activity of car boxy lest erase and one s-transferase in different life-stages of carabid beetle...

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Comparative Biochemistry and Physiology Part C 134 (2003) 501–512

1532-0456/03/$ - see front matter 2003 Elsevier Science Inc. All rights reserved.PII: S 1 532- 0456 Ž0 3.0 0 0 3 9 - 5

Activity of carboxylesterase and glutathione S-transferase indifferent life-stages of carabid beetle (Poecilus cupreus) exposed to

toxic metal concentrations

Grazyna Wilczek , Paulina Kramarz *, Agnieszka Babczynskaa b , a´

Department of Physiology and Ecotoxicology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9,a

Katowice 40-007, Poland

Department of Ecotoxicology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 3, Krakow 30-387, Poland b ´

Received 7 August 2002; received in revised form 10 February 2003; accepted 10 February 2003

Abstract

Among the cytoplasmatic enzymes responsible for neutralization of organic xenobiotics, carboxylesterases (CarE) andglutathione S-transferases (GST) play important roles. Our study tested to what extent dietary Zn or Cd could modifythe activity of CarE and GST at different life-stages of the carabid beetle Poecilus cupreus. Treatment and stage effectsgenerally were statistically significant. For CarE activity in the beetles exposed to cadmium, only treatment was asignificant factor. In all cases, the interaction between studied factors was statistically significant, implying that thephysiological condition of the animals may enhance or reduce enzyme activity. We also observed differences betweenanimals treated with cadmium and zinc in the pattern of enzyme activity, and a difference in GST activity measuredwith two different substrates. Our results confirmed that in studying enzyme activity under metal stress one shouldconsider the animal’s 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 anenvironment altered by industrial contaminationdepends mainly on effective mechanisms of detox-ification of various endo- and exogenic compounds(Jokanovic, 2001). Among the cytoplasmaticenzymes responsible for neutralizing xenobioticscarboxylesterases (CarE) and glutathione S-trans-ferases (GST) play important roles (Toung et al.,1990).

Carboxylesterases are generally characterized bylow substrate specificity and most of them hydro-

*Corresponding author. Tel.: q48-12-2642516x157; fax: q48-12-2690927.

E-mail address: [email protected] (P. Kramarz).

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 izoen-

zymes differing in their affinity for specific sub-

strates (Soderlund and Bloomquist, 1990).

Glutathione S-transferases are a group of structur-

ally similar but functionally different izoenzymeswith differing specificities (Grant et al., 1991).

The activity of CarE and GST may be changed by

allelochemicals (Iio et al., 1993), organophospho-

rous insecticides (Capua et al., 1991) and products

of carcinogenic substances of metabolic activation

(Lemaire-Gony and Lemaire, 1992). Metals in



502 G. Wilczek et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 501–512

toxic concentrations may be also responsible forchanging activity in enzyme activity (Malkensonde et al., 1984; Dowd and Sparks, 1987). Metalions are probably not necessary as cofactors forCarE and GST activity and do not directly modifythe action of the enzymes neither in vivo nor invitro. The toxic effects of metals strongly depend

on their properties, their concentrations in theorganisms, as well as biological features of theorganisms, such as age, sex and developmentalstage (Mathova, 1990; Schmidt et al., 1992; Post-ma et al., 1994).

This study was part of a project on the effectsof prolonged intoxication with metals on carabidbeetles at individual and population levels (e.g.Kramarz, 2000; Maryanski et al., 2001). Thecarabids have been recognized as poor accumula-tors of metals (Laskowski and Maryanski, 1993).Studies by Janssen (1991) and Kramarz (1999)indicate that this may be due to effective mecha-

nisms of metal elimination. In our study, we testedto what extent an excess of Zn or Cd in the dietcould modify the activity of CarE and GST atdifferent life-stages in both sexes of the carabidbeetle Poecilus cupreus. By comparing the effectsof zinc, a biogenic element and cadmium, forwhich a biological role has not been recognized,we can draw conclusions about the ability of thestudy species to detoxify organic xenobiotics.

2. Material and methods

2.1. Animals

Adult carabids were obtained from a laboratorystock culture kept at the Jagiellonian Universityand were prepared for reproduction according toHeimbach (1992). Five males and five femaleswere put into breeding boxes. Eggs were removedevery 3rd day. Eggs were put separately into cellsof tissue culture boxes on wet filter paper. At theoutset, each tissue culture box was assigned to aparticular treatment. Due to expected high mortal-ity, approximately twice as many eggs wereassigned to the highest metal concentration treat-ments than in the control.

Immediately after hatching, the larvae wereexposed to experimental treatments. They were fedon housefly pupae reared on an artificial mediumcontaminated with 0, 40, 160, 640 or 800 mg Cdkg , or else with 0, 400, 1600, 6400 or 8000 mgy1

Zn kg (dry wt.), respectively. The medium fory1

the housefly pupae consisted of 515.6 g rabbitchow (Wytwornia Pasz, Andrzej Morawski, 89-´

240, Kcynia, Poland), 20 g powdered milk (OSMKrotszyn), 10 g sugar, 0.02 g baker’s yeast (DrOetcker) and 1 l distilled water or experimentalsolution (CdCl =2.5 H O or ZnCl ). After hatch-2 2 2

ing, larvae were transferred to plastic vials (30ml) filled with crushed wet garden peat and werefed every 3rd day on three housefly pupae untilpreparation. Twenty-five days after emerging frompupa, the adults in their peat-filled vials wererefrigerated for overwintering (ca. 7 8C, completedarkness), 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 experimentalgroup were done in six replicates. Enzyme activitywas measured in last-stage larvae ( just beforepupation), immature adults of both sexes (20 daysafter emergence) and mature females and males(10 days after overwintering). For zinc treatments,CarE and GST were also measured in pupae. Highmortality at this stage in Cd-treated carabids pre-cluded assays.

Animals (one per sample) from each develop-mental stage were weighed, anaesthetized on iceand then homogenized in a glass–Teflon homog-enizer at 4 8C in 0.05 M buffer Tris–HCl, pH 7.6,using 50-fold initial homogenate dilution. Thehomogenates were centrifuged for 10 min at15 000=g. After decanting and further dilutionthe supernatants were used for assays. Proteincontents was measured according to Bradford(1976) using bovine albumin as the standard.

2.3. Carboxylesterases (CarE ) w EC 3.1.1.1x

Carboxylesterases (EC 3.1.1.1) were measuredspectrophotometrically (Helios Epsilon UNICAM)

in the submitochondrial fraction according to Lju-ngquist and Augustinsson (1971), with p-nitro-phenyl acetate ( p-NPA) as the substrate. Changesin the substrate hydrolysis product ( p-nitrophenol)concentration were measured at ls400 nm.Results were expressed in nmol p-NPA min mgy1

protein .y1



503G. Wilczek et al. / Comparative Biochemistry and Physiology Part C 134 (2003) 501–512

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 betweentreatments (LSD test).

2.4. Glutathione S-transferases (GST ) w EC

2.5.1.18x

GST activity was determined in the postmito-chondrial fraction, as the rate of conjugation of glutathione with 1-chloro-2,4 dinitrobenzene(CDNB, ls340 nm) and 3,4-dichloronitrobenze-ne (DCNB, ls344 nm) according to Lindroth(1989) and Yu (1982), respectively. The productsof glutathione-CDNB and glutathione-DCNB con-

jugation are S(2-chloro-4-nitrophenyl) glutathioneand S(2,4dinitrophenyl) glutathione, respectively.

2.5. Statistical analyses

Statistical analyses were done separately forcadmium, zinc, each enzyme and each substrate inthe case of GST. In the calculations, the nominalconcentrations of metals in the housefly larvae’sdiet were taken to represent total concentrations inthe environment, the common measure in fieldstudies.

As we expected a linear relationship betweenenzyme activity and metal concentration, at thefirst we intended to use regression analysis and tocompare regression lines with developmental stageas a factor. However, because clear linear relation-ships were observed in only a few cases, theseresults were finally analyzed with two-way analy-sis of variance (MANOVA) with treatment andstage as factors. If significant differences were

detected, means were separated by the LSD test.To obtain normal distributions the data were log-transformed (Zar, 1974, CSS-Statistica).

3. Results

Metals and developmental stage-dependenteffects generally were statistically significant (P-

0.005), except for CarE activity in the beetlesintoxicated with cadmium, where only metal wasa significant factor (P-0.005). In all cases, theinteractions between the studied factors were sta-tistically significant (P-0.0001). There weresome differences between animals treated withcadmium and zinc in the pattern of enzyme activ-ity, and a difference in GST activity measuredagainst the two different substrates.

3.1. Carboxylesterases (CarE ) w EC 3.1.1.1x

3.1.1. Cadmium

Cadmium caused an increase in carboxylesteraseactivity in all stages of the beetles (Fig. 1), withthe strongest effects in larvae, mature females andmature males (Fig. 1). Even though the specificstage had no effect on CarE activity (P)0.05,Fig. 2), the statistical significance of interactionsbetween developmental stage and treatmentimplies that the physiological condition of theanimals may affect this enzyme activity.




Fig. 2. CarE activity (against pNPA) vs. life stage of P. cupreus fed with food ( M. domestica larvae) contaminated with differentcadmium concentrations. Bold numbers are nominal Cd concentrations (mg kg ); means (small boxes) and S.D. (whiskers) arey1

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 strongsignificant effects on animals intoxicated with zinc(P)0.00001), there was no clear pattern of car-boxylesterase activity, neither for the metal nor forthe developmental stage (Figs. 3 and 4). CarEactivity was the same as in the controls or slightlyenhanced, except for larvae intoxicated with zincat the highest concentration, where CarE activitywas 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 inpupae (Fig. 4). CarE activity in the remainingstages was similar, irrespective of the metal dose.In immature and mature females overall CarEactivity depended on the zinc concentration (Fig.3).

3.2. Glutathione S-transferases (GST ) w EC

2.5.1.18x

3.2.1. Cadmium

GST activity measured against DCNB in larvaeand immature adults treated with cadmium wasslightly lowered or comparable to that of thecontrol (Fig. 5). In mature females and males theactivity of these enzymes was higher, but therewere no concentration-depended differences (Fig.5). In animals compared between stages under thesame dose of cadmium, untreated larvae and

mature males had the highest GST activity (Fig.6). In other cases, an interesting pattern wasobserved. The highest GST activity, measuredagainst DCNB was found in sexually matureadults. The lowest was observed in immature malesand females, while larvae showed intermediateactivity of the enzyme (Fig. 6).

Less uniform results were obtained for GSTactivity measured against CDNB (Figs. 7 and 8).Significantly higher GST activity was found only

in sexually mature females treated with the twohighest doses of cadmium (Fig. 7). Differencesbetween developmental stages in animals exposedto the same dose of the metal, as in the case of DCNB, were noted only for the control and thetwo highest Cd concentrations (Fig. 8).

3.2.2. Zinc

No general pattern of changes in GST activitywas observed in zinc-intoxicated animals (Figs.9–12). Its activity measured against DCNB vs.the control was enhanced in larvae from the 6400-Zn group, pupae intoxicated with the three highestzinc concentrations (with no difference betweenconcentrations), immature females and males fromzinc treatments and mature females from the 1600-Zn group (Fig. 9). Lowered GST activity vs. thecontrol was found in larvae at the lowest andhighest zinc doses, and mature males from thelowest zinc treatment (Fig. 9). The highest GST




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 (LSDtest).

Fig. 4. CarE activity (against pNPA) vs. life stage of P. cupreus fed with food ( M. domestica larvae) contaminated with different zincconcentrations. Bold numbers are nominal Cd concentrations (mg kg ); means (small boxes) and S.D. (whiskers) are shown. Differenty1

letters indicate significant differences between treatments (LSD test). L: larvae; P: pupae; IF: immature females; MF: mature females;IM: immature males; MM: mature males.

activity was observed in larvae, the only exceptionbeing the 8000-Zn group, where mature malesexhibited the highest GST activity (Fig. 10). GSTactivity measured against CDNB in zinc-intoxicat-ed animals somewhat differed from that measuredagainst DCNB (Figs. 9– 12). The level of GSTwas also increased in immature females and malesexposed to all concentrations of zinc. In contrastto the result with DCNB as a substrate, measured

against CDNB GST activity was enhanced inmature females administered the lowest and twohighest zinc concentrations and in mature malestreated 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. 5. GST activity (against DCNB) in different stages of P. cupreus vs. nominal cadmium concentrations in their food (diet of M.


Fig. 6. GST activity (against DCNB) vs. life stage of P. cupreus fed with food ( M. domestica larvae) contaminated with differentcadmium concentrations. Bold numbers are nominal Cd concentrations (mg kg ); means (small boxes) and S.D. (whiskers) are shown.y1

Different letters indicate significant differences between treatments (LSD test). L: larvae; IF: immature females; MF: mature females;IM: immature males; MM: mature males.

on the metal, (e.g. Kramarz, 1999). Zn-body con-centrations was regulated well: the control beetlescontained ca. 79 mg kg (62% of the 128 mgy1

Zn kg concentration in housefly pupae), andy1

those from the highest nominal treatment reachedonly 110 mg kg , that is, ca. 16% of the concen-y1

tration in the pupae (692 mg Zn kg ) (Maryanskiy1

et al., 2001). In contrast, the final concentrationsin Cd-treated beetles were substantially higher than

in the control beetles. The latter contained 0.36mg kg , that is 39% of the concentration iny1

housefly larvae. Carabids from the highest treat-ment (nominal 800 mg Cd kg ) contained onlyy1

80.5 mg kg , a tenth the concentration in house-y1

fly pupae and the nominal Cd concentration. Thelargest jump in concentration in both the housefliesand the beetles was found for the lowest cadmiumtreatment (nominal 40 mg kg ): the concentrationy1




Fig. 7. GST activity (against CDNB) in different stages of P. cupreus vs. nominal cadmium concentrations in their food (diet of M.


Fig. 8. GST activity (against CDNB) vs. life stage of P. cupreus fed with food ( M. domestica larvae) contaminated with differentcadmium concentrations. Bold numbers are nominal Cd concentrations (mg kg ); means (small boxes) and S.D. (whiskers) are shown.y1

Different letters indicate significant differences between treatments (LSD test). L: larvae; IF: immature females; MF: mature females;IM: immature males; MM: mature males.

in housefly pupae increased by more than 100times vs. the control, and the carabids were ca. 30times more contaminated than control beetles (11.3mg Cd kg ) (Maryanski et al., 2001).y1

Despite the differences between cadmium andzinc detoxication (accumulation vs. efficient elim-ination) that seem to occur in P. cupreus, bothprocesses are probably energy-intensive. This mayexplain why adult body mass decreased withincreasing concentrations of both metals (Kramarz,

2000), and why intoxication with high doses of cadmium caused a decrease in adult body caloricvalue (Maryanski et al., 2001). Intoxication of P.

cupreus with cadmium or zinc also had exertedsome negative effects on population parameters(Kramarz, 2000). Consequences of intoxicationwith cadmium or zinc were also observed at thelevel of enzymatic activity: changes in CarE andGST activity depended on the metals used andtheir doses.




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 (LSDtest).

Fig. 10. GST activity (against DCNB) vs. life stage P. cupreus fed with food ( M. domestica larvae) contaminated with different zincconcentrations. Bold numbers are nominal Cd concentrations (mg kg ); means (small boxes) and S.D. (whiskers) are shown. Differenty1


In general, measured by energy expenditures

(Maryanski et al., 2001), both metals causedincreased enzymatic activity. This holds true par-ticularly for CarE activity at all stages of P. cupreus

and in GST (against DCNB) in mature adults fedpoisoned houseflies. Increased CarE and GSTactivity was also found in the carabid Pterostichus

oblongopunctatus collected in metal-polluted areas(Stone et al., 2002). In our study, groups intoxi-cated 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 weakerinfluence of zinc than cadmium on body caloricvalue (Maryanski et al., 2001), we also foundweaker enzymatic response in zinc-treated beetles.The other sources of variance in enzyme activityobtained in our studies—life-stage and gender—are well known in metal-treated insects (Augus-tyniak and Migula, 2000; Stone et al., 2002).

During development, hormones, (e.g. juvenileand ecdysteroid) control enzyme synthesis, modi-fying their activity, including that of carboxyles-




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

Fig. 12. GST activity (against DCNB) vs. life stage P. cupreus fed with food ( M. domestica larvae) contaminated with different zincconcentrations. Bold numbers are nominal Cd concentrations (mg kg ); mean (small boxes) and S.D. (whiskers) are shown. Differenty1


terases (Ruvolo-Takasuki and Collet, 2000). Thischaracteristic pattern of enzymatic response wasalso noted in our studies in control and metal-intoxicated individuals of P. cupreus. However, thelevel of enzyme activity was higher in beetles fedon metal-poisoned food, and depended on the dose.As in a study by Kedziorski et al. (1996) oncrickets, the differences between beetle stages didnot follow a linear pattern.

Age-dependent changes in enzyme activity mayresult from differences in the expression of the

particular isoenzymes taking part in transformationof endogenous substrates occurring during devel-opment (Woodring and Sparks, 1987). In Lucilia

cuprina, Kotze and Rose (1987) observed increas-ing GST activity with age. In contrast, in insectsageing is often followed by a decrease in proteinsynthesis (Cohen, 1986). In the case of hydrolyticenzymes (CarE), in males of the solitary bee Megachile rotundata the activity of CarEdecreased with age (Frohlich, 1990). Similarly, inour study, CarE activity was the lowest in mature




beetles, while initial stages and mature beetles hadhigher GST activity than immature adults; a studyon L. cuprina (Kotze and Rose, 1987) producedcomparable findings.

The level of detoxifying enzymes in P. cupreus

differed also by sex. In this work, both controland metal-treated adult male beetles had higher

GST activity than females, while CarE activity infemales and males was similar. In a study of carabid beetles P. oblongopunctatus inhabiting ametal-contaminated environment, Stone et al.(2002) found stronger induction of detoxifyingenzyme activity in females than in males. Similargender-related differences were also shown in thegrasshopper Chorthippus brunneus (Augustyniak and Migula, 2000). In our study, the between-sexdifferences are not clear, probably because theanimals were exposed to excess metals only oncein their history. In both cases cited above, the

animals used for assays originated from sites pol-luted with metals for many generations, so thedifferences between males and females may reflectadaptation (Stone et al., 2002). In other studies,Konno and Shishido (1992) observed higher activ-ity of GST in testes of Periplaneta americana thanin ovaries. CarE activity might also differ betweensexes: CarE activity in Aedes aegypti was higherin females than in males (Argentine and James,1995), while females of M. rotundata had loweractivity than males (Frohlich, 1990).

Stimulation of CarE activity by cadmium canalso result from an indirect influence on CarE-gene expression, as with pesticides and allelochem-icals (Malkenson de et al., 1984; Dowd andSparks, 1987). Perhaps, as in some vertebratespecies (Stohs and Biagchi, 1995; Biagchi et al.,1996), cadmium intensified production of metab-olites that are CarE substrates. In the case of GST,at least in vertebrates, glutathione seems to protectcells against the toxic effects of metals irrespectiveof induction of metallothionein synthesis (Susukiet al., 1996). In invertebrates, Smirle and Winston(1988) emphasized the role of GST in defenseagainst the cytotoxic action of metals in the hon-

eybee Apis mellifera. Wilczek et al. (1997) foundincreased GST in a number of spider speciescollected at metal-polluted areas. Similar resultswere obtained by Migula (1997) in cadmiumtreated 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 communalsewage.

Differences between metals in their influenceon enzymatic function are supported by anotheroutcome of our study. Despite the general trend toincreased enzyme activity with increasing metalconcentrations, in immature adults intoxicated with

cadmium a decrease in GST activity was observed,particularly against DCNB, while in immaturezinc-intoxicated adults the effect was quite theopposite. These differences between cadmium andzinc may be due to the physiological condition of the animals during maturation connected with theproduction of hormones and enzymes crucial tosexual development (Ruvolo-Takasuki and Collet,2000).

5. Conclusion

Like other ecotoxicological studies on the sen-sitivity of organisms to toxic substances, (e.g.Kammenga et al., 1996), our results emphasizethat in studying enzymes under metal-stress oneshould also consider the animal’s life-stage andsex. These factors may exert as strong an influenceas the toxic substances themselves.

Acknowledgments

The authors thank Pawel Migula and RyszardLaskowski for their advice during the study.Michael Jacobs helped edit the text. We also thank

two anonymous reviewers for useful suggestionsfor revision of the manuscript. Financial supportcame from the Polish State Committee for Scien-tific Research, Grant Nos. 6 P04F 011 12 and 6P04F 043 18.

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