Metallothionein and Glutathione Content as Biomarkers of Metal Pollution in Mussels and Local Fishermen in Abu Qir Bay, Egypt

Background. When heavy metals accumulate in air, soil, and water, the risk of human exposure increases among industrial workers, as well as in people living near polluted areas. Heavy metals adversely affect a variety of bodily systems such as the cardiovascular, respiratory, endocrine, immune, and reproductive systems. In addition, long-term exposure and accumulation of heavy metals in the body may disturb oxidative stress genes and thus increase the susceptibility to various diseases. Objectives. The aim of this study is to estimate the metallothionein concentration in both mussel samples from Abu Qir Bay, Egypt and the blood of local fishermen as a biomarker of exposure to metal pollution. Methods. Levels of metallothionein and heavy metals were measured in mussels. Blood levels of metallothionein and heavy metals of local fishermen were measured and compared with a control group. The effect of heavy metal exposure on oxidative stress status was investigated through the determination of malondialdehyde (MDA), catalase and glutathione content. Results. The results of this study showed high concentrations of metallothionein in mussels and in fishermen's blood, accompanied by high concentrations of metals such as cadmium (Cd), copper (Cu), lead (Pb), chromium (Cr), and zinc (Zn). At the same time, a significant decrease in glutathione content and catalase enzyme activity was associated with a significant increase in the malondialdehyde concentrations in sera of fishermen. Conclusions. The present study found that the El Maadiya region is polluted with heavy metals, inducing oxidative stress in fishermen in the vicinity. These results reveal the necessity of further environmental monitoring in the study area in order to evaluate other types of pollutants and their effects on human health.


Introduction
Heavy metal contamination in marine ecosystems is of global concern. 1 The level of contamination of the aquatic environment due to heavy metals can be estimated by analyzing water, sediments and marine organisms. The levels of heavy metals in mollusks and other invertebrates are often considerably higher than in other constituents of the marine environment due to their habitat and feeding habits. [2][3][4][5] The most commonly used biomarker for pollution in the marine environment is metallothionein, which has been particularly useful as a monitoring device, namely as a contaminant-specific indicator of metal exposure. 6 Metallothioneins are inducible proteins; heavy metal cations accumulated within the cells stimulate metalloproteinneosynthesis by enhancing metallothionein gene transcription. The metallothionein messenger ribonucleic acid is translated by the cytosolic free ribosome; it leads to an increase in apo-metallothionein that rapidly reacts with the free metal cations that are present in the cytosol. 7 Due to their biochemical and functional characteristics, metallothioneins are able to protect cell structures from non-specific interactions with heavy metal cations and to detoxify the metal excess penetrating into the cell. Due to their inducibility to heavy metals, metallothioneins are usually considered to be important specific biomarkers that detect an organism's response to inorganic pollutants such as cadmium (Cd), mercury (Hg), copper (Cu), and zinc (Zn) that are present in the aquatic environment. Bioindicator organisms that have been commonly employed in the application of metallothioneins as biomarkers are fish, mollusks and crustaceans. Metallothioneins as tools for biomonitoring activities are important as they are ubiquitary proteins and therefore can be studied in most living organisms. 7 The potential of metals in generating reactive oxygen species (ROS) and thus altering cellular reduction-oxidation (redox) states is considered to be the most important mechanism involved in metal-induced carcinogenicity. 8, 9 Recent research suggests that chronic exposure to ROS causes oxidative stress by disrupting the balance between the levels of the produced ROS and the potential of cellular antioxidant systems to remove them. Persistent oxidative stress leads to changes in cellular redox homeostasis and abnormal activation of redoxsensitive signaling molecules. Oxidative stress also damages bio-macromolecules such as deoxyribonucleic acid (DNA), proteins, and lipids; it eventually induces a variety of chronic and degenerative diseases including cancer, cardiovascular disorders, diabetes, rheumatoid arthritis, and Alzheimer's and Parkinson's disease. 10

Research
The toxicity of heavy metals may be attributed to the binding of metals to sulfhydryl groups in proteins such as glutathione (GSH), resulting in an inhibition of activity, interference with structure, or displacement of an essential metal element leading to deficiency effects. 11 Repairing stressdamaged proteins and chelation of metals involving heat shock proteins and metallothionein is thus recognized as a potential mechanism of metal detoxification. Although mechanisms by which heavy metals interact have not been clearly elucidated, a number of biomolecules, including GSH, metallothionein, and heat shock proteins have been predominantly recognized as major interactive mediators when evaluating interactions based on metal mixture exposure. 12 The primary purpose of this study is to obtain quantitative estimations of metallothionein concentrations in mussels as biomarkers of exposure to heavy metals in order to monitor the pollution of Abu Qir Bay, Egypt (El Maadiya region). The second purpose of the study is to examine the impact of heavy metal pollution on residents of the study area through the determination of various metals in the blood of study subjects and the detection of metallothionein and oxidative stress through the estimation of malondialdehyde, blood glutathione and catalase enzyme activity.

Study Area
Abu Qir Bay is a shallow semicircular basin about 35 km east of Alexandria City between Abu Qir peninsula (west) and the Rosetta branch of the Nile (East), with a shoreline extending about 50 km (Figure 1). 13 The bay is adjacent to one of the most populous, industrialized and commercialized coastal metropolitan areas in Egypt. The people of the region put the bay to a wide variety of recreational, commercial, and industrial uses. The bay contributes about 10% of the fish and shrimp catch from Egyptian Mediterranean waters. The bay is subjected to multiple pollution from two point sources: (i) Tabia pumping station; the station pumps to the bay (1.5-2.0) x 106 m 3 /day of industrial waste water get from 20 different factories, mostly textile, food processing and canning, mixed agricultural and domestic waste water and (ii) the outlet of Lake Edku. 13

Mussel Sampling
Andaradulofii mussel samples were collected from the Mediterranean Sea, Abu-Qir Bay, in the El-Maadiya region and acclimated to laboratory conditions at 20°C for three days in ethylenediaminetetraacetic acid-free synthetic seawater, pH 7.9-8.0 and 35 osmolarity and salinity (Viarengo et al, 1997) to determine the presence of metallothionein and five other metals (cadmium, lead, chromium, copper and zinc). 14

Mussel Analysis
Mussel metallothionein concentration was evaluated using a partially purified metalloprotein fraction which was obtained by acidic ethanol/chloroform fractionation of the tissue homogenate and measured colorimetrically using GSH (Viarengo A, et al, 1997). 14 Mussel El-Sikaily, Saad, Kassem

Metal Pollution in Mussels and Local Fishermen in Abu Qir Bay, Eqypt
Abbreviations Research 0990-0279).

Analysis of Human Samples
Metallothionein concentration in human erythrocytes was evaluated using a partially purified metalloprotein fraction obtained by acidic ethanol/ chloroform fractionation of erythrocytes hemolysate. 16 The concentration of metallothionein is expressed as nanomoles of metallothionein per gram of hemoglobin (nmol MT/g Hb). Atomic absorption spectroscopy assay was used to determine the concentration of Cd, Pb, Cr, Cu, and Zn in blood samples of the study subjects. 17 Blood metal content was obtained from a standard curve ranging from 0.25 to 5 mg/l. Determination of serum lipid peroxide was based on the reaction of lipid peroxides with thiobarbituric acid in an acidic medium, which forms a red pigment that is extracted using n-butanol and measured at 530 nmol. 18 The concentration of MDA is expressed as nmol MDA/ml.
Determination of blood glutathione content was based on the reduction of 5,5' dithiobis-(2-nitrobenzoic acid) with glutathione to produce a yellow compound. 19 The reduced chromogen was found to be directly proportional to the GSH concentration and its absorbance measured at 405 nm.
Determination of erythrocytes catalase activity was based on the conversion of hydrogen peroxide to water and oxygen. 20 The apparatus was adjusted at zero using a blank cuvette. The reaction was started by adding hydrogen peroxide and was followed by a decrease in absorbance at intervals of 15 seconds and was measured at 240 nm.

Quality Control
All the glassware and the Teflon cups were previously soaked overnight with 20% nitric acid and then rinsed. Each element concentration was estimated quantitatively according to the standard conditions described in the instrument manual. However, working standards of studied elements were prepared by diluting concentrated stock solutions with deionized water. Reagents of analytical grade were utilized for the blanks and calibration curves; precision was checked against a reference material (IAEA-433, International Atomic Energy Agency; Analytical Quality Control Services) which was analyzed with the digested mussel solutions. The measured concentrations of heavy metals were within the range of certified values with a recovery of 96.9-105.5%, and precision was found to be within 10%. The recoveries of studied metals for four replicates of the standard reference material were 105.5% for Cd; 96.9% for Cu; 105.2% for Cr; and 98.0% for Zn. The absorption wavelength and detection limits of heavy metals were as follows: 228.8 nm and 0.006 μg/g for Cd; 324.7 nm and 0.008 μg/g for Cu; 213.9 nm and 0.004 μg/g for Zn, respectively.
Absolute ethanol, chloroform, hydrochloric acid and nitric acid were of high performance liquid chromatography grade. Sodium citrate, 5,5'-dithiobis-2nitrobenzoic acid, leupeptine, phenylmethylsulphonylfloride, thiobarbituric acid, and reduced glutathione were purchased from Sigma-Aldrich Chemical Co., (St. Louis, MO, USA). Table 1 depicts metallothionein concentrations in mussels collected from the El Maadiya region of the Mediterranean Sea. The results ranged from 1.37-32.6 µg/g wet weight. The concentrations of the five studied metals (Cd, Pb, Cr, Cu, and Zn) in mussels are also shown in Table 1. Table 2 demonstrates the concentrations of metallothionein in the erythrocyte gills and digestive glands were rapidly dissected and then stored at 80 o C for determination of metals content (Cd, Pb, Cr, Cu, and Zn) in mussels. 15 All digested solutions were analyzed and measured using an atomic absorption spectrophotometer (SPECTR plus version) with an air-acetylene flame and deuterium background correction.

Human Population
Sampling A total of 56 male subjects, with an age range of 20-55 years old, and weight ranging from 62-85 kg were divided into two groups: Control (Group I): Included (12) healthy control male subjects living in the El-Maadiya region, but working in jobs other than fishing. They were free from diabetes, liver disease and thyroid dysfunction.
Fishermen (Group II): Included (44) professional fishermen volunteers living in the El-Maadiya region. Information on age, weight, area of residence, smoking history, caffeine consumption, medication use, and history of acute or chronic illness were gathered via questionnaire. The food habits of study subjects were also taken into consideration. Subjects with thyroid dysfunction, diabetes, and liver disease were excluded.
Blood was collected from all subjects enrolled in this study for the assay of metallothionein, Cd, Pb, chromium Cr, and Zn, malondialdehyde (MDA), catalase and GSH content. Research samples of the control and fishermen groups. Concentrations ranged from 6-30 nmol/gHb in the control group and from 11-151 nmol/gHb in fishermen. Differences between variables were measured by independent t-test and are shown in Table 3. Significantly higher levels of metallothionein were found in the fishermen group compared to the control group. The serum malondialdehyde results of the healthy control and fishermen groups are shown in Table 5. They ranged from 0.8-2.6 nmol/ml and from 2.2-8.9 nmol/ml in the control and fishermen group, respectively. Statistical analysis using the independent t-test ( Table 6) demonstrated significantly higher levels of malondialdehyde in the serum of the fishermen group compared to the control group. Table 7 presents the blood glutathione content results, with mean concentrations of 39.6±1 and 24.4±0.6 mg/dL in the control and fishermen groups, respectively. Differences between the variables measured by independent t-test (Table 8) showed a significantly sharp decrease in blood glutathione levels in the fishermen group compared to the control group.

Ethical Approval
Data on erythrocyte catalase activity are presented in Table 9. Its concentration ranged from 805-991 units per gram of hemoglobin (U/gHb) and from 308-702 U/gHb for the control and fishermen groups, respectively. There was a very highly significant decrease in the enzymatic activity of erythrocyte catalase in the fishermen group compared to that of the control group (Table 10).

Discussion
The results of this study indicate that the study area was contaminated with heavy metals such as cadmium, lead, copper, chromium, and zinc. These results are in agreement with previous studies which found that Abu Qir Bay is contaminated with heavy metals such as cadmium, nickel, cobalt, and aluminum. 2,5, 21 Our study demonstrates the presence of measurable amounts of metallothionein in mussel tissues collected from the study area.
Metallothioneins have been implicated in the homeostasis of essential metals such as Cu and Zn, and in the detoxification of excess levels of essential and nonessential metals in marine invertebrates. 22 Several studies have demonstrated that metallothioneins protect organisms from metal toxicity due to their ability to bind metals like Cd, Cu, Zn and Hg. Metallothioneins also have the ability to increase their expression when metal concentrations in tissues rise. 23-27 The findings of the study demonstrate that residents of the El Maadiya region face the immediate environmental impact of heavy metal pollution; however, no study in the available literature has addressed the effects of heavy metal contamination on the human population in this area.
The results of the current study demonstrate the presence of high concentrations of heavy metals such as cadmium, chromium, lead, copper and zinc in the blood of local fishermen. This has been linked to significantly high levels of metallothionein in the fishermen's erythrocytes. The most conspicuous feature of metallothionein is the inducibility of MT-1 and MT-2 genes by a variety of agents and conditions. The regulation of metallothionein biosynthesis occurs primarily at the level of transcription, where the cis-acting elements of DNA respond to transacting transcriptional regulatory proteins.
The MT-1 and MT-2 genes in higher species are rapidly induced in vitro and in vivo by a variety of stimuli including metals, hormones, cytokines, oxidants, stress and irradiation. 28 Owing to their induction by a variety of stimuli, metallothioneins are considered to be valid biomarkers in the medical and environmental fields. 29 Metallothionein is a remarkable protein, with a variety of cellular roles in homeostasis and the management of toxicant exposure. 30 This small, cysteine-rich protein can combine with divalent heavy metal cations via cysteine associated thiols. Under normal circumstances, metallothionein is associated with zinc and copper cations, and thus serves as an important reservoir of these essential metals, donating them to apoenzymes and other apoproteins that require these metals to function. 31 Other metals such as mercury and cadmium can also combine with metallothionein, which limits their toxic effects. Metallothionein's cysteine-associated thiols can also serve as anti-oxidant moieties that protect DNA and other macromolecules from the damaging effects of reactive oxygen species. The regulation of ROS levels can indirectly regulate nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) activity, but metallothionein has also been found to directly interact with NF-κB and may regulate NF-κB-responsive genes via that direct interaction. 32, 33 A hypothesis has been proposed that metallothionein acts as a chaperone during synthesis and modulation of metalloproteins and metallothionein and appears to be stabilized at high cellular GSH concentrations. Metal-requiring apoenzymes can abstract metals from metallothionein as demonstrated in vitro. Glutathione can form a complex with metallothionein. Subsequently, a release of zinc from metallothionein mediated by interactions with GSH and the oxidized form of glutathione through S-thiolation has been reported. 34,35 Evidence suggests that zinc released from metallothionein is facilitated by a directly coupled interaction of GSH and the oxidized form of glutathione with metallothionein (301) via the thiolate ligands that confer redox activity on zinc clusters. This results in an oxido reductive metallothionein-Zn complex. 36 This finding confirms the results of our study, demonstrating the presence of a negative correlation coefficient between metallothionein, glutathione,

Table 3 -Statistical Analysis (Independent t-test)of Erythrocytes Metallothionein Concentrations of the Healthy Control and Fishermen Group
Data expressed as nmol/gHb p< 0.05 indicates significance Abbreviations: SD, standard deviation; SE, standard error

Research
and catalase values (Figures 2 and 3). Metallothionein may be acting as a sensor of the localized intracellular redox balance and may itself influence redox balance through GSH and the known antioxidant properties of zinc. Hartwig suggested that "the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of metallothionein". 37 Metallothionein is thought to play a major role in metal detoxification, supported by extensive evidence from both in vivo and in vitro studies. After exposure to various metals, there is a significant increase of metallothionein in tissues of the kidney, liver and intestine. Likewise, various cell types have been shown to accumulate metallothionein after metal exposure. Some metals such as lead are known to induce and bind to other intracellular proteins, which may also play a role in their detoxification. 34 Glutathione has also been implicated in metal detoxification. Several in vivo and in vitro studies have reported increased sensitivities towards the toxic effects of mercury and cadmium following a depletion in GSH levels. Glutathione, however, appears tobe the first line of defense against cadmium toxicity preceding metallothionein induction. 34 A number of metallothionein inducers, such as glucocorticoids, lipopolysaccharides, steroid hormones, cytokines and tumor necrosis factors, among others, can influence apoptosis in certain cells. This and other experimental data would seem to suggest that metallothionein plays a role in the apoptotic process. 36 The results of the present study found that the fishermen group was exposed to various types of heavy metals as evidenced by the presence of cadmium, chromium, copper, lead and zinc in their blood, generating severe oxidative stress as manifested by the presence of high      13.9 P = 0.000 *

Table 10 -Statistical Analysis (Independent t-test) of Erythrocytes Catalase Enzymatic Activity Levels of the Control and Fishermen Group
Data expressed as U/gHb * p < 0.05indicates significance Abbreviations: SD, standard deviation; SE, standard error

Group Statistics
Test of Significance X n

Research
DNA lesions may increase their steady state levels upon chronic exposure to metal compounds. Because these inhibitions have frequently been observed at far lower concentrations than with the induction of considerable amounts of oxidatively generated DNA lesions, as in the case of nickel and cadmium compounds, they may be particularly relevant to metal-induced carcinogenicity. 39

Conclusion
The present study found that the El Maadiya region is polluted with heavy metals, inducing oxidative stress in fishermen in the vicinity. The risk persists due to a large increase in the levels of malondialdehyde coinciding with a large decrease in the levels of the antioxidant glutathione and the enzymatic activities of catalase. The current results demonstrate the presence of highly significant levels of metallothioneinin the blood of local fishermen along with a positive correlation between metallothionein and malondialdehyde and a negative correlation between metallothionein and the antioxidant glutathione. These results reveal the necessity of further environmental monitoring in the study area in order to evaluate other types of pollutants and their effects on human health.