Cadmium-induced mRNA encoding a nonmetallothionein 33-kDa protein in Enchytraeus buchholzi (Oligochaeta)

ECOTOXICOLOGY

AND ENVIRONMENTAL

SAFETY

29, 93-100 (1994)

Cadmium-Induced mRNA Encoding a Nonmetallothionein Protein in Enchytraeus buchholzi (Oligochaeta) J. WILLUHN,* *Division

H.-P. SCHMITT-WREDE,*

y/ Parasirology. Marh.-Naf. Heinrich-Heine-Universiiaet

H. GREVEN,?

AND F. WUNDERLICH*

Facahy and TDivision of Zoomorphology Daesseldo$ Universitaerssrrasse 1. 4000 Federal Republic ojGermany

Received

July

33-kDa

and Cell Biology, DaesseldocL

27. I993

Enchyfraeas buchhoki (Oligochaeta) were exposed to various concentrations of Cd& in agar and aqueous solution. The Cd uptake was determined by atomic absorption spectroscopy as well as Cd effects on survival, reproduction, and mRNA synthesis by in vifro translation of total RNA in a rabbit reticulocyte lysate system. Although Cd was rapidly accumulated by the worms, any acute toxic Cd effects at concentrations below 4 mg Cd/liter were not detectable. However, such subtoxic Cd concentrations caused the induction of an mRNA species coding for a nonmetallothionein 33-kDa protein as revealed by 2D electrophoresis of in vitro translated proteins using [‘%]methionine. The Cd-induced synthesis of this transcriptionally regulated protein might be a preindicator for a Cd intoxication in enchytraeids. Q 1994 Academic ores. IIIC.

INTRODUCTION

Many oligochaeta species are known for their capability to accumulate Cd in both aquatic and terrestrial habitats and to reduce its toxic potency by several detoxification mechanism such as complexation and sequestering in lysosomes or binding to metallothionein-like proteins (Bauer-Hilty et al., 1989; Back, 1990; Klerks and Bartholomew, 199 1). Among oligochaetes, Cd accumulation is well documented for lumbricid earthworms (Ireland, 1979; Kiewiet and Wei-Chun, 199 1). In these species, a variety of metal-binding proteins were isolated and characterized, which were all suggested to be involved in heavy-metal detoxification (Suzuki et al., 1980; Morgan et al., 1989; Furst and Nguyen, 1989; Nejmeddine et al., 1992). Therefore, lumbricids have been considered as possible indicators of Cd availability and pollution in soil (Weigmann, 199 1) and they are used in several laboratory test systems for determination of the toxicity of chemicals to soil organisms (OECD, 1984; Van Gestel et al., 1989, 1992). Only little attention has been paid, so far, to enchytraeid oligochaetes, although they have high ecological relevance and meet many criteria asked for test organisms or even bioindicators. They are abundant in various terrestrial habitats including forested and cultivated areas, where they often comprise a considerable part of the total animal biomass (Kasprzak, 1982). They are involved directly and indirectly in the decomposition of organic matter and are important members of the food chain. Compared to lumbricids, enchytraeids have several advantages in experimental studies: (i) Many species can easily be cultured under laboratory conditions in artificial soil, in agar, and even in aqueous solution (Roembke, 1989; Westheide et al., 1991; Westheide and Bethke-Beilfuss, 1991). (ii) Generation periods are short. (iii) Reproduction and life cycle data are directly examinable in aqueous media. Thus, enchytraeids as soil inhabiting organisms can be studied in simple aquatic test systems, which are more 93

0147-65 l3/94 $6.00 Copyright 0 1994 by Academic Press. Inc. All rights 01 reproduction in any form reserved.

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convenient for investigations about the effects of environmental pollutants including heavy metals. Moreover, enchytraeids are known to take up considerable amounts of heavy metals, especially Cd (Rtither and Greven, 1990). Recent studies have revealed the occurrence of two Cd-inducible, Cd-binding proteins in Enchytraeus albidus kept in artificial pond water (R&her ef al., 1993). In the present communication, evidence is provided for a new Cd-induced protein, regulated at the transcriptional level, in Enchytraeus buchholzi kept in aqueous solution. MATERIALS

AND

METHODS

Animals Mass cultures of E. buchholzi were grown in large petri dishes half filled with artificial soil ( 100 g spagnum peat, 100 g calcium carbonate, 10 g quartz sand, and 50 g kaolin in distilled water). The cultures were kept in a dark room at 2 1“C. Animals were fed with flaked oats every week. Toxic&) Tests Mortality and reproduction tests were performed in 1% agar medium (Westheide and Bethke-Beilfuss, 1991) and in a modified aquatic system recently developed by Roembke and Knacker (1989). The aqueous solution in both media consisted of 1 g Ca(NO&, 0.25 g MgS04*7Hz0, 0.25 g KN03, 0.25 g KH2P04, pH 5.0, and a trace of FeS04 per 1 liter distilled water. Media were autoclaved at 12 1“C for 20 min. For mortality tests, at least 20 animals of the same size (mature, 3-4 mm; juveniles, > 1 mm) were used for each of the Cd concentrations and the control. Batches of 10 animals were put into a loo-ml glass beaker (aquatic test) or in a 1 IO-mm petri dish (agar test) filled with 40 ml of the appropriate test solution and maintained under mass culture conditions. Reproduction tests were carried out in 1% agar medium supplemented with flaked oats. For each of the Cd concentrations and the control, I2 mature animals of the same size (3-4 mm) were allocated to 4 petri dishes. The cocoons were counted under a dissecting microscope every 2 days. The test was finished when new mature worms were observed in the dishes (after 16 days). Cd Accumulation Worms were incubated in aqueous solution containing I and 4 mg Cd/liter, respectively. For Cd measurements, at least 10 worms per sample were pooled, lyophilized, weighed, digested with HN03 and subjected to Cd analysis using a graphite furnace atomic absorption spectrometer (Perkin-Elmer 50002). Detection of Cd-Induced mRNA Synthesis Enchytraeids were taken from mass culture, incubated with 3 mg Cd/liter in glass beakers, and maintained under mass culture conditions. After 6 days, Cd-treated and control animals were collected and total RNA was isolated according to the method of Chirgwin et al. ( 1979). Aliquots of 20 pg from the total RNA were in virro translated using the Boehringer reticulocyte translations kit type I and [3sS]methionine. After stopping the translation reaction, “S-labeled proteins were precipitated with acetone,

CADMIUM-INDUCED

mRNA IN

Enchytraeus

buchholzi

95

dissolved in gel-loading buffer and subjected to 2D electrophoresis according to the method of O’Farrell ( 1975). Statistics LC5e values were calculated by applying the method of Litchfield and Wilcoxon ( 1948). Data of the reproduction test were expressed as means + SD. Significance was evaluated using Student’s t test. RESULTS Figure 1 presents the effect of different Cd concentrations on mortality of E. buchholzi in agar and aqueous solution. The extreme slope of the concentration-response curve indicates the existence of a threshold value for the lethal concentration of Cd. The 4day L& values were about the same in both aqueous solution and agar. They were estimated to be 8.4 vs 8.0 mg Cd/liter, respectively. However, juvenile enchytraeids responded more sensitive to Cd than adults, the 4-day LCsO values amounted to 6.4 vs 8.4 mg Cd/liter, respectively (P = 0.05). Both mature and juvenile worms were not affected by Cd concentrations less than 6 mg Cd/liter. Even after exposure to Cd for 22 days, all worms survived at Cd concentrations below 4 mg Cd/liter (Fig. 2). The sublethal concentration of 3 mg Cd/liter did not reduce reproduction capacity of E. buchholzi when time of exposure did not exceed 6 days. After 16 days of exposure, the number of cocoons was reduced by 63.4 + 11.8%. However, this inhibition of cocoon production was fully reversible when worms were transferred to fresh, untreated agar (Fig. 3). In this case, cocoon production started after a lag phase of 2 days at the normal rate (0.64 + 0.10 cocoons/worm/day). Furthermore, sublethal injuries such as disintegrations or loss of segments were not observed. Although there were no acute toxic effects at a concentration of 3 mg Cd/liter, Cd was rapidly accumulated by the worms (Fig. 4). Accumulation of worms exposed to

100

60

.c 60 ii 5 E 40 ap 20 0 0

2

4

6 mg Cd/l

6

10

12

FIG. 1. Mortality of E. buchholzi after exposure to Cd for 4 days. No mortality in controls. At least 40 worms were examined at each Cd concentration. 0, juveniles, aquatic test; 0, adults, aquatic test; A, adults, agar test.

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ET AL.

10 .

= s 0

0.

E

.

5: Y

6.

4 .

0

5

10

20

15

time (days) FIG. 2. Decrease in LCSo values for the effect of Cd on mortality of adult E. b~rchholzi in aquatic test medium at prolonged periods of Cd incubation. LCSo values were determined from six different Cd concentrations. At each concentration 20 worms were examined. No mortality in controls.

1 mg Cd/liter was almost linear for 13 days, whereas that of worms exposed to 4 mg Cd/liter tended to saturate after 13 days. After 13 days the Cd concentrations in worm tissues were calculated to be enriched 63-fold ( 1 mg Cd/liter) and 17-fold (4 mg Cd/ liter) in comparison to the test solution.

I

/ /1 %1

Cd-axposure

I

I.

I

.I.II

I

5

IO

15

0

I

I.

5

10

.I. 15

time (days) FIG. 3. The influence of Cd on the cocoon production of E. brtchholzi (mean of four different experiments). The worms were kept for 16 days at 3 mg Cd/liter (left) and then transferred to untreated agar (right). 0, control; A, 3 mg Cd/liter.

CADMIUM-INDUCED

mRNA

IN

Enchyrruerrs

97

brcchhoki

400

0 0

4

8

time FIG. 4. Accumulation

medium.

0. control:

12

(days)

of Cd in E. buclhki after exposure 0, I mg Cd/liter: A, 4 mg Cd/liter.

to different

Cd concentrations

in aquatic

test

For detection of Cd-sensitive mRNA species, worms were incubated at the subtoxic Cd concentration of 3 mg Cd/liter for 6 days, before isolating total RNA. The latter was subjected to in vitro translation using [35S]methionine. Fluorographs of 2D gel electrophoretograms revealed 17 major and about 100 minor spots (Fig. 5). The apparent molecular masses of the proteins were in the range between 10 and 95 kDa and the apparent pl values in the range between 3.8 and 7.9. The intensity of some spots varied slightly from one experiment to the other and slight differences occurred in the intensity of the radioactive label of some minor spots after Cd treatment. However, one new major spot was detected in Cd-treated worms. This new protein had an apparent molecular mass of 33 kDa and an apparent pl of 7.5. Due to the fact that only 120 proteins were detected by 2D electrophoresis, the Cd-induced 33-kDa protein represented the translation product of one of the abundant mRNAs in Cdtreated enchytraeids. DISCUSSION The present study shows that E. bzrchholzi rapidly accumulates Cd in aqueous solution under subtoxic conditions. Indeed, the exposure of worms to 1 mg Cd/liter for 13 days in aqueous solution did not result in any acute toxic effects, although the Cd concentration in worms increased from 6 to 404 pg Cd/g dry weight. This concentration is in the range of 765 rg Cd/g dry weight recently reported for E. buchholzi, living for 12 days in 1 mg/liter Cd-treated agar (Riither and Greven, 1990). In addition, our data demonstrate that reproduction of worms begins to be reduced after 8 days of Cd exposure to 3 mg Cd/liter, but this effect is completely reversible. Incubation of E. buchholzi at subtoxic conditions, i.e., 3 mg Cd/liter for 6 days, resulted in the induction of a new mRNA species encoding a 33-kDa protein with an apparent pl of 7.5. However, the in vitro translation assay was unable to identify mRNA species corresponding to the two Cd-inducible proteins with a molecular mass of 5-8 and 70-80 kDa recently found in the enchytraeid species E. albidus (Ruther et al.. 1993). The reason for this is unknown. It may be due to (i) the limited resolution of low-

98

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PI 3.8

5.6

7.9

6.6

Mr x 103

-67

PI 3.8

5.6

7.9

6.6

Mr x 103

;

.:. ,. -

67

-

45

c-

29

FIG. 5. Two-dimensional electrophoresis of in v&-o translated, [35S]methionine-labeled E. buclhlzi proteins. Gels were subjected to autoradiography for 10 days. (A) Cd-treated worms; (B) controls. Surrounded spot indicates the Cd-induced protein.

molecular-mass proteins in the 2D electrophoretic system used, (ii) a low content of methionine of the two Cd-inducible E. albidus proteins, (iii) a translational regulation of these two proteins, and (iv) a low abundance or even absence of mRNAs encoding the E. afbidus proteins in E. buchholzi. In contrast, the Cd-inducible 33-kDa protein detected in E. buchhofzi must be one of the abundant proteins in Cd-treated worms. The nature of the 33-kDa protein in E. buchholzi is still unknown. Presumably, it is not a metallothionein-like protein because of its high molecular weight and its slightly basic pl. Indeed, metallothioneins are characterized by low molecular masses of 6-7 kDa (Kojima, 1991) and the pl values of metallothioneins were reported to vary between 3.9 to 6.0 (Nordberg, 1991). Moreover, metallothioneins contain only

CADMIUM-INDUCED

mRNA

IN

Enchylraeus

buchholzi

99

one methionine, and it is therefore not likely to detect them by labeling with [35S]methionine. Furthermore, there is some information available that Cd can also induce several nonmetallothionein proteins. For instance, Cd was reported to induce stress proteins (Glaven et al., 1991) and metal-binding proteins (Stone and Overnell, 1985) in diverse invertebrates. To the authors’ knowledge, however, a Cd-inducible protein as the 33-kDa protein in E. buchholzi has not yet been reported in soil invertebrates. Currently, an effort is being made to identify the gene encoding the 33-kDa protein as well as further genes possibly induced by Cd under subtoxic conditions, using a more sensitive method than in vitro translation, i.e., differential screening of a cDNA library constructed from Cd-treated E. buchhofzi. CONCLUSIONS The exposure of E. buchholzi to subtoxic Cd concentrations in an aquatic test system induces the synthesis of a new mRNA species encoding a nonmetallothionein 33-kDa protein. This Cd-induced protein, obviously regulated at the transcriptional level, may be a promising candidate preindicator for a Cd intoxication in enchytraeids. ACKNOWLEDGMENTS The authors thank Dr. J. Abel and Dr. L. Dunemann for help with the AAS and the Deutsche Forschungsgesellschaft for financial support.

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