Quantitative competitive (qc) RT-PCR as a tool in biomarker analysis

All inducible proteins which respond to known pollutants are potential molecular biomarkers. Quantitative competitive (qc) RT-PCR represents a uniquely sensitive tool for measuring the extent of induction of molecular biomarkers such as metallothionein, which is responsive to inducers that include a range of heavy metals. Using the yellowbelly flounder Rhombosolea leporina collected from sites in the Manukau Harbour and Hauraki Gulf (Auckland, New Zealand) as an indicator species, we describe the methodology underpinning the use of qcRT-PCR as a tool in biomarker analysis with reference to the induction of metallothionein. The results show reasonable correlation between the extent of metallothionein induction and the liver burden of Cu and Zn.     

Evidence of a regulatory role of the level of poly (ADP-ribose) in chromosomal proteins in metallothionein gene expression by glucocorticoids but not by heavy metals

The induction capacity of dexamethasone, a synthetic glucocorticoid, for the synthesis of metallothionein was about the same as that of 3-aminobenzamide, which is an inhibitor of ADP-ribosylation of chromosomal proteins, in cultured mouse mammary tumor cells. Both inductions of metallothionein were temporally correlated with a decrease in the amount of endogenous poly (ADP-ribose) on nonhistone high-mobility-group 14 and 17 proteins. In contrast, the extent of cadmium-induced metallothionein synthesis was 2-3-times that of dexamethasone or 3-aminobenzamide. However, cadmium had essentially no effect on de-ADP-ribosylation of these proteins.     

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd,Cu and Pb exposure

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis‐regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full‐length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose‐dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.     

Multiple biomarker response in rainbow trout during exposure to hexavalent chromium

This research investigated hexavalent chromium toxicity in rainbow trout using a panel of biomarkers at different levels of biological organization. A time-course experiment in which rainbow trout were exposed in hard water (63.5 mg/L CaCO3) to a sublethal concentration of hexavalent chromium (10 mg/L) for a period of 28 days was conducted. The responses of multiple biomarkers were measured in gill and liver tissues at varying time points. Significant differences in metallothionein induction, superoxide dismutase activity, lipid peroxidation, cellular morphology, and growth were observed. Results indicated that gill tissues were more sensitive than hepatic tissues to chromium toxicity, yet hepatic tissues appeared to play a larger role in the organism's adaptive response to chromium compared to gill tissues. This study highlights the importance of using a set of integrated biomarkers to assess contaminant exposure and effects.     

Cadmium induces a novel metallothionein and phytochelatin 2 in an aquatic fungus

Cadmium stress response was measured at the thiol peptide level in an aquatic hyphomycete (Heliscus lugdunensis). In liquid culture, 0.1 mM cadmium increased the glutathione (GSH) content and induced the synthesis of additional thiol peptides. HPLC, electrospray ionization mass spectrometry, and Edman degradation confirmed that a novel small metallothionein as well as phytochelatin (PC2) were synthesized. The metallothionein has a high homology to family 8 metallothioneins (http://www.expasy.ch/cgi-bin/lists?metallo.txt). The bonding of at least two cadmium ions to the metallothionein was demonstrated by mass spectrometry (MALDI MS). This is the first time that simultaneous induction of metallothionein and phytochelatin accompanied by an increase in GSH level has been shown in a fungus under cadmium stress, indicating a potential function of these complexing agents for in vivo heavy metal detoxification. The method presented here should be applicable as biomarker tool.     

Bivalve metallothionein as a biomarker of aquatic ecosystem pollution by trace metals: limits and perspectives.

Owing to their induction by metals metallothioneins (MTs) have been proposed as biomarkers of the metallic contamination of the environment. On the other hand, bivalves are regarded as very convenient bioindicators of the aquatic ecosystems and an extensive literature has been dedicated to their response to metals. Among studies supporting the involvement of MTs in metal detoxification some discrepancy appears due to inter- and intra-specific variations, or to heterogeneous exposure conditions. A lesser number of papers are dealing with the use of metallothionein levels as biomarkers, and sometimes they evidence that natural factors influencing metallothionein synthesis have to be taken into account before final conclusions can been drawn. Moreover, there is still a large number of non-intercalibrated protocols used to quantify amounts of metallothioneins in organisms. As comparisons are necessary to assess the relative abundance of metallothioneins in a studied species, more work has to be completed before such comparisons could be validated. In the present paper we wish to establish the limits of the use of mollusc metallothioneins as a biomarker of aquatic ecosystem contamination by trace metals, using published and recent data as support for our conclusions and perspectives.     

Time- and dose-dependent biom arker responses in flounder (Platichthys flesus L.) exposed to benzo[a]pyrene, 2,3,3′,4,4′, 5-hexachlorobiphenyl (PCB-156) and cadmium

Responses in flounder (Platichthys flesus) towards benzo [a]pyrene (BaP), 2,3,3′,4,4′,5-hexachlorobiphenyl (PCB-156), and cadmium (Cd) were investigated in time-course and dose-response studies of selected biomarkers. Measurements of biliary fluorescent BaP metabolites and hepatic concentrations of PCB-156 and cadmium showed that the injected toxicants were rapidly m obilized from the muscle to the liver, but a depot effect was indicated in the highest dose groups of BaP and PCB-156 (12 mg kg^-1 bodyweight). Clearest biomarker responses were found in the induction of hepatic cytochrome P450 1A (CYP1A) enzymes as a response towards BaP and PCB-156 exposure. Maximum induction of CYP1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was observed after 2 and 8 days in BaP and PCB-156-treated flounder, respectively. Positive dose-effect relationships were observed towards both compounds, but the CYP1A induction was more persistent with PCB exposure than with BaP exposure. In Cd-exposed fish, the hepatic level of metallothionein responded more slowly with highest levels observed after 16 days in the time-study. In the combined BaP + Cd treatment, the CYP1A induction was only slightly suppressed. Aspartate aminotransferase in serum appeared to be responsive towards BaP, but also towards the acetone vehicle in controls in the first part of the exposure period. Hematocrit as well as hepatic activities of aldrin epoxidase, glutathione S-transferase, and UDP-glucuronyl transferase were not responsive to any treatm ent in the present study. In general, the results demonstrate that selected biom arkers in flounder are responsive to PAH, PCB, and heavy metal pollutant exposure, indicating the applicability of this species in future environmental pollution monitoring programmes.     

The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location

The promoter region of the Neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (Münger, K., Germann, U. A., and Lerch, K. (1985) EMBO J. 4, 2665-2668). We therefore studied the regulation of expression of the N. crassa metallothionein gene in response to different metal ions (Cu2+, Cd2+, Zn2+, Co2+, and Ni2+) by Northern analysis. Only copper led to the induction of metallothionein mRNA. In N. crassa cultures inoculated and grown in copper-supplemented media, metallothionein mRNA appeared during the late logarithmic growth period (about 30 h after inoculation) and was detectable for a time period of more than 30 h. In response to copper shock, however, rapidly increasing amounts of metallothionein mRNA were detected within minutes after copper administration at any time in vegetatively growing mycelia of N. crassa. Maximum levels were detected about 1 h after addition of copper to the medium. The half-life time of the mRNA was estimated as 2.5 h. The amounts of copper metallothionein reach a maximum level at 3 h after induction and thereafter remain constant. The rapid induction by copper ions of metallothionein mRNA and metallothionein together with the remarkable stability of the native protein intracellularly suggest that this protein serves an important homeostatic role in the copper metabolism in this fungus. The structural gene of N. crassa metallothionein has been located on chromosome VI using restriction fragment-length polymorphisms as genetic markers.     

Comparative proteomics lends insight into genotype‐specific pathogenicity

Comparative proteomic analyses have emerged as a powerful tool for the identification of unique biomarkers and mechanisms of pathogenesis. In this issue of Proteomics, Murugaiyan et al. utilize difference gel electrophoresis (DIGE) to examine differential protein expression between nonpathogenic and pathogenic genotypes of Prototheca zopfii, a causative agent in bovine enteritis and mastitis. Their findings provide insights into molecular mechanisms of infection and evolutionary adaptation of pathogenic genotypes, demonstrating the power of comparative proteomic analyses.     

Effect of growth temperature,induction, and molecular chaperones on the solubilization of over-expressed cellobiose phosphorylase from <Emphasis Type="Italic">Cellvibrio Gilvus</Emphasis> under <Emphasis Type="Italic">in vivo</Emphasis> conditions

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of overexpressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.     

Role of thiocyanate ion in metallothionein induction and in endogenous distribution of essential elements in the rat liver

Thiocyanate is the major toxic metabolite of hydrogen cyanide, a toxic substance the organism may be exposed to as a result of cigarette smoking or industrial pollution. The complex interactions existing between metals and metallothionein induction are well known. However, the possible role of thiocyanate, which is also an anion, has not been established yet. Considering the interactions between metals and the metallothioneins, in this study the relationship between thiocyanate and the in vivo distribution of hepatic metallothionein and zinc, copper, iron, calcium, magnesium, and manganese are investigated in rats. This study implies that thiocyanate has, to some extent, an effect on the in vivo expression of metallothionein and endogenous distribution of essential elements in rat liver. Elevated levels of metallothionein and changes in hepatic concentrations of essential elements have suggested a role for thiocyanate in cellular metabolism and it might reflect a direct role of thiocyanate on alteration of cellular functional activities.     

Differentiating Between Direct (Physiological) and Food-Chain Mediated (Bioenergetic) Effects on Fish in Metal-Impacted Lakes

The objectives of this field project were to test relationships between the physiological and population status of indigenous fish and (a) ecological factors (habitat quality, food resources), (b) toxicological factors (ambient and tissue metal concentrations), and (c) metal detoxification factors (metallothionein induction and subcellular metal partitioning). The sentinel species, yellow perch (YP: Perca flavescens), was collected from lakes with contrasting metal levels located on the Canadian Precambrian Shield, downwind and downstream from metal smelters. In lakes at the high end of our exposure gradient, metals (Cu, Ni, and especially Cd) accumulate in YP to concentrations well above background tissue values; increases in tissue Zn concentrations were much more modest, despite the existence of a very marked gradient in ambient [Zn]. Metal accumulation in YP is accompanied by metallothionein induction, but all evidence to date suggests that metal detoxification by metallothionein is incomplete. Indeed, direct effects of metal toxicity are detected at multiple levels of biological organization, from effects at the cellular level, to effects in organs and tissues, to individuals and populations, in a pattern linked to accumulated metal concentrations (i.e., along the contamination gradient). In addition to direct or physiological effects, we also documented indirect, food-web-mediated effects of metals on YP in the most contaminated lakes. The most common indication of such indirect effects on YP is severely stunted growth coupled with a high degree of zooplankton dependence throughout their life.     

Early biomarkers of cadmium exposure and nephrotoxicity

As the risks of cadmium (Cd)-induced kidney disease have become increasingly apparent, much attention has been focused on the development and use of sensitive biomarkers of Cd nephrotoxicity. The purpose of this review is to briefly summarize the current state of Cd biomarker research. The review includes overviews of the toxicokinetics of Cd, the mechanisms of Cd-induced proximal tubule injury, and mechanistic summaries of some of the biomarkers (N-acetyl-β-d-glucosamidase; β₂-microglubulin, metallothionein, etc.) that have been most widely used in monitoring of human populations for Cd exposure and nephrotoxicity. In addition, several novel biomarkers (kidney injury molecule-1, α-glutathione-S-transferase and insulin) that offer the potential for improved biomonitoring of Cd-exposed populations are discussed.     

Metal-binding proteins of the Syrian hamster ovaries: apparent deficiency of metallothionein

A deficiency of metallothionein, a high-affinity metal-binding protein thought to detoxify cadmium, has been observed in rat and mouse testes, tissues that are highly susceptible to the necrotizing and carcinogenic effects of cadmium. Like the testes, the ovaries undergo a hemorrhagic necrosis when exposed to cadmium, and female Syrian hamsters have recently been shown to be highly susceptible to cadmium. However, the nature of cadmium-binding proteins in the ovary is unknown; thus, this study was undertaken to define the nature of any such proteins in the Syrian hamster ovary. A low molecular weight (Mr) zinc- and cadmium-binding protein was detected in cytosol derived from the ovaries after gel filtration that eluted with a relative elution volume similar to authentic metallothionein. This protein was extractable by heat-treatment and sequential acetone precipitation. When such extracts were further purified with a reverse phase high performance liquid chromatography (HPLC) technique developed for the isolation of metallothionein isoforms, two forms were separated. However, neither of these could be classified as metallothionein on the basis of amino acid composition, since both were particularly low in cysteine, a very common amino acid in metallothionein. The ovarian protein also contained significant amounts of aromatic amino acids, unlike metallothionein--which is devoid of aromatics, and contained much more glutamate than metallothionein. Hamsters were also made resistant to cadmium-induced ovarian necrosis by zinc treatment. Such zinc treatment, however, did not alter levels of this protein, yet caused a marked induction of hepatic metallothionein. Likewise, cadmium treatment did not increase the levels of the ovarian metal-binding protein yet markedly induced hepatic metallothionein.(ABSTRACT TRUNCATED AT 250 WORDS)