Christer Hogstrand

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

The biotic ligand model: a historical overview

During recent years, the biotic ligand model (BLM) has been proposed as a tool to evaluate quantitatively the manner in which water chemistry affects the speciation and biological availability of metals in aquatic systems. This is an important consideration because it is the bioavailability and bioreactivity of metals that control their potential to cause adverse effects. The BLM approach has gained widespread interest amongst the scientific, regulated and regulatory communities because of its potential for use in developing water quality criteria (WQC) and in performing aquatic risk assessments for metals. Specifically, the BLM does this in a way that considers the important influences of site-specific water quality. This journal issue includes papers that describe recent advances with regard to the development of the BLM approach. Here, the current status of the BLM development effort is described in the context of the longer-term history of advances in the understanding of metal interactions in the environment upon which the BLM is based. Early developments in the aquatic chemistry of metals, the physiology of aquatic organisms and aquatic toxicology are reviewed first, and the degree to which each of these disciplines influenced the development of water quality regulations is discussed. The early scientific advances that took place in each of these fields were not well coordinated, making it difficult for regulatory authorities to take full advantage of the potential utility of what had been learned. However, this has now changed, with the BLM serving as a useful interface amongst these scientific disciplines, and within the regulatory arena as well. The more recent events that have led to the present situation are reviewed, and consideration is given to some of the future needs and developments related to the BLM that are envisioned. The research results that are described in the papers found in this journal issue represent a distinct milestone in the ongoing evolution of the BLM approach and, more generally, of approaches to performing ecological assessments for metals in aquatic systems. These papers also establish a benchmark to which future scientific and regulatory developments can be compared. Finally, they demonstrate the importance and usefulness of the concept of bioavailability and of evaluative tools such as the BLM.

The physiology of waterborne silver toxicity in freshwater rainbow trout (Oncorhynchus mykiss) 1. The effects of ionic Ag

Abstract Adult rainbow trout, fitted with arterial catheters, were exposed to AgNO3 for 6 days at a concentration (10 μg Ag l−1, flow-through) close to the 7 day LC50 in moderately hard freshwater. Approximately 35% of total Ag occurred as free ionic Ag+, and the remainder as silver chlorides. Ag accumulated on the gills and increased about 4-fold above control levels in blood plasma, stabilizing by 48 h. Much greater concentrations of Ag accumulated in the liver, but not the kidney, at 6 days. Metallothionein induction did not occur. Plasma [Na+] and [Cl−] declined steadily to 70% of control levels by day 6, accompanied by a progressive metabolic acidosis, a 5-fold increase in blood [glucose], a 40% decrease in relative plasma volume, contraction of the spleen, and marked hemoconcentration. Plasma [Ca2+] and [K+] were largely unaffected. Respiratory suffocation did not occur: plasma [lactate] remained constant, arterial PO2 increased and PCO2 decreased, the latter compensating the metabolic acidosis so arterial pH fell only moderately. Comparably sampled control fish exhibited negligible disturbance. Unidirectional Na+ influx from the water, measured in juvenile trout, was inhibited by 42% immediately, and abolished by 48 h of AgNO3 exposure. These symptoms suggested a similar toxic mechanism of action to that of low environmental pH. We speculate that Ag+ interferes with net Na+ and Cl− uptake at the gills, and causes death by secondary fluid volume disturbance, hemoconcentration, and eventual cardiovascular collapse.

Zinc transporters and cancer: a potential role for ZIP7 as a hub for tyrosine kinase activation

Zinc, which is essential for many cellular processes, is controlled by zinc transporters and through buffering by metallothioneins and glutathione. Although zinc is increasingly implicated in disease states, little is known about how zinc regulates cellular biochemical pathways. Recent seminal articles have revealed discrete zinc-trafficking pathways that are linked to signalling cascades, particularly those involving protein phosphatase inhibition and downstream activation of mitogen-activated protein kinases and tyrosine kinases. Here, we discuss the mechanisms of cellular zinc homeostasis, and we propose an important role for the zinc transporter solute carrier family 39, member 7 (SLC39A7; commonly referred to as ZIP7). ZIP7 releases zinc from the endoplasmic reticulum and might be required for tyrosine kinase activation. These observations position ZIP7 at a critical node in zinc-mediated tyrosine kinase signalling and suggest that this protein might form a novel target for diseases such as cancer where prevention of tyrosine kinase activation would be therapeutically advantageous.

Protein Kinase CK2 Triggers Cytosolic Zinc Signaling Pathways by Phosphorylation of Zinc Channel ZIP7

Phosphorylation of the zinc channel ZIP7 releases zinc from the endoplasmic reticulum and promotes signals associated with proliferation and migration. Regulating Zinc Signals Zinc, which has emerged as a second messenger, is released from intracellular stores, leading to activation of tyrosine kinases and thereby to stimulation of pathways involved in cell proliferation and migration. Here, Taylor et al. uncover evidence implicating the kinase CK2, which plays a prominent role in cell proliferation, in this process through its association with and phosphorylation of the endoplasmic reticulum–resident zinc channel ZIP7. Inhibition of CK2 attenuated zinc release from the endoplasmic reticulum and downstream signals. A functional mutation of ZIP7 that interfered with CK2-ZIP7 interactions prevented ZIP7-mediated zinc signals, decreased downstream signaling events, and attenuated ZIP7-mediated cell migration. Thus, the authors conclude that CK2 plays a crucial role in the activation of ZIP7 and thereby in mediating zinc signals. The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP (ZRT1- and IRT1-like protein) family [also known as solute carrier family 39A (SLC39A)] transiently increase the cytosolic free zinc (Zn2+) concentration in response to extracellular signals. We show that phosphorylation of evolutionarily conserved residues in endoplasmic reticulum zinc channel ZIP7 is associated with the gated release of Zn2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal–regulated kinases 1 and 2. Through pharmacological manipulation, proximity ligation assay, and mutagenesis, we identified protein kinase CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that transition element channels in eukaryotes can be activated posttranslationally by phosphorylation, as part of a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals in these events. The connection with proliferation and migration, as well as the activation of ZIP7 by CK2, a kinase that is antiapoptotic and promotes cell division, suggests that ZIP7 may provide a target for anticancer drug development.

Application of genomics and proteomics for study of the integrated response to zinc exposure in a non-model fish species, the rainbow trout.

The advent of DNA array technology and proteomics has revolutionised biology by allowing global analysis of cellular events. So far, the benefits from these new techniques have primarily been realised for well-characterised species. These organisms are rarely the most relevant for environmental biology and ecotoxicology. Thus, there is a need to explore new ways to exploit transcriptomics and proteomics for non-model species. In the present study, rainbow trout (Oncorhynchus mykiss) were exposed to a sublethal concentration of waterborne zinc for up to 6 days. The response in gill tissue was investigated by differential screening of a heterologous cDNA array and by protein profiling using Surface Enhanced Laser Desorption/Ionisation (SELDI). The cDNA array, which was a high-density spotted library of cDNA from Fugu rubripes gill, revealed differentially expressed genes related to energy production, protein synthesis, paracellular integrity, and inflammatory response. SELDI analysis yielded seven proteins that were consistently present only in zinc-exposed gills, and four proteins unique to gills from control fish. A further 11 proteins were differentially regulated. Identification of these proteins by bioinformatics proved difficult in spite of detailed information on molecular mass, charge and zinc-binding affinity. It is concluded that these approaches are viable to non-model species although both have clear limitations.

Relationship between copper exposure duration, tissue copper concentration, and rainbow trout growth

Abstract Rainbow trout fry were exposed in soft water to sublethal concentrations of copper for 60 days under controlled laboratory conditions. At 20-day intervals, fish were sampled for weight, length, and whole-body copper and metallothionein concentrations. Exposures to waterborne copper concentrations as low as 4.6 μg 1−1 resulted in significantly reduced growth and significantly elevated whole-body copper concentrations after 20 days. Whole-body metallothionein concentrations did not differ significantly from controls. Fish did not recover or return to control growth rates throughout the entire exposure period; a 45% reduction in mean weight relative to controls observed on day 40 in the 9.0 μg 1−1 Cu exposure was sustained through day 60. Whole-body accumulation rates of copper in fish exposed to 4.6 μg 1−1 and higher levels of Cu increased significantly between 0 and 40 days and appeared to reach steady-state after 40 days. Copper accumulation was found to depend on dose and time. Trout exposed to higher copper concentrations accumulated more whole-body copper, with longer times to reach steady-state. Our data suggest that both accumulation capacity and copper depuration rates from a slowly exchangeable pool are concentraion-dependent. A linear model was developed for the relationship between exposure duration, copper accumulation, and fish weight: In (wet wt., μg) = 4.8 + 0.03 (exposure duration, days)—0.04 (whole-body copper, mg g−1 dry wt.) (P

Mechanisms of heavy metal accumulation and toxicity in fish

As a result of mining, forestry, waste disposal and fuel combustion, our environment is becoming increasingly contaminated with heavy metals. The aquatic environment receives waste products from such activities and may be the final depository for these anthropogenically remobilized heavy metals. In order to understand the impact of heavy metals on aquatic biota it is important to characterize the mechanisms available for aquatic life to transport, immobilize and excrete heavy metals.

Picomolar Concentrations of Free Zinc(II) Ions Regulate Receptor Protein-tyrosine Phosphatase β Activity

Background: Protein-tyrosine phosphatases (PTPs) are critical regulators of phosphorylation signaling. Results: Zinc(II) ions are reversible inhibitors of receptor PTPβ with a remarkably low Ki of 21 ± 7 pm. Conclusion: The inhibition implicates cellular zinc(II) ions as physiological regulators of PTPs. Significance: The results suggest that zinc modulates signal transduction in endothelial cells affecting angiogenesis and wound healing. As key enzymes in the regulation of biological phosphorylations, protein-tyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc(II) ions are known to inhibit these enzymes, but the physiological significance of this inhibition has remained elusive. Employing metal buffering for strict metal control and performing a kinetic analysis, we now demonstrate that zinc(II) ions are reversible inhibitors of the cytoplasmic catalytic domain of the receptor protein-tyrosine phosphatase β (also known as vascular endothelial protein-tyrosine phosphatase). The Ki(Zn) value is 21 ± 7 pm, 6 orders of magnitude lower than zinc inhibition reported previously for this enzyme. It exceeds the affinity of the most potent synthetic small molecule inhibitors targeting these enzymes. Inhibition is in the range of cellular zinc(II) ion concentrations, suggesting that zinc regulates this enzyme, which is involved in vascular physiology and angiogenesis. Thus, for some enzymes that are not recognized as zinc metalloenzymes, zinc binding inhibits rather than activates as in classical zinc enzymes. Activation then requires removal of the inhibitory zinc.

Renal Cu and Na excretion and hepatic Cu metabolism in both Cu acclimated and non acclimated rainbow trout (Oncorhynchus mykiss)

Abstract 64Cu and total Cu accumulation were measured in gills, plasma, liver, kidney, bile and urine during 72 h of exposure to 64Cu at 20 μg Cu l−1, in non-acclimated and Cu-acclimated (28 days of pre-exposure) rainbow trout (Oncorhynchus mykiss) fitted with urinary bladder catheters. Renal Cu excretion gradually declined from 0.03 μg Cu kg−1 h−1 in non-exposed fish to 0.01 μg Cu kg−1 h−1 after 28 days of Cu exposure. A comparison of the 64Cu-labelled Cu and the total Cu excretion rates and the corresponding renal clearance revealed apparent differences in Cu binding to plasma protein depending on whether the Cu is derived from recent branchial uptake or is already present in the plasma prior to 64Cu exposure. The plasma Cu pool derived from recent branchial uptake and the Cu pool present in the plasma prior to 64Cu exposure is accessible to renal excretion to different extents, whereas the pools seem equally accessible to hepatic accumulation and elimination. The renal Cu excretion is of minor importance compared with the hepatic Cu excretion, which was estimated to be 0.5–0.75 μg Cu kg−1 h−1 and 1.1–1.6 μg Cu kg−1 h−1 for non-acclimated and Cu-acclimated fish, respectively. Based on the biliary Cu concentration, hepatic Cu elimination appeared to be stimulated in the Cu-acclimated relative to the non-acclimated fish. Only 17% and 12% of the hepatic Cu could be accounted for by metallothionein in the control and Cu-acclimated fish, respectively. Renal Na+ efflux decreased by 40%, which was largely due to increased tubular Na+ reabsorption. Renal compensation for the impaired branchial Na+ uptake, seen during Cu exposure, thus seems to be involved in Cu acclimation in rainbow trout.