Marius Brouwer

Rationale for a new generation of indicators for coastal waters.

More than half the world’s human population lives within 100 km of the coast, and that number is expected to increase by 25% over the next two decades. Consequently, coastal ecosystems are at serious risk. Larger coastal populations and increasing development have led to increased loading of toxic substances, nutrients and pathogens with subsequent algal blooms, hypoxia, beach closures, and damage to coastal fisheries. Recent climate change has led to the rise in sea level with loss of coastal wetlands and saltwater intrusion into coastal aquifers. Coastal resources have traditionally been monitored on a stressor-by-stressor basis such as for nutrient loading or dissolved oxygen. To fully measure the complexities of coastal systems, we must develop a new set of ecologic indicators that span the realm of biological organization from genetic markers to entire ecosystems and are broadly applicable across geographic regions while integrating stressor types. We briefly review recent developments in ecologic indicators and emphasize the need for improvements in understanding of stress–response relationships, contributions of multiple stressors, assessments over different spatial and temporal scales, and reference conditions. We provide two examples of ecologic indicators that can improve our understanding of these inherent problems: a) the use of photopigments as indicators of the interactive effects of nutrients and hydrology, and b) biological community approaches that use multiple taxa to detect effects on ecosystem structure and function. These indicators are essential to measure the condition of coastal resources, to diagnose stressors, to communicate change to the public, and ultimately to protect human health and the quality of the coastal environment.

METAL REGULATION AND MOLTING IN THE BLUE CRAB, CALLINECTES SAPIDUS: METALLOTHIONEIN FUNCTION IN METAL METABOLISM

We recently demonstrated that zinc, copper, and hemocyanin metabolism in the blue crab varies as a function of the molt cycle. To extend these observations, and better delineate metal metabolism in marine crustaceans, we have conducted experiments to determine if environmental temperature and season of the year affect concentrations of hemocyanin and copper in the hemolymph and copper and zinc in the digestive gland. Overwintering, cold water crabs (6°C) had decreased hemocyanin and copper in the hemolymph and normal zinc and copper in the digestive gland with respect to summer crabs collected at 20-30°C. When these crabs were warmed to 20°C and fed fish for three weeks, they showed increases in the concentrations of copper in the digestive gland, and copper and hemocyanin in the hemolymph. In addition, a change from a zinc to a copper-dominated metallothionein was found in a majority of the warmed crabs, suggesting the involvement of copper metallothionein in the resynthesis of hemocyanin. Based on these observations and previous data (Engel, 1987) a conceptual model of copper and zinc partitioning in the blue crab has been constructed. In this model, metallothionein has an important role in metal regulation both during molting and in the changes related to season of the year. Metallothionein-bound copper and zinc appear to be regulated at the cellular level for the synthesis of metalloproteins, such as hemocyanin (copper) and carbonic anhydrase (zinc), both of which are necessary for normal growth and survival. Finally, we present evidence showing that copper metallothionein can directly transfer its metal to the active site of apohemocyanin. Copper insertion seems to precede the formation of viable oxygen binding sites.

Replacement of a cytosolic copper/zinc superoxide dismutase by a novel cytosolic manganese superoxide dismutase in crustaceans that use copper (haemocyanin) for oxygen transport

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.

Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus

The role of copper metallothionein (CuMT) in copper metabolism and metalloenzyme activation is poorly understood. We have chosen marine crustaceans, in which a direct correlation exists between levels of Cu(I)MT and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-251, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homarus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclear-copper sites, which subsequently slowly return to their native stereochemical configuration. Finally, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.

Role of a copper-specific metallothionein of the blue crab, Callinectes sapidus, in copper metabolism associated with degradation and synthesis of hemocyanin

We have identified three MT encoding genes in the blue crab: MT-I, inducible by cadmium, zinc and copper; MT-II, inducible by cadmium and zinc; and MT-III, inducible by copper only [Syring et al., Comp. Biochem. Physiol. C, 125 (2000) 325-332]. To examine the role of the CuMT-I and CuMT-III isoforms in copper metabolism associated with the synthesis and degradation of the oxygen-binding copper protein, hemocyanin, we (1) cloned and sequenced hemocyanin cDNA, (2) examined interaction of the CuMTs with endoplasmic reticulum (ER) vesicles and (3) measured changes in levels of hemocyanin, MT-I, MT-III protein and mRNA that occur in crabs during different stages of the molt cycle. The cDNA-derived hemocyanin amino-acid sequence revealed the presence of a leader peptide indicating that hemocyanin is a secretory protein that is synthesized on the ER. Copper uptake studies show that ER vesicles take up both Cu1+ and Cu2+ in an ATP-independent process. The copper transporter has a Km of 10.8+/-2.4 microM copper and a Vmax of 6.1+/-0.5 nmol Cu/mg protein/10 min. ER vesicles contain hemocyanin, and bind CuMT-I and, preferentially, CuMT-III. However, binding does not result in copper transfer to the ER. There are statistically significant changes in hepatopancreas MT-III and hemocyanin mRNA, and in hemolymph hemocyanin concentrations during the molt cycle. MT-I mRNA remains constant. Changes in MT-III mRNA are positively correlated with changes in hemocyanin mRNA and hemocyanin protein, which points to coordinate control of MT-III and hemocyanin transcription. No CuMT-III protein is observed in hepatopancreas of intermolt crabs when levels of both MT-III and hemocyanin mRNA are high, suggesting rapid utilization of copper bound to MT-III when cells are actively synthesizing hemocyanin. CuMT-III is present in premolt and softshell crabs, and its emergence appears to coincide with a decrease in hemocyanin synthesis and increase in hemocyanin degradation. These results support the hypothesis that the copper-specific metallothionein is intricately involved in copper homeostasis associated with both the synthesis and degradation of hemocyanin.

Effects of chronic nanoparticulate silver exposure to adult and juvenile sheepshead minnows (Cyprinodon variegatus).

The use of nanoparticulate silver (AgNP) is increasingly widespread and recently has been shown to have a plausible release route into aquatic environments. To date, relatively little research has examined the effects of AgNP on estuarine fish. The authors present data indicating that chronic exposure to low levels of AgNP induces significant adverse effects in both juvenile and adult sheepshead minnows (Cyprinodon variegarus; SHMs). Chronic exposure to low levels of AgNP produced significant increases in tissue burdens in both juvenile and adult SHMs, resulting in significant thickening of epithelia gill tissue and in dramatically altered gene expression profiles. The results do not appear to be attributable to the release of silver ions through particle dissolution. The alteration in gene expression was greatest in adult gonads, but no evidence of AgNP-related dysfunction was found at the tissue level. In contrast, the authors found a significant effect on gill morphology, but very little evidence of effect on gill transcription profiles.

Trace metal-binding proteins in marine molluscs and crustaceans

Some marine invertebrates, such as the American oyster, Crassostrea virginica, the blue crab, callinectes sapidus and the American lobster, Homarusamericanus, concentrate trace metals in their tissues. The occurrence of metallothionein, a low molecular weight, sulfur-containing metal-binding protein, has been correlated with elevated levels of trace metals in these organisms. It is our hypothesis that, whilst metallothioneins are involved in the sequestration of elevated levels of trace metals, they primarily function in regulating normal metal metabolism. In this paper we describe recent field and laboratory experiments designed to examine how oysters, blue crabs and lobsters manage accumulated cadmium, copper and zinc. The possible roles of metallothionein, as well as the tissue distribution of metals, are emphasized. Metallothioneins were found in all animals we examined; concentrations of metallothioneins and the the amount of bound trace metals are related to time of exposure. We have also demonstrated that the laboratory developed metallothionein models for blue crabs and lobsters may be useful in predicting the partitioning of trace metals in animals living in environments polluted with trace metals.

Metal-specific induction of metallothionein isoforms in the blue crab Callinectes sapidus in response to single- and mixed-metal exposure

Metallothioneins (MTs) play an important role in the metabolism of copper and zinc during the molt cycle of the blue crab. In this study we examined the hypothesis that MT expression in crabs is metal specific. Anion-exchange chromatography showed one major ZnCuMT (ZnMT-I) in control crabs, two MT isoforms in cadmium-treated crabs (CdMT-I, CdMT-II), and three forms (CuMT-I, CuMT-II, CuMT-III) in copper-treated animals. Amino acid analysis of the carboxymethylated apo-MTs, purified by reversed-phase HPLC, showed minor differences between ZnMT-I, CdMT-I, CdMT-II, CuMT-I, and CuMT-II, while CuMT-III was unique. After combined exposure to cadmium and copper, four MTs with differing copper/cadmium ratios were observed, equivalent to CdMT-II and the three CuMTs. We conclude that the blue crab has four genes that encode different MTs. Transcription of the CdMT-I gene is induced by cadmium, but inhibited by copper. CuMT-I, CdMT-II, and ZnMT-I may be the products of a single gene responsive to copper, cadmium, and zinc. Expression of the CuMT-II and CuMT-III genes is initiated by copper and not by cadmium. We believe that CdMT-I and CuMT-III are important in detoxification, whereas Zn/CuMT-I and CuMT-II are involved in regulatory functions. These results show the importance of the use of mixed-metal exposures in the study of the molecular mechanisms of metal regulation and function.

Brain aromatase in Japanese medaka (Oryzias latipes): Molecular characterization and role in xenoestrogen-induced sex reversal

In female fish estrogen is required for the development of primary and secondary sex characteristics and is derived from the aromatization of androgens by aromatase. There are two isoforms of aromatase in several teleost species, brain and ovarian. The objective of this study was two-fold: clone and sequence the coding and promoter region of brain aromatase in medaka, and determine the effects of exposure to an environmental estrogen (o,p-DDT) on sex determination and brain aromatase transcription and activity. The brain aromatase coding sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR-based genomic DNA walking was used to clone the promoter of the brain aromatase gene. The promoter sequence revealed potential binding sites for the estrogen receptor and for transcription factors involved in primary neurogenesis and sex determination. Medaka fry were exposed to increasing o,p-DDT concentrations (0-5.5 microg/L) from days 1 to 15 after hatch and brain aromatase expression and activity were measured on days 5, 9, and 14. A complete male-to-female sex reversal occurred at 5.5 microg/L o,p-DDT and aromatase activity and expression data showed a significant five-fold increase at this concentration at day 14. This information suggests that brain aromatase is involved in the abnormal sexual differentiation of fish treated with xenoestrogens.

Metallothionein and Metallothionein-Like Proteins: Physiological Importance

Since the initial demonstration of a low-molecular weight cadmium-binding protein in mammals, the research on the function of these proteins, which also can bind Cu, Zn, and Hg, has been focused primarily on their detoxifying properties. Such a direction is understandable, since the synthesis of the proteins can be induced by these potentially toxic metal ions. These proteins, isolated from mammals, have structural characteristics that are very different from those of most proteins, such as having 30% of the amino acid residues as cysteine and having the capacity to bind 7 mol of cadmium, zinc, or mercury or 12 mol copper per mole of protein. The binding of metal to these proteins is through thiol bonding, and therefore, the name metallothionein was proposed and accepted (throughout the text metallothionein will be denoted as MT).