Biomonitoring Heavy Metal Pollution Using an Aquatic Apex Predator,the American Alligator,and Its Parasites

Monitoring the bioaccumulation of chemical elements within various organismal tissues has become a useful tool to survey current or chronic levels of heavy metal exposure within an environment. In this study, we compared the bioaccumulations of As, Cd, Cu, Fe, Pb, Se, and Zn between the American alligator, Alligator mississippiensis, and its parasites in order to establish their use as bioindicators of heavy metal pollution. Concomitant with these results, we were interested to determine if parasites were more sensitive bioindicators of heavy metals relative to alligators. We found parasites collectively accumulated higher levels of As, Cu, Se, and Zn in comparison to their alligator hosts, whereas Fe, Cd, and Pb concentrations were higher in alligators. Interestingly, Fe levels were significantly greater in intestinal trematodes than their alligator hosts when analyzed independently from other parasitic taxa. Further analyses showed alligator intestinal trematodes concentrated As, Cu, Fe, Se, and Zn at significantly higher levels than intestinal nematodes and parasites from other organs. However, pentastomids also employed the role as a good biomagnifier of As. Interestingly, parasitic abundance decreased as levels of As increased. Stomach and intestinal nematodes were the poorest bioaccumulators of metals, yet stomach nematodes showed their ability to concentrate Pb at orders of magnitude higher in comparison to other parasites. Conclusively, we suggest that parasites, particularly intestinal trematodes, are superior biomagnifiers of As, Cu, Se, and Zn, whereas alligators are likely good biological indicators of Fe, Cd, and Pb levels within the environment.     

Sequential changes in Fe,Cu, and Zn in target organs during early Coxsackievirus B3 infection in mice

In Coxsackievirus B3 (CB3) infection, the heart and pancreas are major target organs and, as a general host response, an associated immune activation and acute phase reaction develops. Although iron (Fe), copper (Cu), and zinc (Zn) are involved in these responses, sequential trace element changes in different target organs of infection have not been studied to date. In the present study, Fe, Cu, and Zn were measured through inductively coupled plasma mass spectrometry (ICP-MS) in the plasma, liver, spleen, heart, and pancreas during the early phase (d 1 and 3) of CB3 infection in female Balb/c mice. The severity of the infection was assessed through clinical signs of disease and histopathology of the heart and pancreas, including staining of CD4 and CD8 cells in the pancreas. During infection, the concentrations of Fe, Cu, and Zn changed in the plasma, liver, and pancreas, but not in the spleen and heart. The changes in plasma Cu, Zn, and Fe seemed to be biphasic with a decrease at d 1 that turned into increased levels by d 3. Cu showed similar biphasic changes in the liver, spleen, and pancreas, whereas, for Zn and Fe, this pattern was only evident in the liver. In the pancreas, the reverse response occurred with pronounced decreases in Fe (23%, p < 0.05) and Zn (64%, p < 0.01) at d 3. Although the pathophysiological interpretation of these findings requires further research, the sequential determination of these elements may be of clinical value in enterovirus infections in deciding the stage of disease development.     

Serum and tissue trace elements in patients with breast cancer in Taiwan

The objective of this study was to compare levels of four elements (zinc, copper, selenium, and iron) in the serum and tissue of 68 breast tumor patients (benign and malignant), from a teaching hospital in central Taiwan. Samples of normal tissue (5 cm away from tumor) were also taken from patients with malignant tumors. Only serum was taken from the 25 healthy persons in the control group. Results showed that Zn, Cu, Se, Fe, Cu/Zn, Cu/Se, and Cu/Fe were present in different amounts in the serum of each of the three groups. Zn and Se levels were lower in the serum of the two tumor groups compared to the control group. In tissue samples, Zn, Cu, Se, and Fe concentrations were different in each of the three groups. The malignant tissue had the highest levels of all four elements. In advanced-stage malignant tumors, levels of Cu and the ratios of Cu/Fe and Cu/Zn (in both serum and tissue) were highest. The ratios of serum Cu/Zn, Cu/Fe, and Cu/Se were also higher in malignant patients. The cutoff value of serum Cu/Zn was 1.2 (sensitivity and specificity were both 100%). The Cu/Zn ratio was highest in the advanced stages of cancer and was a better diagnostic tool for breast cancer than Cu/Se and Cu/Fe. The authors suggest that change of trace elements in serum and tissue might be useful and significant as biomarkers involving the initial plastic process.     

Stress-induced suppression of testosterone secretion in male alligators

In order to test the effect of acute stress on gonadal hormone secretion in reptiles, six mature male alligators were captured, and a blood sample was taken within 5 min of capture. Additional blood samples were taken at timed intervals for up to 41 hr, and plasma testosterone and corticosterone were measured by radioimmunoassay. Plasma testosterone declined to 50% of the initial value by 4 hr and dropped to less than 10% of initial by 24 hr. Plasma corticosterone increased during the first 12 hr, declined at 24 hr, and rose again at 40 hr. Blood samples from male alligators collected in North and South Carolina, south Florida, and in south Louisiana in two consecutive breeding seasons were also assayed for testosterone and corticosterone. In these populations there were significant differences in mean plasma testosterone and corticosterone levels. Elevated corticosterone levels were consistently seen in alligators caught in traps and from which a blood sample was taken several hours later. Plasma testosterone, although consistently lower in trapped alligators, did not show a negative correlation with plasma corticosterone. Farm-reared alligators bled once, released, and bled again at 24 hr also showed a highly significant suppression of testosterone secretion. These results demonstrate that stress has a rapid and dramatic effect on testicular steroid secretion in both farm-reared and wild alligators.     

Changes in Trace Metals in Hemolymph of Baculovirus-Infected Noctuid Larvae

We studied how biologically relevant trace metals (i.e., micronutrients) in the hemolymph of larval Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of hemolymph Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, and Zn were measured using inductively coupled plasma-mass spectrometry. H. virescens larvae exhibited greater fluctuations in hemolymph trace metal levels in response to baculovirus infection and development than did H. zea larvae. H. zea single nucleopolyhedrosis virus infection significantly altered the levels of Cu, Fe, Mg, Mn, Mo, and Zn in fourth instar H. virescens larvae. Conversely, in fifth instar H. virescens and both H. zea instar infections, no metal levels were significantly different between infected and uninfected larvae. In fourth instar H. virescens hemolymph, Cu, Fe, Mo, and Zn increased during development. Cu, Fe, Mg, Mn, Mo, and Zn levels changed significantly during development in fifth instar H. virescens as well as both H. zea instars. Based on this analysis, metals were identified whose levels changed during development in both species and during the immune response of H. virescens larvae.     

Serum Levels of Trace Elements and Heavy Metals in Patients with Acute Hemorrhagic Stroke

Trace elements are essential components of biological structures, but alternatively, they can be toxic at concentrations beyond those necessary for their biological functions. Changes in the concentration of essential trace elements and heavy metals may affect acute hemorrhagic stroke. The aim of this study was to measure serum levels of essential trace elements [iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and magnesium (Mg)] and heavy metals [cobalt (Co), cadmium (Cd), and lead (Pb)] in patients with acute hemorrhagic stroke. Twenty-six patients with acute hemorrhagic stroke and 29 healthy controls were enrolled. Atomic absorption spectrophotometry (UNICAM-929) was used to measure serum Fe, Cu, Pb, Cd, Zn, Co, Mn and Mg concentrations. Serum Cd, Pb and Fe levels were significantly higher in patients with acute hemorrhagic stroke than controls (p < 0.001), while serum Cu, Zn, Mg and Mn levels were significantly lower (all p < 0.001). However, there was no significant difference between the groups with respect to serum Co levels (p > 0.05). We first demonstrate increased Cd, Pb, and Fe levels; and decreased Cu, Zn, Mg, and Mn levels in patients with acute hemorrhagic stroke. These findings may have diagnostic and prognostic value for acute hemorrhagic stroke. Further studies are required to elucidate the roles of trace elements and heavy metals in patients with acute hemorrhagic stroke.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long-Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long–Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Application of trace metal analysis to basic problems in neurobiology studies of patients with Reye’s syndrome

Reye’s syndrome (Rs) is an acute illness in children manifested by encephalopathy and fatty degeneration of the liver. The syndrome may be secondary to injury of mitochondria following a toxic insult in a susceptible individual with a viral illness. Since the response to infection often involves a change in trace metals, we investigated the metal status of patients with Rs. Decreased levels of serum and liver selenium (Se) and copper (Cu) were demonstrated via PIXE analysis, in addition to an increase in serum iron (Fe) and zinc (Zn). In a subsequent study using a rat animal model of Rs, the hepatotoxin 4-pentenoic acid (4-PA) produced similar changes in serum and liver trace metals. Serum and liver Se levels were also significantly depleted in rats exposed to another toxin, valproic acid (VPA). Aspirin, known to complex metals, may also be associated with Rs. Rats chronically exposed to aspirin had decreased serum Se, Fe, and Zn compared to controls. Selenium was also decreased in liver, as was Cu. Atomic absorption spectrophotometric analysis of serum and liver Cu for mice exposed to aspirin and influenza A virus were also studied. In liver, Cu was significantly decreased in mice on Cu-deficient diets but, not in control mice exposed to virus, or aspirin and virus. For the Se-deficient animals, liver Cu was not different from controls, but there was an increase in tissue Cu for Se-sufficient mice exposed to virus and aspirin; Cu levels were decreased in sera of this latter group. Serum Cu was increased in Cu-sufficient mice exposed to virus and aspirin. The above data are of biologic and toxicologic interest because of metalloenzyme localization in the mitochondrial matrix, the cellular compartment showing the greatest degree of pathologic change in Rs. In particular, Se-dependent gluthathione peroxidase is a major deterent of peroxidative damage of lipid membranes. The accumulated evidence suggests that alteration of trace metals, e.g., decrease in Se, may promote peroxidation of mitochondrial membranes in patients with Rs.     

Zinc is a constituent of bovine heart cytochrome c oxidase preparations

Cytochrome c oxidase preparations from bovine heart muscle contain 1 zinc per 2 irons. Metal contents of nine preparations determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) show that Cu, Fe and Zn are the only metals present in significant amounts with average Cu/Fe, Fe/Zn, and Cu/Zn atom ratios of 1.3, 2.1 and 2.8, respectively. Removal of adventitious copper results in a Cu:Fe:Zn stoichiometry of 2:2:1. The zinc is tightly bound. Dialysis against a solution of 1,10-phenanthroline at pH 7.4 or an acidic buffer (pH 4.4) does not remove Zn. Dialysis against 0.8 M KCN at pH 10 causes partial loss of both Cu and Zn. This is the first evidence for the presence of Zn in a cytochrome c oxidase.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.     

Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography–inductively coupled plasma atomic emission spectroscopy

Analytical methods which are capable of determining the plasma or serum metalloproteome have inherent diagnostic value for human diseases associated with increased or decreased concentrations of specific plasma metalloproteins. We have therefore systematically developed a method to rapidly determine the major Cu-, Fe-, and Zn-containing metalloproteins in rabbit plasma (0.5 mL) based on size-exclusion chromatography (SEC; stationary phase Superdex 200, mobile phase phosphate-buffered saline pH 7.4) and the simultaneous online detection of Cu, Fe, and Zn in the column effluent by an inductively coupled plasma atomic emission spectrometer (ICP-AES). Whereas most previous studies reported on the analysis of serum, our investigations clearly demonstrated that the analysis of plasma within 30 min of collection results in the detection of one more Cu peak (blood coagulation factor V) than has been previously reported (transcuprein, ceruloplasmin, albumin-bound Cu, and small molecular weight Cu). The average amount of Cu associated with these five proteins corresponded to 21, 18, 21, 30 and 10% of total plasma Cu, respectively. In contrast, only two Fe metalloproteins (ferritin and transferrin, corresponding to an average of 9 and 91% of total plasma Fe) and approximately five Zn metalloproteins (α₂-macroglobulin and albumin-bound Zn, which corresponded to an average of plasma Zn) were detected. Metalloproteins were assigned on the basis of the coelution of the corresponding metal and protein identified by immunoassays or activity-based enzyme assays. The SEC-ICP-AES approach developed allowed the determination of approximately 12 Cu, Fe, and Zn metalloproteins in rabbit plasma within approximately 24 min and can be applied to analyze human plasma, which is potentially useful for diagnosing Cu-, Fe-, and Zn-related diseases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Parts of the work described in this paper were presented at HPLC 2007 in Ghent, Belgium. An erratum to this article can be found at     

The role of trace elements on hepatitis virus infections: a review

The significance of the nutritional roles of trace metals (includes some heavy metals) is widely recognized, since these elements are as constituent components of many metal proteins and metalloenzymes serum trace metals levels, and their ratios are frequently reported to be good marker for diagnosing various diseases. Trace metals play an important role in liver disease particularly liver degeneration. Influence of trace elements has been studied in a large number of viruses belonging to different groups. This review reported the role of some trace elements iron (Fe), copper (Cu), cobalt (Co), manganese (Mn) and zinc (Zn) as well as toxic elements Pb on hepatitis virus infections.     

Heavy Metal Contamination of Soil,Plant and Air of Scrapyard of Discarded Vehicles at Zarqa City,Jordan

Ninety soil samples, forty plant samples (Anabasis articulata), and twenty air samples were collected from the scrap yard of discarded vehicles near Zarqa city, Jordan. These samples were analyzed for heavy metals: Cd, Pb, Zn, Cu, Mn, Al, and Fe. Longitudinal and vertical profiles of soil samples were studied. Generally, the levels of all heavy metals studied in the scrap yard area were found to be higher than those of the control samples. The levels of heavy metals decreased with depth until reaching a constant value at 9 cm depth. The levels of heavy metals also decreased at distances farther away from the scrap yard area. A significant difference in heavy metal concentrations was found between washed and unwashed plant samples. On the other hand, no significant differences have been found between plant samples from inside and outside the scrap yard area. Air samples showed wide variations in heavy metal levels among the sampling sites. The enrichment factors for non-crustal elements such as Pb, Cd, Cu, and Zn, in both soil and particulate matter, were found to be more than 10, indicating anthropogenic sources such as dust, rust, and exhaust emissions from the scrap yard area, whereas the crustal elements such as Fe and Mn showed enrichment factors of less than 10.     

Alterations of serum selenium, zinc, copper, and iron concentrations and some related antioxidant enzyme activities in patients with cutaneous leishmaniasis

The aim of this study was to measure the alterations in serum selenium (Se), copper (Cu), zinc (Zn), and iron (Fe) concentrations and their carrier proteins, ceruloplasmin (Cp), transferrin (Tf) albumin, and related antioxidant enzyme activities, erythrocyte Cu-Zn Superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities in patients with cutaneous leishmaniasis (CL). Erythrocyte Cu-Zn SOD activities, serum Cu concentrations, and Cp levels were found to be significantly higher in the patients group than those of controls. However, GSH-Px and CAT activities and Se, Zn, Fe, and Tf levels were lower in patients than in the control subjects. There were positive important correlation’s between Cu-Zn SOD and Cp, Cu-Zn SOD and Cu, Cp and Cu, GSH-Px and Se, and Fe and CAT in the patients group. Our results showed that serum essential trace elements Se, Zn, Cu, and Fe concentrations and their related enzymes Cu-Zn SOD, GSH-Px, and CAT activities change in CL patients. The changes may be a part of defense strategies of organism and are induced by the hormonelike substances.     

Inhibition of iron and copper uptake by iron, copper and zinc

Interactions of micronutrients can affect absorption and bioavailability of other nutrients by a number of mechanisms. In aqueous solutions, and at higher uptake levels, competition between elements with similar chemical characteristics and uptake process can take place. The consequences of these interactions may depend on the relative concentrations of the nutrients. In this work, we measure the effects of increasing concentrations of iron, zinc, and copper on iron and copper uptake in Caco-2 cells. Intracellular Fe or Cu levels were affected by incubating with increased concentrations of metals. However, when the cells already had different intracellular metal concentration, the uptake of Fe or Cu was nor affected. In competition studies, we showed that Cu and Zn inhibited Fe uptake, and while Fe inhibited Cu uptake, Zn did not. When the three metals were given together (1:1:1 ratio), Fe or Cu uptake was inhibited approximately 40%. These results point to a potential risk in the absorption and bioavailability of these minerals by the presence of other minerals in the diet. This aspect must be considered in food supplementation and fortification programs.     

Effects of pretreatment with cadmium on the discriminative uptake of subsequent cadmium, copper or zinc by the liver

1. Effects of pretreatment with cadmium (Cd) on the uptake by the liver of subsequent Cd, copper (Cu) and zinc (Zn) were examined at two different time intervals to elucidate the biological discrimination mechanism among metals of similar chemical properties. 2. Pretreatment with 0.3 mg Cd/kg body wt 6 hr but not 24 hr before a subsequent dose of 0.8 mg metal/kg body wt enhanced the disappearance rate from plasma and accumulation rate in the liver of Cu (and Zn) but not of Cd. 3. Synthesis of metallothionein was induced with different time-courses depending on the time interval between the pretreatment and subsequent treatment, which coincided with the accumulation curves for Cu (and Zn) but not for Cd. 4. Although uptake of Cd was not enhanced by any pretreatment, metallothionein synthesis was enhanced depending on the timing of pretreatment.     

Relevance, essentiality and toxicity of trace elements in human health

The metals Mn, Fe, Cu, and Zn, and the non-metal Se are considered "trace elements" (TE) because of their essentiality and very limited quantity in humans. The biological activities of Cu, Fe, Mn, and Se are strongly associated with the presence of unpaired electrons that allow their participation in redox reactions. In biological systems these metals are mostly bound to proteins, forming metalloproteins. Many of the metals in metalloproteins are part of enzymatic systems, have structural and storage functions, or use the protein to be transported to their target site in the organism. In humans Mn, Fe, Cu, Zn, and Se accomplish decisive functions to maintain human health. Deficiency in any of these TE leads to undesirable pathological conditions that can be prevented or reversed by adequate supplementation. In sufficiently nourished persons, supplementation should be carefully controlled, given the toxic effects ascribed to TE when present in quantities exceeding those required for accomplishing their biological functions. The dietary reference intakes provided by national regulatory agencies are guides to define intake, supplementation and toxicity of Mn, Fe, Cu, Zn, and Se, as well other elements considered micronutrients for humans.