University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, P.O. Box 38, Solomons, Maryland 20688
Received June 27, 2000; accepted August 31, 2000
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ABSTRACT |
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Key Words: metallothionein; cadmium; copper; zinc; antisense; phosphorothioate; oligodeoxynucleotide; lysosome; hemocyte; mollusc; oyster.
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INTRODUCTION |
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While studies on the biology and chemistry of MT in invertebrate species are numerous (reviewed by Roesijadi, 1992), there is still little direct evidence of a role for MT in protection against metal toxicity in the varied taxa represented in this group. Extension of such function to species of lower phylogenetic status could shed light on the general question of metal detoxification as a function of MT. Class I MTs similar to those of vertebrates have been reported in several crustacean and molluscan species (Brouwer et al., 1995; Dallinger et al., 1993
; Lerch et al., 1982
; Pedersen et al., 1994
; Unger et al., 1991
). Putative metal regulatory elements exhibiting homology with mammalian counterparts have also been reported in an insect, sea urchin, nematode, and a mollusc (Freedman et al., 1993
; Harlow et al., 1989
; Khoo and Patel, 1999
; Otto et al., 1987
). Furthermore, the notion that MT is a protein involved in metal detoxification remains central to the field of ecotoxicology, despite the current lack of direct evidence. Thus, it is of interest to investigate the function of MT in species directly relevant to this field. MTs in aquatic species such as fish, molluscs, arthropods, and annelids are induced by and, in turn, bind metals such as cadmium, copper, mercury, or zinc (reviewed by Roesijadi, 1992). Individuals in metal-contaminated aquatic environments often possess elevated concentrations of MT, and MT is considered a potential biomarker of effects or exposure to metals (Stegeman et al., 1992
). However, its use as a practical measure for assessing metal exposure has yet to be realized, due in part to unresolved questions relating to function and factors that affect induction.
We examined the effect of cadmium on hemocytes, the blood cells involved in the molluscan nonspecific defense response (reviewed by Cheng, 1996), in the eastern oyster Crassostrea virginica, hypothesizing that MT expression is necessary to protect against cadmium toxicity. C. virginica (Unger et al., 1991), like other molluscan species studied to date (Barsyte et al., 1999
; Berger et al., 1995
; Dallinger et al., 1997
; Engelken and Hildebrandt, 1999
; Khoo and Patel, 1999
; Mackay et al., 1993
), possesses class I MT resembling those present in higher organisms. This species is commercially and ecologically important and has been the subject of monitoring programs for metal contamination of the coastal environment (Lauenstein et al., 1990
; NOAA, 1987
). Phosphorothioate-substituted oligodeoxynucleotides antisense to the oyster MT mRNA (aMT S-ODN) were used to determine whether disruption of MT expression would influence the toxicity of cadmium. Both basal and cadmium-induced MT expression have been described for these cells (Roesijadi et al. 1997a
). We now report that treatment of hemocytes with the aMT S-ODN inhibited levels of both basal and cadmium-induced MT mRNA and that low cadmium concentrations that normally induce MT, but do not elicit symptoms of sublethal cytotoxicity, became toxic when MT expression was disrupted by administration of aMT S-ODN. These findings represent direct evidence that induction of MT by cadmium confers protection against cadmium toxicity in cells of this mollusc.
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MATERIALS AND METHODS |
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Isolation of hemocytes.
The procedure for isolation of hemocytes was based on a previously described protocol (Anderson et al., 1992). Hemolymph was withdrawn from the adductor muscle sinus using a syringe with a 23-gauge needle and transferred to plastic petri dishes. After 30 min, adherent hemocytes were washed and resuspended in culture medium composed of 25 ppt artificial seawater, 0.5% antibiotic/antimycotic solution (penicillin, streptomycin, and amphotericin B; Sigma A-9909), and 2 mg/ml glucose. Hemocytes were pelleted at 250 x g, resuspended in culture medium and diluted to a density of 4 x 106 cells/ml, then aliquoted to various experimental treatments. Cells isolated from 12 to 16 oysters were pooled to provide sufficient numbers of cells to conduct experiments in which MT mRNA was determined by RT-PCR or in which antisense was used to disrupt MT expression; numbers of replicate pools are noted with the results of individual experiments.
Metal exposure.
Hemocytes were exposed for 20 h to cadmium, copper, or zinc in 1.5 ml polypropylene microcentrifuge tubes at room temperature (2426°C) in darkness. Stock solutions of CdCl2, CuCl2, and ZnCl2 dissolved in 0.01 N nitric acid were first diluted in culture medium to make fresh working stocks. Volumes of the working stocks added to samples were the same in all cases, and the small volumes added had no detectable effect on pH.
Cytotoxicity assays.
Hemocyte viability was measured by supravital staining with neutral red (Borenfreund and Puerner, 1985, 1987
) during a 20-min incubation or by trypan blue exclusion.
Sublethal cytotoxicity was measured as reduced lysosomal neutral red retention time, which is diagnostic of lysosomal membrane instability of stressed cells (Lowe et al., 1992). Measurement of lysosomal neutral red retention time was conducted according to a procedure developed for isolated mussel hemocytes (Lowe et al., 1995
), modified by omitting dimethylsulfoxide in the solvent for neutral red. The neutral red incubation solution was prepared in culture medium just prior to use. Cells were monitored under oil immersion at 1000x magnification. Following uptake and concentration of neutral red in lysosomes, the proportion of cells exhibiting loss of dye from lysosomes was recorded until loss was observed in greater than 50% of the cells. Observations for neutral red retention in cells exposed to cadmium or zinc were recorded at 20-min intervals. With copper, observations were made every 5 min for the initial 20 min, due to the more rapid release, then at 20-min intervals thereafter. The neutral red retention time was estimated as the time at which 50% of hemocytes released their accumulated neutral red (ET50). Measurements were made on cells of individual oysters.
Total RNA isolation and quantification.
Total RNA was isolated using Tri Reagent (Molecular Research Center, Cincinnati, OH) and quantified fluorometrically (Schmidt and Ernst, 1995) using SYBR Green II RNA dye (Molecular Probes Inc., Eugene, OR). RNA was incubated with dye in white microtiter plates (Packard Optiplate) for 30 min at room temperature, then analyzed on an FCA fluorescence plate reader (IDEXX, Portland, ME) at 485 nm excitation/535 nm emission. Quantification was based on standard curves derived from fluorescence of yeast transfer RNA standards.
Antisense oligodeoxynucleotides.
An antisense phosphorothioate-substituted oligodeoxynucleotide (5'-GGACCGGTGCACTTCTCCCCA-3') corresponding to nucleotides 201 to 221 of the oyster MT cDNA (Unger et al., 1991) was synthesized by Oligos Etc. (Wilsonville, OR) and further purified in our laboratory by anion exchange high-performance liquid chromatography (HPLC) (Warren and Vella, 1993
). The sample was applied to a TSK DEAE-5PW column (7.5 mm x 7.5 cm; Toyo Soda) at initial buffer conditions (73% buffer A [25 mM Tris, pH 8, 10% acetonitrile], 27% buffer B [3 M NaCl in buffer A]), and aMT-S-ODN was eluted with a 45-min gradient from 27 to 100% buffer B at 1 ml/min. HPLC-purified material was recovered by ethanol precipitation overnight at 20°C and centrifugation for 15 min at 12,500 x g, 4°C. The pellet was washed twice in 80% ice-cold ethanol, resuspended in water, quantified by measuring absorbance at 260 nm, aliquoted, lyophilized, and stored at 70°C.
Antisense S-ODN was administered to the cells 2 h prior to initiation of 20-h metal exposures. A fully phosphorothioate-substituted oligonucleotide of identical length and similar nucleotide composition (5'-GCCGAGGTCCATGTCGTACGC-3') was used as a control for potential nonspecific S-ODN toxicity.
Quantitative RT-PCR..
MT mRNA was quantified by competitive RT-PCR (Becker-Andre and Hahlbrock, 1989; Wang et al., 1989
), using an external standard derived from a cDNA deletion clone (
6 bases) of the oyster MT coding region. This
6 clone was created by in vitro mutagenesis (ExSiteTM PCR-based Site-Directed Mutagenesis Kit, Stratagene, LaJolla, CA) of the native cDNA sequence cloned in pGEM-3Z (Fuentes et al., 1994
). The external standard was synthesized using T7 RNA polymerase (Ausubel et al., 1993
).
For the competitive RT-PCR, 0.025 µg total sample RNA was co-reverse transcribed with serial dilutions of the 6 standard. Reverse transcription was conducted in 1 x Tris buffer solution (10 mM TrisHCl pH 9, 50 mM KCl, 0.1% Triton X-100), 5 mM MgCl2, 1 mM dNTP mix, 5 µM reverse primer (5'-CACTTCTTGCAGCTGCAGCC-3'), and 0.33 U Prime RNase Inhibitor (5-Prime3-Prime Inc., Boulder, CO) in a final volume of 10 µl. For PCR, samples were supplemented with additional 1 x Tris buffer, MgCl2 to 1.5 mM final concentration, 5 µM biotinylated (*) forward primer (5'-*TGTATTGAGACTGGCACCTG-3'), and 1.25 U Taq polymerase, adjusted to a final volume of 50 µl, and amplified for 21 cycles. Amplification conditions were denaturation for 5 min at 94°C initially, 1 minute thereafter; annealing for 2 min at 50°C; elongation for 3 min at 72°C, with a 10-min elongation step in the last amplification cycle.
RT-PCR product detection was based on a microtiter plate-based chemiluminescent assay. Duplicate, amplified, biotinylated cDNA subsamples were bound to streptavidin-coated microtiter plates, denatured, and hybridized to fluorescein-labeled oligonucleotide probes specific for either the native MT mRNA or the external standard. The hybridization buffer was 6 x SSC, 5 x Denhardt's solution, 0.1% Tween-20, 100 µg/ml sheared, denatured, calf thymus DNA. Alkaline phosphatase-conjugated antifluorescein antibody (Tropix Inc, Bedford, MA) was bound to the hybridized probes and analyzed for chemiluminescence after addition of substrate (CSPD; Tropix Inc.) and enhancer solution (Sapphire II, Tropix Inc). A Top Count liquid scintillation counter (Packard Instrument Co.) in single photon counting mode was used for chemiluminescence detection. MT mRNA was estimated at log(Tn/Sn) = 0, the point of equivalence of target and standard, in a plot of log(Tn/Sn) versus logSo (Raeymaekers, 1993). Tn/Sn is the ratio of target to standard signal after RT-PCR, and So is the initial amount of each external standard in the standard dilution series.
Data analysis.
Data were analyzed using ANOVA. Post hoc comparisons used Student-Newman-Keul's test. The level of significance was set at p 0.05.
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RESULTS |
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The relative effectiveness of cadmium, copper, and zinc as inducers of MT expression and cytotoxicants was determined for oyster hemocytes. Dose-response curves indicated large differences in cadmium-, copper-, and zinc-induced MT mRNA (Fig. 1). With all three metals, dose-response curves exhibited an increase in MT mRNA to maximal levels as dose increased. Increasing dose above these levels attenuated induction. Cadmium was the most effective inducer of the metals investigated, followed by copper and zinc. This was evident in both magnitude of induction and sensitivity to dose: induction by cadmium reached higher levels and required lower doses. Estimations of the effectiveness of MT mRNA induction by the three metals, which took into account both the magnitude of induction and sensitivity to dose, supported a rank order of Cd > Cu > Zn (Table 1
). When both were taken into account, cadmium was 13.7-fold more effective than copper and 258-fold more effective than zinc in inducing MT mRNA. In this case, maximal levels of induction in response to CdCl2 reflected a 9.3-fold increase in MT mRNA over basal levels, in comparison with 5.9- and 3.8-fold over basal levels in response to CuCl2 and ZnCl2, respectively.
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Fully substituted aMT S-ODN was used in this study. As antisense at concentrations used here had no adverse effect on viability (data not shown), any changes in lysosomal membrane stability would reflect sublethal cytotoxic effects rather than nonspecific responses associated with cell death. Partially substituted antisense S-ODN for MT (i.e., 4 sulfur substitutions at each end of the oligonucleotide) was also tested and found to be ineffective in downregulating MT, causing little or no reduction of MT mRNA at the concentrations found to be effective with the fully substituted form. Treatment with 5 and 10 µM aMT-S-ODN resulted in dose-dependent reductions in MT mRNA induction (not shown), with no additional reduction when the dose was increased to 20 µM. Thus, 10 µM aMT-S-ODN was selected for routine disruption of MT expression. This concentration of control and aMT S-ODN did not affect viability of untreated and cadmium-exposed cells. Treatment of cells with 10 µM control S-ODN affected neither basal nor cadmium-induced MT mRNA levels (Fig. 3). Treatment with 10 µM aMT-S-ODN reduced both basal and cadmium-induced MT mRNA concentrations by approximately 55 and 50%, respectively (Fig. 3
).
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DISCUSSION |
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The increase in cadmium toxicity that resulted from disruption of MT expression with antisense oligodeoxynucleotides was consistent with the large numbers of studies implicating MT in protection against cadmium toxicity in higher animals (reviewed in Klaassen et al., 1999). For example, induction of MT by pretreatment with low doses of cadmium results in adaptive tolerance to the toxicity of higher concentrations (Klaassen and Liu, 1998); overexpression of MT as a result of gene amplification results in increased cadmium resistance in cultured mammalian cells (Beach and Palmiter, 1981
; Gick and McCarty, 1982
; Hildebrand et al., 1979
); overexpression of MT in MT transgenic mice results in increased cadmium resistance (Liu et al., 1995
); and disruption of MT expression in knockout mice results in loss of resistance to cadmium toxicity (Masters et al., 1994
; Michalska and Choo, 1993
). Increased sensitivity to cadmium in MT knockout mice is the strongest evidence to date that loss of protection against cadmium toxicity is an important phenotype associated with MT gene disruption in animals. Recent studies also suggest that MT induction by cadmium may be a specific cellular response not mediated by pathways involving zinc. They describe cadmium-specific regulation of MT gene expression in cells lacking the zinc-responsive metal transcription factor MTF-1 (Chu et al., 1999
) and a signal transduction pathway that involves the upstream regulatory factor (USF) (Andrews, 2000
; Datta and Jacob, 1997
; Li et al., 1998
). Such observations lend credibility to the notion that induction of MT by cadmium follows pathways specific for this metal and that the induction functions to protect against cadmium toxicity.
A recent study describes the first reported genomic MT sequences of a mollusc, MT-Ia1 and MT-Ia2 of the green mussel Perna viridis (Khoo and Patel, 1999). MT-Ia1 possesses a TATA box and putative sequences for two metal responsive elements, two glucocorticoid receptor sites, and three AP1 binding sites. MT-Ia2 has a TATA box and putative sequences for an MRE, two AP1 sites, and an enhancer E box promoter region. This complexity mirrors that seen in higher animals (Palmiter, 1999
) and suggests responsiveness of MT expression to zinc and factors other than metals, such as hormones and reactive oxygen species. However, the significance of such responsiveness remains to be clarified in higher animals (Klaassen et al., 1999
; Palmiter, 1998
, 1999
), and has yet to be investigated in molluscs.
Our findings contribute to a mechanistic justification for continued evaluation of molluscan MT as a biomarker for potential biological effects in cadmium-contaminated aquatic environments: where MT-bound cadmium is detected in organisms such as oysters, individuals are likely protected from this fraction of accumulated cadmium. Thus, the biological significance of such binding would be the lack of adverse effects from this MT-bound fraction of cadmium, a notion that has value for interpreting responses of individuals in metal-contaminated environments (reviewed in Roesijadi, 1992). Our findings of a strong inverse relationship between MT expression and the toxicity of cadmium will need to be extended to an understanding of how MT is expressed and functions in natural populations.
Our study showed that, of the metals examined, cadmium was the most effective in inducing MT in isolated oyster hemocytes and that expression of MT conferred protection against cadmium toxicity. The latter conclusion was deduced from a loss-of-function experiment in which cadmium concentrations normally not cytotoxic became highly toxic after MT expression was disrupted with antisense oligodeoxynucleotides. Thus, in molluscs, as in higher animals, MT protects against cadmium toxicity. However, the finding that MT is important in protection against cadmium does not preclude the possibility that MT is also involved in other important biological functions.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Florida Atlantic University, Department of Biological Sciences, 777 Glades Road, P.O. Box 3091, Boca Raton, FL 334310991. E-mail: groesija{at}pop.fau.edu.
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REFERENCES |
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Andrews, G. K. (2000). Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95104.[ISI][Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1993). Current Protocols in Molecular Biology. Current Protocols, Massachusetts General Hospital, Harvard Medical School.
Barsyte, D., White, K. N., and Lovejoy, D. A. (1999). Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 122, 287296.[Medline]
Beach, L. R., and Palmiter, R. D. (1981). Amplification of the metallothionein-I gene in cadmium-resistant mouse cells. Proc. Natl. Acad. Sci. U. S. A. 78, 21102114.[Abstract]
Becker-Andre, M., and Hahlbrock, K. (1989). Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 17, 94379446.[Abstract]
Berger, B., Hunziker, P. E., Hauer, C. R., Birchler, N., and Dallinger, R. (1995). Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum. Biochem. J. 311, 951957.[ISI][Medline]
Borenfreund, E., and Puerner, J. A. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119124.[ISI][Medline]
Borenfreund, E., and Puerner, J. A. (1987). Short-term quantitative in vitro cytotoxicity assay involving an S-9 activating system. Cancer Lett. 34, 243248.[ISI][Medline]
Brouwer, M., Enghild, J., Hoexum-Brouwer, T., Thogersen, I., and Truncali, A. (1995). Primary structure and tissue-specific expression of blue crab (Callinectes sapidus) metallothionein isoforms. Biochem. J. 311, 617622.[ISI][Medline]
Calabrese, A., Collier, R. S., Nelson, D. A., and MacInnes, J. R. (1973). Toxicity of heavy metals to embryos of the American oyster Crassostrea virginica. Mar. Biol. 18, 162166.[ISI]
Calabrese, A., MacInnes, J. R., Nelson, D. A., and Miller, J. E. (1977). Survival and growth of bivalve larvae under heavy-metal stress. Mar. Biol. 41, 179184.[ISI]
Cheng, T. C. (1996). Hemocytes: forms and functions. In The Eastern Oyster Crassostrea virginica (V. S. Kennedy, R. I. E. Newell, and A. F. Eble, Eds.). pp. 29993333. Maryland Sea Grant College, College Park, Maryland.
Chu, W. A., Moehlenkamp, J. D., Bittel, D., Andrews, G. K., and Johnson, J. A. (1999). Cadmium-mediated activation of the metal response element in human neuroblastoma cells lacking functional metal response element-binding transcription factor-1. J. Biol. Chem. 274, 52795284.
Dallinger, R., Berger, B., Hunziker, P., and Kagi, J. H. (1997). Metallothionein in snail Cd and Cu metabolism. Nature 388, 237238.[ISI][Medline]
Dallinger, R., Berger, B., Hunziker, P. E., Birchler, N., Hauer, C. R., and Kagi, J. H. (1993). Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A. Eur. J. Biochem. 216, 73946.[Abstract]
Datta, P. K., and Jacob, S. T. (1997). Activation of the metallothionein-I gene promoter in response to cadmium and USF in vitro. Biochem. Biophys. Res. Commun. 230, 159163.[ISI][Medline]
Engelken, J., and Hildebrandt, A. (1999). cDNA cloning and cadmium-induced expression of metallothionein mRNA in the zebra mussel Dreissena polymorpha. Biochem. Cell Biol. 77, 237241.[ISI][Medline]
Freedman, J. H., Slice, L. W., Dixon, D., Fire, A., and Rubin, C. S. (1993). The novel metallothionein genes of Caenorhabditis elegansstructural organization and inducible, cell-specific expression. J. Biol. Chem. 268, 25542564.
Fuentes, M. E., Unger, M. E., and Roesijadi, G. (1994). Individual variability in the 3' untranslated region of metallothionein mRNAs in a natural population of the mollusc Crassostrea virginica. Mar. Mol. Biol. Biotechnol. 3, 141148.
Gick, G. G., and McCarty, K. S., Sr. (1982). Amplification of the metallothionein-I gene in cadmium- and zinc-resistant Chinese hamster ovary cell. J. Biol. Chem. 15, 90499053.
Hamer, D. H., Thiele, D. J., and Lemontt, J. E. (1985). Function and autoregulation of yeast copperthionein. Science 228, 685690.[ISI][Medline]
Harlow, P., Watkins, E., Thornton, R. D., and Nemer, M. (1989). Structure of an ectodermally expressed sea urchin metallothionein gene and characterization of its metal-responsive region. Mol. Cell Biol. 9, 54455455.[ISI][Medline]
Hildebrand, C. E., Tobey, R. A., Campbell, E. W., and Enger, M. D. (1979). A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity. Exp. Cell Res. 124, 237246.[ISI][Medline]
Kägi, J. H. R., and Vallee, B. L. (1960). Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem. 235, 34603465.[ISI][Medline]
Kägi, J. H. R., and Vallee, B. L. (1961). Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. II. Physicochemical properties. J. Biol. Chem. 236, 24352442.[ISI][Medline]
Kelly, E. J., Quaife, C. J., Froelick, G. J., and Palmiter, R. D. (1996). Metallothionein I and II protect against zinc deficiency and zinc toxicity in mice. J. Nutr. 126, 17821790.[ISI][Medline]
Khoo, H. W., and Patel, K. H. (1999). Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis. J. Exp. Zool. 284, 445453.[ISI][Medline]
Klaassen, C. D., and Liu, J. (1998). Induction of metallothionein as an adaptive mechanism affecting the magnitude and progression of toxicological injury. Environ. Health Perspect. 106(suppl.), 297300.[ISI][Medline]
Klaassen, C. D., Liu, J., and Choudhuri, S. (1999). Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39, 267294.[ISI][Medline]
Lauenstein, G. G., Robertson, A., and O' Connor, T. P. (1990). Comparison of trace metal data in mussels and oysters from a mussel watch programme of the 1970s with those from a 1980s programme. Mar. Pollut. Bull. 21, 440447.[ISI]
Lerch, K., Ammer, D., and Olafson, R. W. (1982). Crab metallothionein. Primary structures of metallothionein 1 and 2. J. Biol. Chem. 257, 24202426.
Li, Q., Hu, N., Daggett, M. A., Chu, W. A., Bittel, D., Johnson, J. A., and Andrews, G. K. (1998). Participation of upstream stimulator factor (USF) in cadmium induction of the mouse metallothionein-I gene. Nucleic Acids Res. 26, 51825189.
Liu, Y. P., Liu, J., Iszard, M. B., Andrews, G. K., Palmiter, R. D., and Klaassen, C. D. (1995). Transgenic mice that overexpress metallothionein-I are protected from cadmium lethality and hepatotoxicity. Toxicol. Appl. Pharmacol. 135, 222228.[ISI][Medline]
Lowe, D. M., Fossato, V. U., and Depledge, M. H. (1995). Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar. Ecol. Prog. Ser. 129, 189196.[ISI]
Lowe, D. M., Moore, M. N., and Evans, B. M. (1992). Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from Dab Limanda-Limanda. Mar. Ecol. Prog. Ser. 91, 135140.[ISI]
Mackay, E. A., Overnell, J., Dunbar, B., Davidson, I., Hunziker, P. E., Kägi, J. H. R., and Fothergill, J. E. (1993). Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis. Eur. J. Biochem. 218, 183194.[Abstract]
Masters, B. A., Kelly, E. J., Quaife, C. J., Brinster, R. L., and Palmiter, R. D. (1994). Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U. S. A. 91, 584588.[Abstract]
Michalska, A. E., and Choo, K. H. A. (1993). Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mice. Proc. Natl. Acad. Sci. U. S. A. 90, 80888092.
NOAA (1987). National Status and Trends Program for Marine Environmental Quality. National Oceanic and Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.
Otto, E., Allen, J. M., Young, J. E., Palmiter, R. D., and Maroni, G. (1987). A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn. Mol. Cell. Biol. 7, 17101715.[ISI][Medline]
Palmiter, R. D. (1998). The elusive function of metallothioneins. Proc. Natl. Acad. Sci. U. S. A. 95, 84288430.
Palmiter, R. D. (1999). Metallothionein facts and frustrations. In Metallothionein IV (C. Klaassen, Ed.). pp. 215221. Birkhäurser Verlag, Basel.
Pedersen, K. L., Pedersen, S. N., Højrup, P., Andersen, J. S., Roepstorff, P., Knudsen, J., and Depledge, M. H. (1994). Purification and characterization of a cadmium-induced metallothionein from the shore crab Carcinus maenas (L.). Biochem. J. 297, 609614.[ISI][Medline]
Piscator, M. (1964). On cadmium in normal human kidney together with a report on the isolation of metallothionein from livers of cadmium exposed rabbits. Nord. Hyg. Tidskr. 45, 7682.[Medline]
Raeymaekers, L. (1993). Quantitative PCR: theoretical considerations with practical implications. Anal. Biochem. 214, 582585.[ISI][Medline]
Roesijadi, G. (1992). Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22, 81114.[ISI]
Roesijadi, G., Brubacher, L. L., Unger, M. E., and Anderson, R. S. (1997a). Metallothionein mRNA induction and generation of reactive oxygen species in molluscan hemocytes exposed to cadmium in vitro. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 118C, 171176.[ISI][Medline]
Roesijadi, G., Hansen, K. M., and Unger, M. E. (1997b). Concentration-response relationships for Cd, Cu, and Zn exposure and metallothionein mRNA induction in larvae of Crassostrea virginica. Comp. Biochem. Physiol. 118, 267270.[ISI]
Schmidt, D. M., and Ernst, J. D. (1995). A fluorometric assay for the quantification of RNA in solution with nanogram sensitivity. Anal. Biochem. 232, 144146.[ISI][Medline]
Stegeman, J. J., Brouwer, M., Di Giulio, R. T., Förlin, L., Fowler, B. A., Sanders, B. M., and Van Veld, P. A. (1992). Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress (R. J. Huggett, R. A. Kimerle, P. M. Mehrle, and H. L. Bergman, Eds.). pp. 235335. Lewis Publishers, Chelsea, MI.
Thiele, D. J., Walling, M. J., and Hamer, D. H. (1986). Mammalian metallothionein is functional in yeast. Science 231, 854856.[ISI][Medline]
Unger, M. E., Chen, T. T., Murphy, C. M., Vestling, M. M., Fenselau, C. C., and Roesijadi, G. (1991). Primary structure of molluscan metallothioneins deduced from PCR-amplified DNA and mass spectrometry of purified proteins. Biochim. Biophys. Acta 1074, 371377.[ISI][Medline]
Vallee, B. L. (1995). The function of metallothionein. Neurochem. Int. 27, 2333.[ISI][Medline]
Wang, A. M., Doyle, M. V., and Mark, D. F. (1989). Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. U. S. A. 86, 97179721.[Abstract]
Warren, W. J., and Vella, G. (1993). Analysis of synthetic oligodeoxyribonucleotides by capillary gel electrophoresis and anion-exchange HPLC. Biotechniques 14, 598606.[ISI][Medline]