Metallothionein-bound cadmium in the gut of the insect
Orchesella cincta
(Collembola) in relation to dietary
cadmium exposure
Paul J. Hensbergen
a,*, Martin J.M. van Velzen
a, Rully Adi Nugroho
b,
Marianne H. Donker
a, Nico M. van Straalen
aaDepartment of Ecology and Ecotoxicology,Vrije Uni6ersiteit,De Boelelaan1087,1081HV Amsterdam,Netherlands bSatya Wacana Christian Uni6ersity,Jalan Diponegoro52-60,Salatiga50711,Indonesia
Received 21 January 1999; received in revised form 3 September 1999; accepted 9 September 1999
Abstract
Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtailOrchesella cinctaby determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0mmol Cd/dry food. Cadmium is
primarily located within the gut ofO.cinctaand isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115mg metallothionein/g fresh gut. TheO.cinctametallothionein gives
the possibility of using this protein as a biomarker for heavy metal exposure in soil insects. © 2000 Elsevier Science Inc. All rights reserved.
Keywords:Biomarker; Cadmium; Collembola; Metal detoxification; Metallothionein; Heavy metals
1. Introduction
There is increasing understanding that exposure of soil animals to heavy metals can not directly be derived from total concentrations present in the environment due to the fact that a variable frac-tion of the total concentrafrac-tion is bioavailable
(Van Gestel, 1992). More accurate information can be obtained from direct measurements of heavy metals within the animal in combination with detailed studies about the distribution and cellular storage mechanisms of these potentially toxic metals.
Metallothioneins can account for storage of a substantial amount of the body burden of metals. These proteins are characterized by their relatively small size (2 – 11 kD) and a high cysteine content (necessary for the binding of metal ions) and in
* Corresponding author. Tel.: +31-20-4447066; fax: + 31-20-4447123.
E-mail address:[email protected] (P.J. Hensbergen)
addition to heavy metals they are induced by a wide variety of stress factors (Ka¨gi and Scha¨ffer, 1988). Although the metal-ions directly bound to metallothionein are not considered as the most potentially harmful fraction, the amount of metal-lothionein and the respective metal-ions bound to it can give important information about the flux of metals into the body and the biological re-sponse elicited. This role of metallothionein as a biomarker has therefore been proposed (Roesi-jadi, 1992; Berger et al., 1995). One of the obvious requirements for the use of metallothionein as a biomarker is the concentration dependency of its induction in combination with a reliable quantifi-cation method.
Quantification of metallothionein can be per-formed with different detection methods either on the protein (McCormick and Lin, 1991; Richards, 1991; Berger et al., 1995; Viarengo et al., 1997) or mRNA level (Kaplan et al., 1995; Roesijadi et al., 1997). One of the detection methods on the protein level focuses on measuring the metal bound to it. The metallothionein concentration can then be estimated from knowledge about the metal stoichiometry of the metallothionein. The advantage of direct measurements of heavy metals bound to metallothionein is the high sensitivity of these measurement and it opens the possibility of measuring metallothionein-bound cadmium in very small insects, like Collembola.
There is very little knowledge about metalloth-ionein in insects, especially with respect to isola-tion procedures of the proteins and their primary structure. Studies in Drosophila have shown that this fly contains two metallothionein genes, Mtn andMto, but detection of Mtn at the protein level has still been unsuccessful (Lastowski-Perry et al., 1985; Mokdad et al., 1987). Expression of metal-lothionein genes was primarily detected in the gut (Durliat et al., 1995). Other studies on metal binding proteins in insects did not result in a primary structure elucidation (Everard and Swain, 1983; Aoki et al., 1984; Kasai et al., 1993).
Orchesella cincta (Collembola) is a very com-mon species of insect, especially in the litter layer of pine forests. Upon exposure to cadmium, O. cincta is able to excrete part of the assimilated cadmium at every moult by renewing the gut epithelium and shedding the old one. This gut pellet contains about 35% of the total cadmium present within the animal prior to the moult. This results in an equilibrium of the internal
concentra-tion when excreconcentra-tion balances uptake. We have recently been successful in the isolation and char-acterisation of a metallothionein from this insect using protein isolation and characterisation tech-niques in combination with a molecular approach (Hensbergen et al., 1999). We showed that upon exposure to cadmium two metallothionein pep-tides could be isolated which are probably en-coded by one gene.
In this study we investigated the concentration dependence of metallothionein-bound cadmium using O. cincta individuals exposed to different concentrations of cadmium. Furthermore, we studied in more detail where cadmium, as well as metallothionein-bound cadmium, is located within this animal.
2. Material and methods
2.1. Animals and rearing conditions
O. cincta (Apterygota: Collembola) individuals from the laboratory culture at the Vrije Univer-siteit in Amsterdam, originating from a pine forest (Roggebotszand) in the Netherlands, were used in the experiments. Animals were held in PVC-jars with a moist layer of plaster of Paris and were fed green algae (mainly Desmococcus spp.) supplied on filter paper. Food was renewed twice a week. Animals were kept in a climate room (Temperature: 18°C, RH: 75%, light/dark: 12/12). At the end of the feeding period, animals were allowed to empty their gut for 48 h on a plaster of Paris substrate without food and were stored at −20°C afterwards.
2.2. Cadmium exposure
dry weight for the algae) were 0, 0.25, 0.38, 0.50, 0.75, 1.0, 1.5 and 2.0 mmol Cd/g dry algae.
In another experiment, fifteen specimens of O. cincta per concentration were exposed individu-ally to the above mentioned cadmium levels and their weight increase during the time period of the experiment was determined.
2.3. Quantification of metallothionein-bound cadmium
Metallothionein was quantified by measuring cadmium in fractions collected after chromato-graphic isolation of metallothionein from Cd ex-posed animals.
For this, approximately 150 animals per sample (total average weight (9SD) 70.4912.5 mg fresh weight (n=16, 8 concentrations in duplicate) were homogenised in 3 ml of ice-cold 45% acetone and 55% 20 mM Tris – HCl buffer pH 8. This homogenate was centrifuged at 12 000×g for 10 min. After this step, the supernatant was adjusted to 80% acetone and centrifuged again at 12 000×
g. The resulting pellet was dried under a stream of nitrogen gas and subsequently taken up in 300ml of 20 mM Tris – HCl. 200 ml of this supernatant was applied to gel filtration chromatography us-ing a Superose 12 column (Pharmacia Biotech) connected to an FPLC system (Pharmacia Bio-tech) using 0.1 M NH4HCO3pH 7.9 at a flow rate of 0.6 ml/min.
After this separation step cadmium containing fractions eluting between 24 and 26 min were taken and applied to reversed phase HPLC at neutral pH using a PEP-RPC column (Pharmacia Biotech). Elution was performed with buffer A being 50 mM ammonium acetate pH 6.0, and buffer B being 50 mM ammonium acetate con-taining 60% acetonitril, in a gradient of 0 – 5 min 0% B, 5 – 25 min from 0 – 53% B, 25 – 30 min from 53 – 100% B, 30 – 40 min 100% B. Cadmium was determined in the fractions and the cadmium elut-ing between 16 – 18 min corresponds to metalloth-ionein-bound cadmium (Hensbergen et al., 1999). The total amount of cadmium measured in the collected fractions after HPLC was taken as a measure of the metallothionein concentration. This was multiplied by 3/2 (only 200 ml of the initial homogenate was applied to gel filtration chromatography) and divided by the total weight of the animals used to determine the metalloth-ionein-bound cadmium concentration based on the fresh weight basis.
2.4. Cadmium and metallothionein-bound cadmium in guts from O. cincta
After exposure for 2 weeks to 0.5 mmol Cd/g dry algae, the ratio between cadmium in the gut and the rest of the body was determined by dissecting guts after narcotising animals with CO2, and measuring total cadmium in the gut and the rest of the body. For this, two pairs of tweezers were used, one to hold the animal, the other to gently pull out the gut from the caudal end of the individual. The fresh weight of dissected guts was also determined and related to the fresh weight of the animal.
To show that the metallothionein detected in whole body homogenates is actually originating from the guts, 1500 guts were dissected from animals one week exposed to 1.0 mmol Cd/g dry algae. Guts were dissected and collected in groups of 40 in 40 ml of ice cold 20 mM Tris – HCl (pH 8.0) on ice, and subsequently frozen in liquid nitrogen prior to storage at −20°C. Homogenisa-tion was performed by adding ice-cold acetone (−20°C) to the guts to a final concentration of 45% and further steps of homogenisation, cen-trifugation and gel-permeation chromatography and reversed phase chromatography were per-formed as described above.
2.5. Metal analysis
Cadmium concentrations in fractions derived from chromatographic separations were measured directly, using a graphite furnace atomic absorp-tion spectrometer (Perkin Elmer, model 1100B) at a wavelength of 228.8 nm. The internal cadmium concentrations in the animals, guts and in the food were measured after complete digestion
us-ing a technique previously described (Van
Straalen et al., 1987). Cadmium measurements were calibrated with a certified standard using bovine liver (298925 mg Cd/g) (CRM 185). When amounts of biomass digested were between 0.61 – 1.27 mg the concentration of Cd measured was 268921 mg/g Cd. Since the deviation was within 90% of the certified values, no corrections were made.
2.6. Statistical analysis
One way ANOVA (PB0.05) was used to
different treatments using the Systat 5.1 software package.
3. Results
O. cincta was exposed to different concentra-tions of cadmium to quantify the metallothionein-bound cadmium at these concentrations. Actual concentrations were found to be 71% of the nom-inal concentration for all concentrations (r=
0.996) except for the non-contaminated algae (actual conc. 0.002 mmol Cd/g dry algae). Actual concentrations will be used in the presentation of the results.
After 2 weeks of exposure total internal cad-mium and metallothionein-bound cadcad-mium were measured (Fig. 1). Increasing exposure concentra-tions resulted in an increase in the total internal cadmium concentration until approximately 1 mmol Cd/g dry algae (Fig. 1A). This pattern was also observed for metallothionein-bound cad-mium (Fig. 1B). The variation in the metalloth-ionein-bound cadmium concentration determined
Fig. 2. Cadmium elution profile after gelfiltration chromatog-raphy from an homogenate of guts from O. cincta dietary exposed to 1mmol Cd/g dry algae for a period of 2 weeks.
in pooled animals is of course lower than the variation found in individual internal concentra-tions. We have to point out that from the expo-sure concentration of 1.05 mmol Cd/g dry algae only one measurement of metallothionein-bound cadmium could be performed.
Individual collembolans were also exposed to the concentrations ranging from 0.002 to 1.42 mmol Cd/g dry algae and their weight in-crease during 2 weeks of exposure was
deter-mined. Weight increase varied between an
average 0.14 and 0.20 mg/animal without any significant differences between the different treat-ments.
Guts were dissected from 2 weeks-exposed ani-mals to determine the amount of cadmium in the gut in relation to the cadmium in the whole animal. The fresh weight of a gut that had been collected after dissecting an animal was on aver-age 3.891.2% (n=9) of the total weight of an animal while 9296.4% (n=13) of the cadmium was measured within this organ. This suggests that the cadmium which is bound to metalloth-ionein detected in total body homogenates is actu-ally present within the gut. To confirm this, metallothioneins were isolated from dissected guts using the same procedure applied with whole ani-mal homogenates. Fig. 2 shows the elution profile of cadmium found after gel filtration chromatog-raphy. Again, most of the cadmium is found to elute in fractions around 25 – 26 min (Fig. 2), the
same fractions which are used in the quantifica-tion of metallothionein-bound cadmium from to-tal body homogenates. Upon further separation of these fractions the metallothioneins could be identified based on the absorbance at 254 nm (Fig. 3). The cadmium elution coincided with the absorbance peaks indicated in Fig. 3.
The above results show that upon exposure to cadmium metallothionein is primarily induced within the gut resulting in the fact that concentra-tions in this primary organ of expression are approximately 25 times (about 4% is gut fresh weight) higher than the concentration based on the total fresh weight of the animal shown in Fig. 1B.
Calculation of the actual metallothionein con-centration from the cadmium data determined in this experiment requires knowledge about the ex-act mass and metal stoichiometry of theO. cincta metallothionein. Within O. cincta two metalloth-ionein peptides have been isolated with a mass of 2989 Da and 4139 Da, respectively. Both are probably encoded by the same gene and are the result of processing of the primary translation product (Hensbergen et al., 1999). However, no cadmium:protein ratio could be determined for the small peptides isolated from animals in these studies, but a study with recombinant metalloth-ionein showed that the complete protein is able to bind 7 – 8 cadmium molecules (unpublished re-sults). If we take a ratio of 7:1 and a total mass of the most probable complete protein of 7110 Da (2989+4139 Da minus 18 Da (H2O)) we can estimate the actual metallothionein concentration in the gut. For the highest exposure concentration this then results in a concentration of about 115 mg metallothionein/g gut fresh weight.
4. Discussion
After exposingO.cinctato different concentra-tions of cadmium a steady increase in the internal cadmium concentration and metallothionein-bound cadmium was found. Both reach a plateau at an exposure concentration of about 1.05 mmol Cd/g dry algae. If we calculate the percentage of cadmium in the fractions from the contaminated animals after reversed phase chromatography in comparison with the total amount of cadmium an average percentage (9SD) of 1592% is found without any concentration dependent differences. If at higher concentrations the ratio between cad-mium found in reversed phase chromatography fractions and the total cadmium should become lower this could possibly be the concentration where significant effects would be expected. In this experiment this was not found, as shown also by the fact that no effects on weight increase were found between the treatments. Significant effects of cadmium on growth of females of O. cincta were found at concentrations similar to the ones used in this experiment (Van Straalen et al., 1989). However, in these experiments 3-day old individu-als were used and effects appeared only after 4 weeks of exposure.
The 15% cadmium retrieved after reversed phase chromatography will actually be an under-estimate of the actual metallothionein-bound cad-mium present in the animal. This is due to losses during homogenisation, gel filtration and reversed phase chromatography. In gills of Crassostrea
6irginicait was found that on average 22% of the
total gill cadmium was bound to metallothionein (Roesijadi, 1994). Other sources of cadmium in
O. cincta are related to the higher molecular weight proteins indicated by the first peak found after gel filtration chromatography (Fig. 2). Non-protein bound cadmium is presumably located in granular structures, like the ones described for other species (Hopkin, 1989; Ko¨hler et al., 1995). Studies on granular structures in the midgut of Collembola show that they also contain these type of structures (Pawert et al., 1996; Posthuma, 1992).
In this study we have also shown that990% of the cadmium is present in the gut and that isola-tion of metal binding proteins from these guts show the same pattern as from total body ho-mogenates (Hensbergen et al., 1999). In insects the digestive tract seems to be the major organ for storage of heavy metals (Aoki et al., 1984; Maroni and Watson, 1985; Postma et al., 1996). Metal kinetics inO.cincta is strongly connected with its physiology because the gut epithelium is renewed at every moult, and the old one is excreted as a gut pellet. Measurements of cadmium in the gut pellet and the animal after a moult gives an impression of the amount of cadmium that is excreted in the gut pellet. It is interesting to point out that although990% of the cadmium in the animals is present within the gut, only935% is excreted at every moult (Posthuma et al., 1992). This implies that more than half of the cadmium is not present in the one cell layer epithelium but also in the basal membrane or that cad-mium is resorbed from the old gut epithelium to the new gut epithelium. This second possibility would be interesting, especially if it turns out that only a specific fraction of the cadmium (granular
versus metallothionein-bound) is excreted
cq. resorbed. Electron microscopic studies in O. cincta have shown that the old gut epithelium contains a high number of granular structures but at present there is no of information about their content (Posthuma, 1992; Van Straalen et al., 1987).
The experiment shows that with a minimal amount of material (on average only 70 mg of total body fresh weight) it is possible to measure metallothionein-bound cadmium concentrations. In this study we used the direct measurement of cadmium after partial purification of the protein. Other quantification methods using metalloth-ionein-bound cadmium as a measure for the metallothionein concentrations, however, try to take into account the saturation of the
metalloth-ionein and the problem of simultaneous binding of other metals. This is done by saturating the protein before the measurement with an appropri-ate heavy metal ion (Ag, Cd) and binding the excess of non-metallothionein-bound cadmium with a high binding capacity substance (Martı´nez et al., 1993; Berger et al., 1995). The level of saturation after long term cadmium exposure in snails was 58% (Berger et al., 1995). A more direct measurement of metallothionein is the determina-tion of SH-groups either by polarography or
spec-trophotometrically. A comparison of these
methods with the direct measurement of cadmium
without the saturation assay, showed that
they were in good agreement with each other (Wageman et al., 1994; Pedersen and Lundebey, 1996).
The estimation of the real metallothionein con-centration (about 115 mg/g) at the highest expo-sure level may actually be an underestimation of the real concentration due to the above mentioned uncomplete saturation or simultaneous binding of other metals in combination with the losses due to the isolation procedure. In fish liver, however, a similar concentration was found (Jessen-Eller and Crivello, 1998), while in snails concentrations even reach a concentration of 900 mg/g in the midgut gland (Berger et al., 1995).
The metallothionein system from O. cincta gives the first possibility of using this protein as a biomarker for cadmium exposure in soil insects. Additional studies have to be performed to im-prove the detection method at the protein level, possibly also in combination with a saturation assay. The question is sometimes raised whether there is an advantage of measuring metalloth-ionein over the quantification of internal metal concentrations. We believe that measuring only metallothionein is not sufficient but that at least the ratio of total metal load and metallothionein bound metal, possibly in combination with fur-ther subcellular distributions, should be
incorpo-rated. The recent identification of the
metallothionein cDNA from O. cincta opens the possibility of studying actual induction of metal-lothionein, by determining quantitatively the ex-pression on the mRNA level.
concentration. This metallothionein is primarily present within the gut of this species.
Acknowledgements
The authors wish to thank Lorraine Leahy and Jojanneke van de Graaf for technical assistance. This work was supported by the Netherlands Organisation for Scientific Research (N.W.O.), project 805-33.401-P.
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