Metallothionein: a multifunctional protein from toxicity to cancer.

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.

Focal adhesion kinase expression is not a prognostic predictor in colon adenocarcinoma patients.

AIM: Focal adhesion kinase (FAK) is an enzyme of the tyrosine kinase group linked to signaling pathways between cells and their extracellular matrix. FAK expression in tumor cells in vitro may correlate with their ability for invasion and metastasis. METHODS: FAK protein expression was examined immunohistochemically in 80 cases of colon adenocarcinoma, and correlated with clinicopathological parameters; tumor proliferative capacity, reflected by Ki-67 antigen expression; and survival. RESULTS: All tumor samples were FAK positive compared to normal colonic mucosa. FAK protein overexpression was seen in 32 out of 80 cases. FAK protein overexpression did not correlated with tumor histological grade, stage, Ki-67 positivity or survival. CONCLUSIONS: Raised FAK protein expression was noted by immunohistochemistry in human colon carcinoma cases. The implication are discussed.

Occupational exposure to cholinesterase inhibiting pesticides: a Greek case.

The determination of plasma or serum cholinesterase is absolute and it is considered as a reliable index of exposure in workers of organophosphorus pesticides industries. In the present study the plasma cholinesterase of 28 persons working in the packaging of an ortho-thio-phosphate was determined, before and after their exposure to this agent. The results of this study showed a plasma cholinesterase depression of 37%, a decrease which was statistically significant (P<0.001).

Assessing food additive toxicity using a cell model.

Food additives are widely used for technological purposes and their presence is often substantial daily diet. They have also been accused forvarious toxic reactions in humans. The toxicity of the food color tartrazine, the preservatives sodium nitrate and sodium benzoate, and the antioxidant BHT, was studied using the protozoan Tetrahymenapyriformis as a toxicological model. The 4 food additives were added to Tetrahymena cultures and DNA content of the protozoan nuclei measured by an image analysis system. These food additives caused a statistically significant increase in DNA content suggesting stimulation of the mitotic process. This system may contribute to the investigation of the cellular action of food additives, since mitogenic stimuli substantially alter susceptibility to chemical carcinogenesis.

Expression of metallothionein in lung carcinoma: correlation with histological type and grade.

AIMS: Over-expression of cellular metallothionein occurs frequently in human tumours but the underlying mechanism remains unknown. The aim of this study was to assess metallothionein expression in cases of lung carcinoma and to correlate it with histopathological parameters. METHODS AND RESULTS: Tumour tissue samples from 89 patients with lung carcinoma were immunostained by the streptavidin-biotin-peroxidase technique, using a monoclonal antibody against both metallothionein-1 and -2 isoforms. Positive matallothionein immunostaining was prominent in 44 out of 89 (49%) and negative in 45 out of 89 (51%) cases of lung carcinoma examined. Metallothionein positivity was prominent in 32 out of 43 (74%) cases of squamous cell lung carcinoma, and in 12 out of 35 (34%) cases of adenocarcinoma, while it was negative in all 11 cases of small-cell lung carcinoma examined, presenting a statistically significant difference between the different histological types. The intensity of metallothionein staining revealed a statistically significant difference between the squamous cell and adenocarcinoma cases examined. The pattern and extent of metallothionein staining in tumour cells and the expression of metallothionein in stromal cells were not correlated with histopathological parameters (type and grade) in metallothionein-positive cases of lung carcinoma examined. No association was found between metallothionein expression and lymph node status in the examined cases of lung carcinoma. CONCLUSIONS: Our findings indicate that expression of metallothionein was evident in squamous cell lung carcinoma and adenocarcinoma, but absent in small-cell lung carcinoma, supporting evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung.

Countrywide estimation of the burden of injuries in Greece: a limited resources approach.

BACKGROUND: We have assessed the ability of the Emergency Department Injury Surveillance System (EDISS), a registration network operating with limited resources, to predict the burden and pattern of injuries in the whole Greece. METHODS: We have compared hospitalizations calculated on the basis of EDISS with those routinely recorded by the National Statistical Service countrywide. EDISS relies on data collected in the Emergency Departments of four hospitals, two located in the Greater Athens area, and two district hospitals in the remaining Greece. EDISS data concern hospitalized and non-hospitalized patients of all ages, with all types of injuries irrespectively of their etiology or intent. RESULTS: Over a three-year period 148 835 subjects with injuries were interviewed. Using sampling ratios of 2.2% for all injuries outside Greater Athens and for adults in Greater Athens but 28.9% for childhood injuries, the total annual number of injuries in Greece was estimated at 1.53 million (95% confidence interval: 1.48-1.57 million). Of those, about 18% concern children less than 15 years old. The difference between the EDISS estimated and the actually recorded hospitalized injuries was, in preliminary terms, acceptable. DISCUSSION: Of the ten major categories of injuries, seven among children and five among adults have shown deviations of less than 20%. For the remaining categories deviations were larger but in only one instance did the difference exceed 50%. It is concluded, that for a small and relatively homogeneous country, like Greece, injury data provided by four large hospitals can generate reasonably reliable estimates for large categories of injuries.

Lead content of fresh milk samples from different sites in Athens.

Milk and dairy products constitute a major food, especially for infants and children, and relatively low levels of toxic elements can contribute significantly to dietary intakes and be hazardous for public health. The purpose of this survey was to define the levels of lead in milk samples of different origin, to establish the presence or absence of contamination. The milks from different sites of the metropolitan area of Athens contained negligible traces of lead and thus are safe for the public health.

Immunomodulative effects of aflatoxins and selenium on human natural killer cells.

The possible immunomodulative effects of aflatoxin B1 and selenium (as sodium selenite) at concentrations lower than 5.10(-11) M or 0.05ppb and 10(-5) M, respectively on natural killer (NK) cells from healthy volunteers were evaluated by a 51Cr release natural killer assay. Aflatoxin B1 and selenium separately had an immunosuppressive effect on NK cell activity. When they were studied in combination, they caused a statistically significant increase of the cytotoxic activity of NK cells in the presence of the lowest concentrations of aflatoxin B1 (0.005ppb) and of sodium selenite (10(-8)M).

Analysis of total mercury in human tissues prepared by microwave decomposition using a hydride generator system coupled to an atomic absorption spectrometer.

A fast and efficient procedure has been developed for the analysis of total mercury in human tissues and blood using a hydride vapor generator system coupled to an atomic absorption spectrometer (HVG-AA). Tissue and blood samples were digested in a pressurized microwave decomposition system and the digest diluted prior to formation of free mercury vapor and analysis by atomic absorption. Recovery studies performed on 10 spiked/unspiked pairs of human liver and on 10 spiked/unspiked pairs of human blood samples yielded average recoveries of 99.7% (CV=0.4%) and 101.2% (CV=0.5%), respectively. The method detection limit for liver and blood was 50 microg Hg/kg and 12.5 microg Hg/l, respectively. The "normal" concentrations of mercury in human liver and blood are 33-490 microg Hg/kg and 0.6-59 microg Hg/l, respectively [1]. This method is able to determine mercury poisoning levels and may also be applied to detect mercury near the lower levels of these "normal" ranges, using the standard addition method approach.

Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study.

Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl(4)) administration. A single intraperitoneal injection of 1 ml CCl(4)/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl(4) administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl(4), in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl(4) administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl(4), being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes.

The inferiorly based gastrocnemius muscle flap: anatomic aspects.

The arterial communication between the gastrocnemius muscle heads through their lowest anastomotic arteriole bundle alone was examined in specimens from 14 fresh cadavers. In 3 specimens, the larger vessels in close vicinity to the lowest vessels were preserved as well. Distinct communication between the arterial networks of the heads was demonstrated in all cases after injecting dyes through both sural arteries or into the lateral sural artery and the lowest anastomotic arteriole in 11 and 3 specimens, respectively. Therefore, it seems that one head can be adequately supplied from the contralateral one through their lowest anastomotic arteriole(s); nevertheless, the location of this vessel varies significantly and cannot be detected preoperatively. Measurements demonstrated that although this vessel is not found at a constant level, it is invariably detected in the lower third of the medial gastrocnemius head's length and, in 93 percent of cases, in the lower fourth. Thus, rough preoperative planning becomes feasible. Given that the venous communication between the heads has been documented as well, the authors think that an inferiorly based flap of the medial gastrocnemius head for defects of the middle third of the tibia might be both reliable and applicable; however, for reasons of safety, the muscle heads should remain attached along their lower third.

The importance of performing in vitro cytotoxicity testing before immunomodulation evaluation.

This study uses the well known sensitive and reproducible human peripheral blood lymphocyte (PBL) assay as a model for the possible discrimination between cytotoxic and/or immunomodulative properties of 5 reference compounds: cadmium chloride (CdCl2): didodecyl-dimethyl ammonium bromide (DDAB); cyclosporin (CsA); dexamethasone (DEXA); and theophylline. Two colorimetric cytotoxicity assays were employed, the thiazolyl blue tetrazolium bromide and neutral red, and the H-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated PBL was used, as a parameter of cell mediated immunity. This study demonstrated the importance of performing colorimetric or other cytotoxicity assays before any investigation of mechanisms of action of different substances on immunomodulation and relative human risk assessment. This study showed that CdCl2 and DDAB have a cytotoxic rather than immunomodulative effect on PBL and that CsA, DEXA and to a lesser extent theophylline have immunomodulative rather than cytotoxic effects on PBL within the range of the concentrations studied.

Cytotoxic and immunomodulative effects of flunitrazepam, zipeprol and trihexyphenidyl on human peripheral blood lymphocytes.

The cytotoxic effects of flunitrazepam, zipeprol and trihexyphenidyl on cell viability of peripheral blood mononuclear cells from healthy volunteers were studied at concentrations from 10(-2) to 10(-8) M by 2 colorimetric in vitro assays: the neutral red uptake assay and thiazolyl blue tetrazolium bromide assay. All tested drugs of abuse were non-cytotoxic at concentrations lower than 10(-5) M. Possible immunomodulative effects of these substances were evaluated through phytohemagglutinin induced lymphocyte proliferation ([3H]-thymidine DNA incorporation assay) and by a 51Cr release natural killer assay. The 3 drugs studied produced statistically significant immunomodulative alterations on both immunological parameters.

Liver metallothionein expression in thioacetamide-intoxicated rats.

Metallothioneins (MT), a group of ubiquitous low molecular weight proteins, implicated primarily in metal ion detoxification, are known to be expressed during hepatocellular proliferation after partial hepatectomy in rats. In the present study, we investigated the expression of MT in a rat model of liver injury and regeneration, induced by intraperitoneal administration of thioacetamide (TAA). The animals were killed at 0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours after TAA administration. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase, and the assessment of the mitotic index in hepatocytes were used as indices of liver regeneration. Liver MTs were detected immunohistochemically. TAA administration caused severe hepatic injury, followed by regeneration. MT expression became prominent in hepatocytes as early as 12 hours post-TAA administration. At 24 and 36 hours post-TAA administration intense nuclear and cytoplasmic staining of hepatocytes was found in the vicinity of necrotic areas. The maximal nuclear and cytoplasmic MT expression coincides with the peak of hepatocyte proliferative capacity, occurring at 48 and 60 hours post-TAA administration. MT expression correlated positively with the Zn content of liver tissue, but negatively with serum one, at the time of maximum hepatocyte proliferative capacity. This study suggests that MT participates in hepatocyte replication after toxin-induced liver injury.

Identification of an alleged offender of murder by VNTR analysis: case report.

DNA typing was used to demonstrate that three human body pieces found disposed of in the countryside around Athens and the suspicions about the perpetrator's identity were connected. Reverse paternity testing was attempted by comparative typing of three variable number tandem repeats loci in the remains, as well as in the presumptive parents and sister of the decedent, demonstrating Mendelian inheritance of the alleles of the loci analyzed. Confronting the results of the abovementioned analysis, the suspect accepted the accusation.

Delat9-tetrahydrocannabinol content in cannabis plants of greek origin.

The delta9-tetrahydrocannabinol (delta9-THC) content was identified and determined quantitatively using a Gas Chromatography Detector (Gas Chromatography-Electron Ion Detector) instrument in samples of illicit herbal cannabis. Law enforcement authorities sent the samples to the Department of Forensic Medicine and Toxicology, University of Athens, for toxicological analysis. The concentrations of delta9-THC in these samples ranged from 0.08% to 4.41%. Such concentrations suggest that Greece might be at high risk, as an area for the illicit cultivation of "pedigree" cannabis plants. The forensic aspects of cannabis classification are discussed.

The venous communication between the gastrocnemius muscle heads.

This investigation establishes the venous communication between the gastrocnemius muscle heads. Fourteen fresh (with the exception of one) cadaveric specimens were examined. Anastomotic veins were dissected along the raphe after perfusion of each muscle with 0.1 % methylene blue solution. A distally based musculocutaneous perforating vein of the medial head was the site of perfusion in 13 specimens, whereas one specimen received injection through the accompanying vein of the medial sural cutaneous nerve. Additional perfusion of dye from different sites was required in two specimens. Communicating veins were dissected in all 14 muscles. Direct anastomotic veins were detected in eight specimens; whereas in six the accompanying vein of the medial sural cutaneous nerve that had been included proved to be the intermediate pathway of venous communication between the muscle heads. Obstructive valves were encountered in most cases; nevertheless anastomotic veins were always recognized. The role of valves and the clinical implications are discussed.

The arterial communication between the gastrocnemius muscle heads: a fresh cadaveric study and clinical implications.

The purpose of this investigation was to describe the anatomy of the communicating (anastomotic) vessels between the gastrocnemius muscle heads and to record the extent of their supply potential. Ensuing clinical implications are discussed. Fourteen fresh cadaveric gastrocnemius muscles were examined. Detailed dissections of the communicating vessels were facilitated after injections of methylene blue or cadaveric blood solutions through the medial, lateral, or both sural arteries. The extent of the arterial cross-supply between the muscles' heads through these vessels was determined in-eight specimens after methylene blue perfusions through the lateral sural arteries, while one specimen was examined after injection of methylene blue and yellow ink through the lateral and medial sural arteries, respectively. Communicating vessels were detected in all 14 specimens. A mean number of 5.8 vascular bundles and single vessels was found. The bundles consisted of arterioles and, as all indications suggested, of concomitant venules as well. Regarding arterial cross-supply, it was clearly evident that each head could be vascularized solely from the contralateral one, mostly through these bundles. However, even if only a part of the bundles was preserved intact, vasculature was not affected.

Metallothionein and heat shock protein expression during acute liver injury and regeneration in rats.

Metallothioneins (MT) are cytosolic proteins rich in cysteine which play a physiological role in metal ion homeostasis. Heat shock proteins (HSPs) are expressed in various organs in response to different stress stimuli. The purpose of the present study was to examine the intrahepatic distribution of MT and HSP-27, -70 and -90 in two different experimental models of acute liver injury and regeneration, induced by either thioacetamide, or carbon tetrachloride administration in male Wistar rats. Toxicological endpoints and markers of hepatocellular regeneration were assessed at various time points following toxin administration. The enzymatic activities of aspartate and alanine aminotransferases in serum, and histological findings in the liver were used to estimate toxin-induced injury. Tritiated thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index were used to estimate liver regeneration. MT and HSPs were detected immunohistochemically. At the time of maximum liver injury, moderate MT and intense HSPs expression was prominent in hepatocytes in the vicinity of necrotic areas. At the time of maximum hepatocellular proliferation, intense MT and HSP-90 staining was evident in all hepatocytes, while at the same time, mild HSP-27 and HSP-70 immunoreactivity was noted. Our findings indicate that the differential distribution of MT and HSPs in the liver after toxin-induced injury, in common with the observed pattern of staining, reflect liver proliferating capacity.