to what extent dietary Zn or Cd could modify the activity of CarE and GST at different life-stages of the carabid beetle Poecilus cupreus. Treatment and stage effects generally were statistically significant. For CarE activity in the beetles exposed to cadmium, only treatment was a significant factor. In all cases, the interaction between studied factors was statistically significant, implying that the physiological condition of the animals may enhance or reduce enzyme activity. We also observed differences between animals treated with cadmium and zinc in the pattern of enzyme activity, and a difference in GST activity measured with two different substrates. Our results confirmed that in studying enzyme activity under metal stress one should consider the animals life-stage and sex. 2003 Elsevier Science Inc. All rights reserved.
Keywords: Zinc; Cadmium; Pupa; Larva; Adult; Male; Female; Detoxification; Organic xenobiotics

1. Introduction The ability of an organism to adapt to an environment altered by industrial contamination depends mainly on effective mechanisms of detoxification of various endo- and exogenic compounds (Jokanovic, 2001). Among the cytoplasmatic enzymes responsible for neutralizing xenobiotics carboxylesterases (CarE) and glutathione S-transferases (GST) play important roles (Toung et al., 1990). Carboxylesterases are generally characterized by low substrate specificity and most of them hydro*Corresponding author. Tel.: q48-12-2642516x157; fax: q 48-12-2690927. E-mail address: uxkramar@cyf-kr.edu.pl (P. Kramarz). 1532-0456/03/$ - see front matter PII: S 1 5 3 2 - 0 4 5 6 0 3 . 0 0 0 3 9 - 5

lyze both ester and amide bonds of xenobiotics (Jokanovic, 2001). Detailed studies, based on the electrophoresis method, and analyses of the kinetic properties of invertebrate carboxylesterases showed that these enzymes form a group of izoenzymes differing in their affinity for specific substrates (Soderlund and Bloomquist, 1990). Glutathione S-transferases are a group of structurally similar but functionally different izoenzymes with differing specificities (Grant et al., 1991). The activity of CarE and GST may be changed by allelochemicals (Iio et al., 1993), organophosphorous insecticides (Capua et al., 1991) and products of carcinogenic substances of metabolic activation (Lemaire-Gony and Lemaire, 1992). Metals in

2003 Elsevier Science Inc. All rights reserved.

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toxic concentrations may be also responsible for changing activity in enzyme activity (Malkenson de et al., 1984; Dowd and Sparks, 1987). Metal ions are probably not necessary as cofactors for CarE and GST activity and do not directly modify the action of the enzymes neither in vivo nor in vitro. The toxic effects of metals strongly depend on their properties, their concentrations in the organisms, as well as biological features of the organisms, such as age, sex and developmental stage (Mathova, 1990; Schmidt et al., 1992; Postma et al., 1994). This study was part of a project on the effects of prolonged intoxication with metals on carabid beetles at individual and population levels (e.g. Kramarz, 2000; Maryanski et al., 2001). The carabids have been recognized as poor accumulators of metals (Laskowski and Maryanski, 1993). Studies by Janssen (1991) and Kramarz (1999) indicate that this may be due to effective mechanisms of metal elimination. In our study, we tested to what extent an excess of Zn or Cd in the diet could modify the activity of CarE and GST at different life-stages in both sexes of the carabid beetle Poecilus cupreus. By comparing the effects of zinc, a biogenic element and cadmium, for which a biological role has not been recognized, we can draw conclusions about the ability of the study species to detoxify organic xenobiotics. 2. Material and methods 2.1. Animals Adult carabids were obtained from a laboratory stock culture kept at the Jagiellonian University and were prepared for reproduction according to Heimbach (1992). Five males and five females were put into breeding boxes. Eggs were removed every 3rd day. Eggs were put separately into cells of tissue culture boxes on wet filter paper. At the outset, each tissue culture box was assigned to a particular treatment. Due to expected high mortality, approximately twice as many eggs were assigned to the highest metal concentration treatments than in the control. Immediately after hatching, the larvae were exposed to experimental treatments. They were fed on housefly pupae reared on an artificial medium contaminated with 0, 40, 160, 640 or 800 mg Cd kgy1, or else with 0, 400, 1600, 6400 or 8000 mg

Zn kgy1 (dry wt.), respectively. The medium for the housefly pupae consisted of 515.6 g rabbit chow (Wytwornia Pasz, Andrzej Morawski, 89240, Kcynia, Poland), 20 g powdered milk (OSM Krotszyn), 10 g sugar, 0.02 g bakers yeast (Dr Oetcker) and 1 l distilled water or experimental solution (CdCl2=2.5 H2O or ZnCl2). After hatching, larvae were transferred to plastic vials (30 ml) filled with crushed wet garden peat and were fed every 3rd day on three housefly pupae until preparation. Twenty-five days after emerging from pupa, the adults in their peat-filled vials were refrigerated for overwintering (ca. 7 8C, complete darkness), to reach sexual maturity. After 93 days, all beetles were transferred to long-day conditions (16-h light) at 20 8C. 2.2. Enzyme preparation and assays The enzymatic assays for each experimental group were done in six replicates. Enzyme activity was measured in last-stage larvae ( just before pupation), immature adults of both sexes (20 days after emergence) and mature females and males (10 days after overwintering). For zinc treatments, CarE and GST were also measured in pupae. High mortality at this stage in Cd-treated carabids precluded assays. Animals (one per sample) from each developmental stage were weighed, anaesthetized on ice and then homogenized in a glassTeflon homogenizer at 4 8C in 0.05 M buffer TrisHCl, pH 7.6, using 50-fold initial homogenate dilution. The homogenates were centrifuged for 10 min at 15 000=g. After decanting and further dilution the supernatants were used for assays. Protein contents was measured according to Bradford (1976) using bovine albumin as the standard. 2.3. Carboxylesterases (CarE) wEC 3.1.1.1x Carboxylesterases (EC 3.1.1.1) were measured spectrophotometrically (Helios Epsilon UNICAM) in the submitochondrial fraction according to Ljungquist and Augustinsson (1971), with p-nitrophenyl acetate (p-NPA) as the substrate. Changes in the substrate hydrolysis product (p-nitrophenol) concentration were measured at ls400 nm. Results were expressed in nmol p-NPA miny1 mg proteiny1.

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Fig. 1. CarE activity (against pNPA) in different stages of P. cupreus vs. nominal cadmium concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

2.4. Glutathione 2.5.1.18x

S-transferases

(GST)

wEC

detected, means were separated by the LSD test. To obtain normal distributions the data were logtransformed (Zar, 1974, CSS-Statistica). 3. Results Metals and developmental stage-dependent effects generally were statistically significant (P0.005), except for CarE activity in the beetles intoxicated with cadmium, where only metal was a significant factor (P-0.005). In all cases, the interactions between the studied factors were statistically significant (P-0.0001). There were some differences between animals treated with cadmium and zinc in the pattern of enzyme activity, and a difference in GST activity measured against the two different substrates. 3.1. Carboxylesterases (CarE) wEC 3.1.1.1x 3.1.1. Cadmium Cadmium caused an increase in carboxylesterase activity in all stages of the beetles (Fig. 1), with the strongest effects in larvae, mature females and mature males (Fig. 1). Even though the specific stage had no effect on CarE activity (P)0.05, Fig. 2), the statistical significance of interactions between developmental stage and treatment implies that the physiological condition of the animals may affect this enzyme activity.

GST activity was determined in the postmitochondrial fraction, as the rate of conjugation of glutathione with 1-chloro-2,4 dinitrobenzene (CDNB, ls340 nm) and 3,4-dichloronitrobenzene (DCNB, ls344 nm) according to Lindroth (1989) and Yu (1982), respectively. The products of glutathione-CDNB and glutathione-DCNB conjugation are S(2-chloro-4-nitrophenyl) glutathione and S(2,4dinitrophenyl) glutathione, respectively. 2.5. Statistical analyses Statistical analyses were done separately for cadmium, zinc, each enzyme and each substrate in the case of GST. In the calculations, the nominal concentrations of metals in the housefly larvaes diet were taken to represent total concentrations in the environment, the common measure in field studies. As we expected a linear relationship between enzyme activity and metal concentration, at the first we intended to use regression analysis and to compare regression lines with developmental stage as a factor. However, because clear linear relationships were observed in only a few cases, these results were finally analyzed with two-way analysis of variance (MANOVA) with treatment and stage as factors. If significant differences were

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Fig. 2. CarE activity (against pNPA) vs. life stage of P. cupreus fed with food (M. domestica larvae) contaminated with different cadmium concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); means (small boxes) and S.D. (whiskers) are shown; ns: not statistically significant. L: larvae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

3.1.2. Zinc Even though the two studied factors had strong significant effects on animals intoxicated with zinc (P)0.00001), there was no clear pattern of carboxylesterase activity, neither for the metal nor for the developmental stage (Figs. 3 and 4). CarE activity was the same as in the controls or slightly enhanced, except for larvae intoxicated with zinc at the highest concentration, where CarE activity was lower than in the controls (Fig. 3). In animals compared between stages under the same dose of zinc, in all cases CarE activity was enhanced in pupae (Fig. 4). CarE activity in the remaining stages was similar, irrespective of the metal dose. In immature and mature females overall CarE activity depended on the zinc concentration (Fig. 3). 3.2. Glutathione 2.5.1.18x S-transferases (GST) wEC

mature males had the highest GST activity (Fig. 6). In other cases, an interesting pattern was observed. The highest GST activity, measured against DCNB was found in sexually mature adults. The lowest was observed in immature males and females, while larvae showed intermediate activity of the enzyme (Fig. 6). Less uniform results were obtained for GST activity measured against CDNB (Figs. 7 and 8). Significantly higher GST activity was found only in sexually mature females treated with the two highest doses of cadmium (Fig. 7). Differences between developmental stages in animals exposed to the same dose of the metal, as in the case of DCNB, were noted only for the control and the two highest Cd concentrations (Fig. 8). 3.2.2. Zinc No general pattern of changes in GST activity was observed in zinc-intoxicated animals (Figs. 912). Its activity measured against DCNB vs. the control was enhanced in larvae from the 6400Zn group, pupae intoxicated with the three highest zinc concentrations (with no difference between concentrations), immature females and males from zinc treatments and mature females from the 1600Zn group (Fig. 9). Lowered GST activity vs. the control was found in larvae at the lowest and highest zinc doses, and mature males from the lowest zinc treatment (Fig. 9). The highest GST

3.2.1. Cadmium GST activity measured against DCNB in larvae and immature adults treated with cadmium was slightly lowered or comparable to that of the control (Fig. 5). In mature females and males the activity of these enzymes was higher, but there were no concentration-depended differences (Fig. 5). In animals compared between stages under the same dose of cadmium, untreated larvae and

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Fig. 3. CarE activity (against pNPA) in different stages of P. cupreus vs. nominal zinc concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

activity was observed in larvae, the only exception being the 8000-Zn group, where mature males exhibited the highest GST activity (Fig. 10). GST activity measured against CDNB in zinc-intoxicated animals somewhat differed from that measured against DCNB (Figs. 912). The level of GST was also increased in immature females and males exposed to all concentrations of zinc. In contrast to the result with DCNB as a substrate, measured

against CDNB GST activity was enhanced in mature females administered the lowest and two highest zinc concentrations and in mature males treated with the three highest zinc concentrations (Fig. 11). 4. Discussion P. cupreus efficiently regulates the body load of metals, but the effectiveness of the process depends

Fig. 4. CarE activity (against pNPA) vs. life stage of P. cupreus fed with food (M. domestica larvae) contaminated with different zinc concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test). L: larvae; P: pupae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

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Fig. 5. GST activity (against DCNB) in different stages of P. cupreus vs. nominal cadmium concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

on the metal, (e.g. Kramarz, 1999). Zn-body concentrations was regulated well: the control beetles contained ca. 79 mg kgy1 (62% of the 128 mg Zn kgy1concentration in housefly pupae), and those from the highest nominal treatment reached only 110 mg kgy1, that is, ca. 16% of the concentration in the pupae (692 mg Zn kgy1) (Maryanski et al., 2001). In contrast, the final concentrations in Cd-treated beetles were substantially higher than

in the control beetles. The latter contained 0.36 mg kgy1, that is 39% of the concentration in housefly larvae. Carabids from the highest treatment (nominal 800 mg Cd kgy1) contained only 80.5 mg kgy1, a tenth the concentration in housefly pupae and the nominal Cd concentration. The largest jump in concentration in both the houseflies and the beetles was found for the lowest cadmium treatment (nominal 40 mg kgy1): the concentration

Fig. 6. GST activity (against DCNB) vs. life stage of P. cupreus fed with food (M. domestica larvae) contaminated with different cadmium concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test). L: larvae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

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Fig. 7. GST activity (against CDNB) in different stages of P. cupreus vs. nominal cadmium concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

in housefly pupae increased by more than 100 times vs. the control, and the carabids were ca. 30 times more contaminated than control beetles (11.3 mg Cd kgy1) (Maryanski et al., 2001). Despite the differences between cadmium and zinc detoxication (accumulation vs. efficient elimination) that seem to occur in P. cupreus, both processes are probably energy-intensive. This may explain why adult body mass decreased with increasing concentrations of both metals (Kramarz,

2000), and why intoxication with high doses of cadmium caused a decrease in adult body caloric value (Maryanski et al., 2001). Intoxication of P. cupreus with cadmium or zinc also had exerted some negative effects on population parameters (Kramarz, 2000). Consequences of intoxication with cadmium or zinc were also observed at the level of enzymatic activity: changes in CarE and GST activity depended on the metals used and their doses.

Fig. 8. GST activity (against CDNB) vs. life stage of P. cupreus fed with food (M. domestica larvae) contaminated with different cadmium concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test). L: larvae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

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Fig. 9. GST activity (against DCNB) in different stages of P. cupreus vs. nominal zinc concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

In general, measured by energy expenditures (Maryanski et al., 2001), both metals caused increased enzymatic activity. This holds true particularly for CarE activity at all stages of P. cupreus and in GST (against DCNB) in mature adults fed poisoned houseflies. Increased CarE and GST activity was also found in the carabid Pterostichus oblongopunctatus collected in metal-polluted areas (Stone et al., 2002). In our study, groups intoxicated with cadmium showed higher activity of detoxifying enzymes than those treated with zinc, likely due to differences between cadmium and

zinc in their biological roles or concentrations (Maryanski et al., 2001). Along with the weaker influence of zinc than cadmium on body caloric value (Maryanski et al., 2001), we also found weaker enzymatic response in zinc-treated beetles. The other sources of variance in enzyme activity obtained in our studieslife-stage and gender are well known in metal-treated insects (Augustyniak and Migula, 2000; Stone et al., 2002). During development, hormones, (e.g. juvenile and ecdysteroid) control enzyme synthesis, modifying their activity, including that of carboxyles-

Fig. 10. GST activity (against DCNB) vs. life stage P. cupreus fed with food (M. domestica larvae) contaminated with different zinc concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test). L: larvae; P: pupae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

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Fig. 11. GST activity (against CDNB) in different stages of P. cupreus depending on nominal zinc concentrations in their food (diet of M. domestica larvae). Means (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test).

terases (Ruvolo-Takasuki and Collet, 2000). This characteristic pattern of enzymatic response was also noted in our studies in control and metalintoxicated individuals of P. cupreus. However, the level of enzyme activity was higher in beetles fed on metal-poisoned food, and depended on the dose. As in a study by Kedziorski et al. (1996) on crickets, the differences between beetle stages did not follow a linear pattern. Age-dependent changes in enzyme activity may result from differences in the expression of the

particular isoenzymes taking part in transformation of endogenous substrates occurring during development (Woodring and Sparks, 1987). In Lucilia cuprina, Kotze and Rose (1987) observed increasing GST activity with age. In contrast, in insects ageing is often followed by a decrease in protein synthesis (Cohen, 1986). In the case of hydrolytic enzymes (CarE), in males of the solitary bee Megachile rotundata the activity of CarE decreased with age (Frohlich, 1990). Similarly, in our study, CarE activity was the lowest in mature

Fig. 12. GST activity (against DCNB) vs. life stage P. cupreus fed with food (M. domestica larvae) contaminated with different zinc concentrations. Bold numbers are nominal Cd concentrations (mg kgy1 ); mean (small boxes) and S.D. (whiskers) are shown. Different letters indicate significant differences between treatments (LSD test). L: larvae; P: pupae; IF: immature females; MF: mature females; IM: immature males; MM: mature males.

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beetles, while initial stages and mature beetles had higher GST activity than immature adults; a study on L. cuprina (Kotze and Rose, 1987) produced comparable findings. The level of detoxifying enzymes in P. cupreus differed also by sex. In this work, both control and metal-treated adult male beetles had higher GST activity than females, while CarE activity in females and males was similar. In a study of carabid beetles P. oblongopunctatus inhabiting a metal-contaminated environment, Stone et al. (2002) found stronger induction of detoxifying enzyme activity in females than in males. Similar gender-related differences were also shown in the grasshopper Chorthippus brunneus (Augustyniak and Migula, 2000). In our study, the between-sex differences are not clear, probably because the animals were exposed to excess metals only once in their history. In both cases cited above, the animals used for assays originated from sites polluted with metals for many generations, so the differences between males and females may reflect adaptation (Stone et al., 2002). In other studies, Konno and Shishido (1992) observed higher activity of GST in testes of Periplaneta americana than in ovaries. CarE activity might also differ between sexes: CarE activity in Aedes aegypti was higher in females than in males (Argentine and James, 1995), while females of M. rotundata had lower activity than males (Frohlich, 1990). Stimulation of CarE activity by cadmium can also result from an indirect influence on CarEgene expression, as with pesticides and allelochemicals (Malkenson de et al., 1984; Dowd and Sparks, 1987). Perhaps, as in some vertebrate species (Stohs and Biagchi, 1995; Biagchi et al., 1996), cadmium intensified production of metabolites that are CarE substrates. In the case of GST, at least in vertebrates, glutathione seems to protect cells against the toxic effects of metals irrespective of induction of metallothionein synthesis (Susuki et al., 1996). In invertebrates, Smirle and Winston (1988) emphasized the role of GST in defense against the cytotoxic action of metals in the honeybee Apis mellifera. Wilczek et al. (1997) found increased GST in a number of spider species collected at metal-polluted areas. Similar results were obtained by Migula (1997) in cadmium treated ants Formica aquilonia, and Kaaya et al. (1999) in bivalves, Macoma balthica Perna perna and Mytilus galloprovincialis living in an environ-

ment contaminated with industrial and communal sewage. Differences between metals in their influence on enzymatic function are supported by another outcome of our study. Despite the general trend to increased enzyme activity with increasing metal concentrations, in immature adults intoxicated with cadmium a decrease in GST activity was observed, particularly against DCNB, while in immature zinc-intoxicated adults the effect was quite the opposite. These differences between cadmium and zinc may be due to the physiological condition of the animals during maturation connected with the production of hormones and enzymes crucial to sexual development (Ruvolo-Takasuki and Collet, 2000). 5. Conclusion Like other ecotoxicological studies on the sensitivity of organisms to toxic substances, (e.g. Kammenga et al., 1996), our results emphasize that in studying enzymes under metal-stress one should also consider the animals life-stage and sex. These factors may exert as strong an influence as the toxic substances themselves. Acknowledgments The authors thank Pawel Migula and Ryszard Laskowski for their advice during the study. Michael Jacobs helped edit the text. We also thank two anonymous reviewers for useful suggestions for revision of the manuscript. Financial support came from the Polish State Committee for Scientific Research, Grant Nos. 6 P04F 011 12 and 6 P04F 043 18. References
Argentine, J.A., James, A.A., 1995. Characterization of a salivary gland-specific esterase in the vector mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 25, 621630. Augustyniak, M., Migula, P., 2000. Body burden with metals and detoxifying abilities of the grasshopperChorthippus brunneus (Thunberg) from industrially polluted areas. In: Markert, B., Friese, K. (Eds.), Trace ElementsTheir Distribution and Effects in the Environment. Elsevier Science B.V, pp. 423454. Biagchi, D., Biagchi, M., Hassoun, E.A., Stohs, S.J., 1996. Cadmium induced excretion of urinary lipid metabolites, DNA damage, glutathione depletion and hepatic lipid peroxidation in spraqueDawley rats. Biol. Trace Element. Res. 52, 143154.

G. Wilczek et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 501512 Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248254. Capua, S., Cohen, E., Gerson, U., 1991. Induction of aldrin epoxidation and glutathione S-transferase in the mite Rhizoglyphus robini. Entomol. Exp. Appl. 59, 4350. Cohen, E., 1986. Glutathione S-transferase activity and its induction in several strains of Tribolium castaneum. Entomol. Exp. Appl. 41, 3944. Dowd, P.F., Sparks, T.C., 1987. Inhibition of trans-permethrin hydrolysis in Pseudoplusia includens (Walker) and use of inhibitors as pyrethroid synergists. Pest. Biochem. Physiol. 27, 237245. Frohlich, D.R., 1990. Substrate specificity of esterases in a solitary bee Megachile rotundata (Hymenoptera: Megachilidae) variability in sex, age and life stage. Biochem. Syst. Ecol. 18, 539547. Grant, D.F., Dietze, E.C., Hammock, B.D., 1991. Glutathione S-transferase isoenzymes in Aedes aegypti: purification, characterization, and izoenzyme specific regulation. Insect Biochem. 21, 421443. Heimbach, U., 1992. Laboratory method to test effects of pesticides on Poecilus cupreus (Coleoptera, Carabidea). IOBSyWPRS Bull. 15, 103109. Iio, M., Kawaguchi, H., Sakota, Y., Otonari, J., Nitahara, H., 1993. Effects of polyphenols, including flavonoids, on glutathione S-transferase and glutatione reductase. Biosci. Biotech. Biochem. 57, 16781680. Janssen M.P.M. 1991. Comparison on cadmium kinetics in four soil arthropods species. Ph.D. Thesis, Vrije Univeriteit, Amsterdam, pp. 7589. Jokanovic, M., 2001. Biotransformation of organophosphorus compounds. Toxicology 166, 139160. Kaaya, A., Najimi, S., Ribera, D., Narbonne, J.F., Moukrim, A., 1999. Characterisation of glutatione-S-transferases (GST) activities in Perna perna and Mytilus galloprovincialis used as biomarkers of pollution the Agadir Marine Bay (South of Morocco). Bull. Environ. Contam. Toxicol. 62, 623629. Kammenga, J.E., Busschers, M., Van Straalen, N.M., Jepson, P.C., Bakker, J., 1996. Stress-induced fitness reduction is not determined by the most sensitive life-cycle trait. Funct. Ecol. 10, 106111. Kedziorski, A., Nakonieczny, M., Migula, P., Grzesiak, M., 1996. Age-dependent activity patterns of carboxylesterases in insect exposed to cadmium. Stud. Soc. Sciantorum Torunensis 4, 7176. Konno, Y., Shishido, T., 1992. Distribution of glutathione Stransferase activity in insect tissues. Appl. Entomol. Zool. 27, 391397. Kotze, A.C., Rose, H.A., 1987. Glutathione S-transferase in the Australian sheep blowfly, Lucilia cuprina (Wiedemann). Pest. Biochem. Physiol. 29, 7786. Kramarz, P., 1999. The dynamics of accumulation and decontamination of cadmium and zinc in carnivorous invertebrates. 1. The ground beetle, Poecilus cupreus L. Bull. Environ. Cont. Toxicol. 4, 531538. Kramarz, P., 2000. Cadmium and zinc accumulation and its demographic effects in invertebrates. In: Kammenga, J., Laskowski, R. (Eds.), Demography in Ecotoxicology. John Wiley & Sons, pp. 91108.

511

Laskowski, R., Maryanski, M., 1993. Heavy metals in epigeic fauna: trophic-chain and physiological hypotheses. Bull. Environ. Contam. Toxicol. 59, 232240. Lemaire-Gony, S., Lemaire, P., 1992. Interactive effects of cadmium and benzo(a)pyrene on cellular structure and biotransformation enzymes of the liver of the European eel Anquilla anquilla. Aquatic Toxicol. 22, 145160. Lindroth, R.L., 1989. Differential esterase activity in Papilio glaucus subspecies: absence of cross-resistance between allelochemicals and insecticides. Pestic. Biochem. Physiol. 35, 185191. Ljungquist, A., Augustinsson, K.B., 1971. Purification and properties of two carboxylesterases from rat-liver microsomes. Eur. J. Biochem. 23, 303313. Malkenson de, N.C., Wood, E.J., Zerba, E.N., 1984. Isolation and characterization of an esterase of Triatoma infestans with a critical role in the degradation of organophosphorus esters. Insect Biochem. 14, 481486. Maryanski, M., Kramarz, P., Laskowski, R., Niklinska, M., 2001. Decreased energetic reserves, morphological changes and accumulation of metals in carabid beetles (Poecilus cupreus L.) exposed to zinc- or cadmium-contaminated food. Ecotoxicology 11, 127139. Mathova, A., 1990. Biological effects and biochemical alterations after long-term exposure of Galleria mallonella (Lepidoptera: Pyralidae) larvae to cadmium containing diet. Acta Entomol. Bohemoslov. 87, 241248. Migula, P., 1997. Molecular and physiological biomarkers in insects as the tools in environmental hazard assessment. Acta Phytopathol. Entomol. Hung. 32, 231243. Postma, J.F., Buckert-de Jong, M.C., Staats, N., Davids, C., 1994. Chronic toxicity of cadmium to Chironomus riparius (Diptera: Chironomidae) at different food levels. Arch. Environ. Contam. Toxicol. 26, 143148. Ruvolo-Takasuki, M.C.C., Collet, T., 2000. Characterization of Nasutitermes globiceps (Isoptera: Termitidae) esterases. Biochem. Genetics. 38, 367375. Schmidt, G.H., Ibrahim, N.M.M., Abdallah, M.D., 1992. Longterm effects of heavy metals in food on developmental stages of Aiolopus thalassinus (Saltatoria: Acrididae). Arch. Environ. Contam. Toxicol. 23, 375382. Smirle, M.J., Winston, M.L., 1988. Detoxifying enzyme activity in worker honey bees: an adaptation for foraging in contaminated ecosystems. Can. J. Zool. 66, 19381942. Soderlund, D.M., Bloomquist, J.R., 1990. Molecular mechanisms of insecticide resistance. In: Roush, R.T., Tabashnik, B.E. (Eds.), Pesticide Resistance in Arthropods. Routledge Chapman&Hall Inc, New York, pp. 5896. Stohs, S.J., Biagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Rad. Biol. Med. 18, 321336. Stone, D., Jepson, P., Laskowski, R., 2002. Trends in detoxification enzymes and heavy metal accumulation in ground beetles (Coleoptera: Carabidae) inhabiting a gradient of pollution. Comp. Biochem. Physiol. 132, 105112. Susuki, T., Morimura, S., Dicciani, M.B., Yamada, R., Hochi, S.I., Hirabayashi, M., et al., 1996. Activation of glutatione transferase P gene by lead requires glutathione transferase P enhancer I. J. Biol. Chem. 271, 16261632. Toung, Y.-P.S., Hsieh, T.-S., Tu, C.-P.D., 1990. Drosophila glutathione S-transferase L-L shares a region of a sequence homology with the maize glutathione S-transferase III. Proc. Natl. Acad. Sci. USA 87, 3135.

512

G. Wilczek et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 501512 adult stages of the house cricket. Insect. Biochem. 17, 751758. Yu, S.J., 1982. Host plant induction of glutathione S-transferase in the fall armyworm. Pestic. Biochem. Physiol. 18, 101106. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall, Inc, Englewood Cliffs, N.J.

Wilczek G., Majkus Z., Migula P., Bednarska K., Swierczek E. 1997. Heavy metals and detoxifying enzymes in spiders from coal and metallurgic dumps near Ostrava (Czech Republic). Proceedings of the 16th European Colloidial Arachnology Siedlce, pp. 317328. Woodring, J.P., Sparks, T.C., 1987. Juvenile hormone esterase activity in the plasma and body tissue during the larval and