997 resultados para Biological specimens
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Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.
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Research expeditions into remote areas to collect biological specimens provide vital information for understanding biodiversity. However, major expeditions to little-known areas are expensive and time consuming, time is short, and well-trained people are difficult to find. In addition, processing the collections and obtaining accurate identifications takes time and money. In order to get the maximum return for the investment, we need to determine the location of the collecting expeditions carefully. In this study we used environmental variables and information on existing collecting localities to help determine the sites of future expeditions. Results from other studies were used to aid in the selection of the environmental variables, including variables relating to temperature, rainfall, lithology and distance between sites. A survey gap analysis tool based on 'ED complementarity' was employed to select the sites that would most likely contribute the most new taxa. The tool does not evaluate how well collected a previously visited site survey site might be; however, collecting effort was estimated based on species accumulation curves. We used the number of collections and/or number of species at each collecting site to eliminate those we deemed poorly collected. Plants, birds, and insects from Guyana were examined using the survey gap analysis tool, and sites for future collecting expeditions were determined. The south-east section of Guyana had virtually no collecting information available. It has been inaccessible for many years for political reasons and as a result, eight of the first ten sites selected were in that area. In order to evaluate the remainder of the country, and because there are no immediate plans by the Government of Guyana to open that area to exploration, that section of the country was not included in the remainder of the study. The range of the ED complementarity values dropped sharply after the first ten sites were selected. For plants, the group for which we had the most records, areas selected included several localities in the Pakaraima Mountains, the border with the south-east, and one site in the north-west. For birds, a moderately collected group, the strongest need was in the north-west followed by the east. Insects had the smallest data set and the largest range of ED complementarity values; the results gave strong emphasis to the southern parts of the country, but most of the locations appeared to be equidistant from one another, most likely because of insufficient data. Results demonstrate that the use of a survey gap analysis tool designed to solve a locational problem using continuous environmental data can help maximize our resources for gathering new information on biodiversity. (c) 2005 The Linnean Society of London.
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This study demonstrates a novel approach to characterizing hydrated bone's viscoelastic behavior at lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique for analyzing local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying subsurface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods. © 2010 Elsevier Ltd.
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New designer drugs are constantly emerging onto the illicit drug market and it is often difficult to validate and maintaincomprehensive analytical methods for accurate detection of these compounds. Generally, toxicology laboratories utilize a screening method, such as immunoassay, for the presumptive identification of drugs of abuse. When a positive result occurs, confirmatory methods, such as gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), are required for more sensitive and specific analyses. In recent years, the need to study the activities of these compounds in screening assays as well as to develop confirmatory techniques to detect them in biological specimens has been recognized. Severe intoxications and fatalities have been encountered with emerging designer drugs, presenting analytical challenges for detection and identification of such novel compounds. The first major task of this research was to evaluate the performance of commercially available immunoassays to determine if designer drugs were cross-reactive. The second major task was to develop and validate a confirmatory method, using LC-MS, to identify and quantify these designer drugs in biological specimens.^ Cross-reactivity towards the cathinone derivatives was found to be minimal. Several other phenethylamines demonstrated cross-reactivity at low concentrations, but results were consistent with those published by the assay manufacturer or as reported in the literature. Current immunoassay-based screening methods may not be ideal for presumptively identifying most designer drugs, including the "bath salts." For this reason, an LC-MS based confirmatory method was developed for 32 compounds, including eight cathinone derivatives, with limits of quantification in the range of 1-10 ng/mL. The method was fully validated for selectivity, matrix effects, stability, recovery, precision, and accuracy. In order to compare the screening and confirmatory techniques, several human specimens were analyzed to demonstrate the importance of using a specific analytical method, such as LC-MS, to detect designer drugs in serum as immunoassays lack cross-reactivity with the novel compounds. Overall, minimal cross-reactivity was observed, highlighting the conclusion that these presumptive screens cannot detect many of the designer drugs and that a confirmatory technique, such as the LC-MS, is required for the comprehensive forensic toxicological analysis of designer drugs.^
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The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
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Many of the 5,500 threatened species of vertebrates found worldwide are highly protected and generally unavailable for scientific investigation. Here we describe a noninvasive protocol to visualize the structure and size of brain in postmortem specimens. We demonstrate its utility by examining four endangered species of kiwi (Apteryx spp.). Frozen specimens are thawed and imaged using MRI, revealing internal details of brain structure. External brain morphology and an estimate of brain volume can be reliably obtained by creating 3D models. This method has facilitated a comparison of brain structure in the different kiwi species, one of which is on the brink of extinction. This new approach has the potential to extend our knowledge of brain structure to species that have until now been outside the reach of anatomical investigation.
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The ribosomal DNA internal transcribed spacer region was amplified and sequenced from a selection of specimens of the Sporobolus smut Ustilago sporoboli-indici. Phylogenetic comparison with other Ustilago and Sporisorium species revealed strong support for an evolutionary radiation of Ustilago species infecting the Chloridoideae and Pooideae, of which U. sporoboli-indici forms a major lineage. Comparisons are made with other groups of plant pathogenic fungi, and it is concluded that phylogenetic analyses of potential biocontrol agents are useful for identifying pathogens that are derived from evolutionary lineages that parasitize a wide range of unrelated plants. Such pathogens are less desirable as biocontrol agents as they may have a greater likelihood of infecting plants outside their normal host ranges.
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With the cooperation of several of the executive departments, and of the Field Museum of Natural History, a party of about 10 naturalists was accordingly sent to the zone, and the results so far accomplished have been very satisfactory. Large collections of biological material have been received, including specimens of a considerable number of genera and species new to science. It also seemed important to determine exactly the geographical distribution of the various organisms inhabiting the Isthmus, which is one of the routes by which the animals and plants of South America have entered North America and vice versa. The estimated cost of the survey which would have to be met by the Institution is $11,000...
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The FOB-3, anew type fiber optic biosensor, is designed to rapidly detect a variety of biological agents or analytes with better stability, sensitivity and specificity. In order to detect Y. Pestis, a sandwich immunoassay was developed by using the purified antibody against antigen FI immobilized on polystyrene probes as the capture antibody and the monoclonal antibody-Cy5 conjugate as the detector. After a series of optimization for the stability, sensitivity and specificity of the FOB-3, 50-1000 ng/ml of antigen FI and 6 x 10(1)-6 x 10(7) CFU/ml Y. pestis could be detected constantly in about 20 min, and Y pestis could be detected specifically from Y. pseudotuberculosis, Y. enterocolitica, B. anthracis and E. coli. Then, 39 blind samples, including 27 tissues of mice infected with Y pestis and 12 tissues of healthy mice as negative control, were detected with the FOB-3. 92.6% infected tissues were identified from the tissues of healthy mice and the tissues containing more than 100 CFU/ml bacteria could be detected by the biosensor. The results demonstrated the feasibility of the FOB-3 as an effective method to detect Y. pestis rapidly and directly from the infected animal specimens with the advantage of portability, simple-operation as well as high sensitivity and specificity. (c) 2006 Elsevier B.V. All rights reserved.
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This study was conducted to determine reproduction characteristics, diet regime, age structure and population dynamics parameters of the vimba vimba persa (Pallas, 1811) in Mazandaran waters of the Caspian Sea, from October 2008 to September 2009. A total of 994 specimens were monthly collected by beach seine and cast net from six fish landings of Ramsar, Tonekabon, Chaloos, Mahmood Abad, Sari and Behshahr. Biometric characters were measured for each specimen at the laboratory. Scales were used for age determination. Sex determination and fecundity were determined. Population dynamic parameters as well as stock assessment including cohort analysis were estimated using FISAT software. The finding showed that the mean of fork length and body weight of the Caspian Vimba were 168.4±2.6 mm and 71.94±32.24 g respectively. Strong correlation was found between these two variables (a= 0.012; b = 3.047; r2 = 0.955). 92 specimens were studied from the fecundity point of view. This species was found to have more abundance in spring (esp. Apr-May). The samples composed of 397(42.6%) male, 537(57.4%) female; Overall sex ratio (M: F =1: 1.35) was significantly different from the expected 1:1 ratio (p ≤0.05). The advanced stages of maturity (4th & 5th) were found in April and May. The highest Gonadosomatic Index in female was in May and the lowest one was in July. This fish is therefore a spring spawner. The maximum absolute and relative fecundities were 34640 and 260.9, respectively; the minimum absolute and relative fecundities were 5400 and 94.5 respectively. The averages of absolute and relative fecundities were 17198±7710 and 171.85±48.8, respectively. Coefficient vacuity index was 59.2% which indicates that this fish is mesophagous. Among of living creature consumes by Caspian Vimba mollusks, 76 arthropods, worms, plants, detritus and fishes were found 32.9% , 26.7% , 13.4% , 17% , 4.4% and 1.6% respectively. The infinite fork lengths were 261 mm for females, 25mm for males and 261 mm for both sexes respectively. For population growth and mortality parameters; K ( 0.28 per year for both sexes, 0.3 per year for males, 0.33 per year for females); t0 ( -0.65 year for both sexes, -0.23 year in females, -0.51 year in males ); Φ' ( 2.28 ); Z ( 0.98 per year ); M ( 0.59 per year); F ( 0.39 per year) and Exploitation coefficient was 0.4. The analysis showed that total biomass and MSY were 1336 and 528.8 tonnes respectively.
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The Great lizardfish is one of the commercial demersal fishes in the Persian Gulf and Oman Sea. In this study the food preference index (FPI), Fullness index (FI) and stomach contents of Saurida tumbil were evaluated to assess the quantity and kind of food which this species consumes in the study area. The samples were collected monthly from commercial bottom trawl fisheries in main fishing grounds of eastern Strait of Hormuz. A total of 346 specimens were collected during years 2009-2010 of which 269 specimens were female and 77 specimens were male. The minimum and maximum total length were 161 and 590 mm, respectively with the highest frequency in length group of 350-370mm. Results showed that 20.9% of stomachs were full, 18.9% were semi-full and 60.2% were empty. The main stomach contents (FPI=80.2%) were fishes and minor stomach contents (FPI=25.7%) were crustacean (Specially crab and shrimp) and cephalopods were found as random food. The minimum and maximum FIs were 2.5 and 42.5 in December and September respectively; and the overall CV was 60.2. The results indicate that this fish is a relatively frugal species which consumes fishes as main food and there is no difference in feeding of male and female.
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This study was conducted to determine biological characteristics and population dynamics parameters of threadfin bream (Nemipterus japonicus) in Persian Gulf (Bushehr Province), during November 2006 and October 2007. The minimum and maximum specimens were 75-273 mm FL and their weight was 7.6 - 351.9 g. Based on the exponential relationship between fork length and weight, slope (b) for individuals, males and females was 2.987321, 2.992546 and 3.007314, respectively. The emptiness value (V) was 45.6% and it shows that N. japonicus is a moderate feeder. The results of Fp indicates that crustacean with 78.2% are main foods, mollusca (27.7%), fishes (20.7%), polychaeta (19.2%) and Foraminifera (11.7%) were identified as minor foods and phytoplanktons (9.9%), nematoda(8.0%), echinodermata (2.3%) and sea weeds (0.3%) were random foods. The reproduction studies showed the spawning season extended within 2 peaks, from April- May and September and main spawning occurs in spring season.The mean absolute and relative fecundities were 472388±42633 and 3817±293 (X±SE), respectively. The maximum, minimum and mean of oocyte diameter were 0.448, 0.022 and 0.221mm (SE=0.071), respectively.The fork length at 50% maturity estimated to be 20.25 cm for females. The growth coefficient (K) , length infinity (L∞ ) and ɸ' was estimated 0.42/yr , 34.17 cm and 2.69, respectively. The coefficient of total mortality, fishing mortality, natural mortality and E was 1.37, 0.43, 0.94 and 0.31, respectively.
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Biological studies and heavy metal (Ni, v, Fe, Pb,Cd) determination in liver, gonad and muscle of Scomberomorus commerson were carried out from Oct 2006 to Sept 2007 in Hormozgan coastal waters. 599 Samples were gutted for reproduction and nutrition studies, fork length and weight were measured to nearest cm and g respectively. Meanwhile 40 samples were also investigated for heavy metal studies. All specimens were collected from two major landing sites (Bandar Lengeh & Bandar Abbas). Minimums & maximum fork length & weight were 29, 128 cm & 235 and 15350 g respectively.Isometric growth was shown according to our study and b was estimated 2.9 (overall), 2.91(male) & 2.89 (female). The average relative gut length was 0.52± 0.007 and it was determined that S. commerson is a carnivorous. More than 99 percent of gut content was different teleost fishes. Gastro somatic index had two peaks in Nov & Jan (before spawning) and with a decreased trend in July, the spawning period. Occurrence of empty stomach was estimated % 65.77. Maximum amount of condition factor was in Dec. Spawning season was started from June. The average of Absolute & relative fecundity (to weight unit) was 1217149±179315 and 178.2±15.58 respectively. Lm50% was estimated 75 cm for females. Sex ratio was 0.97: 1 (male: female). Chi- Square test showed no significant difference (p>0.05). Maximum amount of hepatosomatic index was estimated in March.Metal concentrations were determined using either Flame Atomic Absorption Spectroscopy (for Fe) or Graphite Furnace Atomic Absorption Spectroscopy (for Pb,Cd,Ni and V). The mean concentration (μg/g dry weight)of Pb,Cd,Ni,V and Fe in the liver were 0.0309, 0.0268, 0.0672, 0.0077, 2.5159 in the gonad 0.0440 ,0.0295, 0.1096, 0.0000, 1.4449 and in the muscle 0.0244, 0.0324, 0.0656, 0.0128, 1.6138 respectively. The maximum metal concentrations were below the maximum permissible limits for human consumption recommended by the USEPA, WHO and the UK. The results of Kendall's Tau-b correlation coefficient were as follows: The Liver tissue: There were significant positive linear relationships between accumulation of V, Fe, and Pb with Fork length, Pb and Fe with weight, GSI with Pb, Cd, V and 109 Fe, and a negative linear relationships between HSI with accumulation of V and Fe, Fork length, weight and GSI. The Gonad tissue: There were significant positive linear relationships between GSI with accumulation of Pb, Cd, Fe, Fork length and weight, a negative linear relationship between HSI with Fork length, weight and GSI. The Muscle tissue: There were significant positive linear relationships between accumulations of V, Fe with Fork length and weight factors and as well as GSI with Cd, V, Fe, Pb, Fork length and weight,a negative linear relationship between HSI with Fork length, weight, Cd, Fe and GSI. The results of Mann-Whitney U tests (P≤0.05) show that there were significant differences between summer and autumn from heavy metal contents in the studied tissues point of view. The only exceptions were for Ni in the liver, gonads and muscle and as well as there were significant differences between male and female from heavy metal contents in the studied tissues. The only exceptions were for Pb in muscle, Ni in liver, gonad and muscle, V in muscle, and Cd and Fe in gonads.