Multiple Biomarker Comparison in <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> from the Greece Coast: “Lysosomal Membrane Stability,Neutral Red Retention,Micronucleus Frequency and Stress on Stress”

The lysosomal membrane stability test applied on the digestive cells (LMS) and the neutral red lysosomal retention assay (NRR) performed on hemocytes have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (Nothern Greece) in June and December 2000. The correlation analysis indicated that there was a strong positive relationship between the above mentioned biomarkers in both sampling seasons, supporting the validity of their application in water quality assessment. The evaluation of micronucleus frequency (MN) has been also performed in hemocytes of the same mussels and, according to the results, it requires additional validation before using it as a stress index. The results obtained in parallel by the stress on stress (SOS) technique do not support the application of this biomarker in biomonitoring, showing, however, negative correlation with LMS and NRR in one sampling season. Finally, morphological observations were performed on cryosections stained for the enzyme N-acetyl-β-hexosaminidase, revealing differences in the epithelial cell-layer thickness, as well as changes in the digestive lysosomal system of mussels, obtained from different sampling sites in the two sampling seasons.     

Non-selective retention of PSP toxins by the mussel Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels, Mytilus galloprovincialis, were contaminated by paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense. Temporal variations in the toxin content and the profile of mussels during the feeding experiment were monitored by high-performance liquid chromatography (HPLC). The toxin profile of mussels was compared with that of A. tamarense to clarify the mechanism of uptake of toxins in mussels. The prominent toxins in mussels and A. tamarense were N-sulfocarbamoyl toxins (C1,2) and carbamate toxins, gonyautoxin-1,4 (GTX1,4). The toxin profiles of both mussels and A. tamarense were almost constant throughout the experimental period. There were no remarkable differences in the toxin proportion between mussel and A. tamarense. These results indicate that mussels do not selectively accumulate particular toxins.     

Biomarkers of environmental contaminants in field population of green mussel (Perna viridis) from Karnataka–Kerala coast (South West coast of India)

The green mussel Perna viridis was sampled from relatively clean and contaminated sites along the Kartanata–Kerala coast (south west coast of India) to study the tissue concentration of trace metals and biological responses to stress (biomarkers) such as sister chromatid exchange (SCE), chromosomal aberration, micronucleus (MN) test, hemic neoplasia (HN), Chromotest (Ames test) and comet assay. In general, mean tissue concentrations of toxic trace metals collected from 25 sampling sites were found to be below the World Health Organisation (WHO) permissible concentration given for seafood. The digestive gland extract of mussels from all 25 sampling sites showed negative reaction for mutagenic activity (Ames test) in the absence of metabolic activation. Very low levels of chromosomal aberration, SCE, MN, HN and comet cells were observed in mussels collected from both the urban associated and relatively clean sites. This study seems to indicate that that the coastal waters of Karnataka and Kerala are minimally contaminated with genotoxic and carcinogenic chemicals.Special Issue on Biomarkers of Marine Pollution and Bioremediation     

Genotyping and in vitro antifungal susceptibility of Neoscytalidium dimidiatum isolates from different origins

This study evaluated the genetic variability and in vitro susceptibility patterns of isolates of Neoscytalidium dimidiatum and Scytalidium hyalinum from different geographical origins. Partial sequences of four loci (the ITS region and D1/D2 domains of the 28S rRNA gene and the tubulin and chitin synthase genes) were analysed. Among a total of 1970 bp sequenced in 24 isolates, 7 polymorphic positions (0.36%) were detected, representing five different sequence types (ST1–ST5), from which two (ST2 and ST3) were detected exclusively in isolates from plants, two (ST1 and ST5) were found only in clinical isolates and one (ST4) was observed in isolates from humans and from a mango tree. We propose subordinating S. hyalinum as a variety of N. dimidiatum. Amphotericin B was the most active drug, but low minimum inhibitory concentrations were also detected for voriconazole, terbinafine and anidulafungin.     

Metallothioneins and heat shock proteins 70 in marine mussels as sensors of environmental pollution in Northern Adriatic Sea

In an attempt to assess the intensity of environmental pollution in industrial zones of Kvarnerian Bay in Northern Adriatic Sea and the reactivity of Mytilus galloprovincialis to these changes, in this study we estimated the concentration of heavy metals at four locations in both sea-sediment and in the mussels. Further we tried to correlate these changes with seasonal variations in environmental temperature, pH and salinity, as well as with the expression of metallothioneins (MTs) and heat shock proteins (HSPs) in the digestive tract of the mussels. Sampling in vivo was performed monthly, during the year 2008, while under the laboratory conditions the reactivity of acclimated mussels were tested to increasing concentrations of CdCl₂ and to thermal stress. The data have shown that the induction of MTs and HSP isoforms of the 70-kDa size class were highly affected by model agents treatment including contamination of sea-sediment by Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution.     

Cytotoxicity of Au,ZnO and SiO2 NPs using in vitro assays with mussel hemocytes and gill cells: Relevance of size,shape and additives

Metal-bearing nanoparticles (NPs) possess unique physico-chemical characteristics that make them useful for an increasing number of industrial products and applications, but could also confer them a higher toxicity due to their higher reactivity compared to bulk forms of the same materials. There is a considerable interest in the use of in vitro techniques in environmentally relevant species, such as marine mussels, to evaluate NPs toxicity. In the present work, mussel hemocytes and gill cells were used to assess the potential toxic effects of Au, ZnO and SiO₂ NPs with different sizes and shapes in parallel with their respective ionic and bulk forms and additives used in the NPs preparations. Cytotoxicity (neutral red and MTT assays) was screened at a wide range of concentrations, and LC50 values were calculated. Uptake of fluorescently labeled SIO₂ NPs of 27?nm by hemocytes was also investigated. Au, ZnO and SiO₂ NPs were less toxic than the corresponding ionic forms but more toxic than the bulk forms. ZnO NPs were the most toxic NPs tested which could be related with their capacity to release free ions. SiO₂ NPs were not taken up by hemocytes and were not toxic to either hemocytes or gill cells. Size-dependent toxicity was found for Au NPs. Shape influenced the cytotoxicity of ZnO NPs. Finally, the presence of the additives Na-citrate and Ecodis P90 contributed to the toxicity of Au and ZnO NPs, respectively. As a general conclusion, solubility appears to play a key role in NPs toxicity to mussel cells.     

The effect of Nepeta cataria extract on adherence and enzyme production of Staphylococcus aureus

Nepeta cataria L., commonly known as catnip, is a perennial herb with a considerable folkloric reputation. A diethyl ether extract of this plant has been shown to have antimicrobial activity against fungi and Gram-positive bacteria. The aim of this work was to study the activity of N. cataria extract on 44 Staphylococcus aureus strains, some resistant to methicillin, and S. aureus 6538P (American Type Culture Collection) by evaluating the effect of subminimum inhibitory concentrations on coagulase, DNAse, thermonuclease and lipase production, and on in-vitro adherence. DNAse, thermonuclease and lipase were inhibited by concentrations equal to 1/2 and 1/4 MIC. A reduction of adherence was also observed.     

Clinical characteristics of infections caused by Tsukamurella spp. and antimicrobial susceptibilities of the isolates

To investigate the clinical and microbiological characteristics of infections caused by Tsukamurella spp., the computerised database of the Bacteriology Laboratory at National Taiwan University Hospital (Taipei, Taiwan) was reviewed retrospectively to identify patients with infections caused by this species during the period January 1997 to December 2008. All of the isolates had been initially misidentified as Rhodococcus spp. Identification of Tsukamurella isolates to species level was carried out by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the heat shock protein gene (hsp65) as well as 16S rRNA gene sequencing. During the study period, a total of eight patients with Tsukamurella infection and two patients with Tsukamurella colonisation were identified. Tsukamurella tyrosinosolvens (n = 6) was the most prevalent species, followed by Tsukamurella spumae (n = 3) and Tsukamurella pulmonis (n = 1). Keratitis was the most common type of infection (n = 3), followed by catheter-related bloodstream infection (n = 2). One of the patients with Tsukamurella infection died due to bacteraemia; the other seven patients with Tsukamurella infection had favourable outcomes. The three species had different drug susceptibility patterns; T. pulmonis was the most resistant pathogen, with higher minimum inhibitory concentrations of clindamycin (>2 mg/L), erythromycin (2 mg/L) and tetracycline (8 mg/L) than those for the other Tsukamurella spp. In conclusion, strains of Tsukamurella spp., including T. spumae, are uncommon causative agents of ocular infections and bacteraemia in cancer patients. Molecular diagnostic methods are essential to distinguish species in the Tsukamurella genus from species in other phylogenetically related genera such as Rhodococcus.     

Distinction of water-soluble constituents between some Paecilomyces (= Isaria) and Cordyceps fungi by capillary electrophoresis

In order to reveal the differences between Cordyceps, Paecilomyces (= Isaria) and Nomuraea, we collected seven entomogenous fungi grown in natural field and analyzed the profiles of water-soluble constituents derived from some different sources of Cordyceps, Paecilomyces and Nomuraea by determination using capillary electrophoresis. C. sinensis and C. kyushuensis showed similar peak clusters of protein migrated at 5–7, 8–9, and 12–20 min. The peak clusters obtained from N. atypicola was similar to those of C. sinensis and C. kyushuensis. The water-soluble constituent clusters of C. militaris migrated at 5–9 and 10–15 min were partly different from those of other Cordyceps. It was also revealed that the P. tenuipes and P. cicadae showed lesser peak clusters rather than Cordyceps. These results indicated that the profiles of protein of these entomogenous fungi by capillary electrophoretic analysis could serve as fingerprints for classification and medicinal quality control of Cordyceps.     

The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: The tunicate Pyura chilensis (piure)

The tunicate Pyura chilensis (Molina, 1782); Phylum Chordata; Subphylum Urochordata; Class Ascidiacea, common local name “piure” or sea squirt; a filter-feeder (plankton and suspended particles) sessile species; may play an important role in monitoring domoic acid (DA) the principal toxic component of Amnesic Shellfish Poisoning (ASP). Significant DA concentrations have been determined in tunicate samples, collected during a recent ASP outbreak in Bahía Inglesa, an important scallop (Argopecten purpuratus) farming area. Several infaunal species were tested for the presence of DA, in addition to the usual scallop monitoring programme. DA was found at sub-toxic levels in filtering bivalves such as mussels (Mytilus chilensis), large mussels (Aulacomya ater) and clams (Protothaca thaca) (6.4, 5.4 and 4.7 μg DA/g tissue respectively). Of particular interest was the observation of significant accumulations of toxic Pseudo-nitzschia sp. diatoms in the internal siphon and atrium spaces of the tunicate.Toxin distribution within major tunicate organs was heterogeneous with 8.7–15.5 μg DA/g in edible tissues, 14.9–17.9 μg DA/g in the fecal material and 13.6–32.7 μg DA/g in the gut content. DA was determined by HPLC–UV and confirmed by diode-array detection and LC–MS/MS analysis. This is the first report of the presence of DA in a tunicate that is regularly consumed by coastal populations. These results confirm the need to include these organisms in sanitation programs for marine toxins.     

An in vivo study of nanorod,nanosphere, and nanowire forms of titanium dioxide using Drosophila melanogaster: toxicity,cellular uptake,oxidative stress,and DNA damage

ABSTRACT

The biological impact of nanomaterials (NMs) is determined by several factors such as size and shape, which need to be taken into consideration in any type of analysis. While investigators often prefer to conduct in vitro studies for detection of any possible adverse effects of NMs, in vivo approaches yield more relevant data for risk assessment. For this reason, Drosophila melanogaster was selected as a suitable in vivo model to characterize the potential risks associated with exposure nanorods (NRs), nanospheres (NSs), nanowires (NWs) forms of titanium dioxide (TiO₂), and their microparticulated (or bulk) form, as TiO₂. Third instar larvae (72 hr old larvae) were fed with TiO₂ (NRs, NSs, or NWs) and TiO₂ at concentrations ranging from 0.01 to 10 mM. Viability (toxicity), internalization (cellular uptake), intracellular reactive oxygen species (ROS) production, and genotoxicity (Comet assay) were the end-points evaluated in hemocyte D. melanogaster larvae. Significant intracellular oxidative stress and genotoxicity were noted at the highest exposure concentration (10 mM) of TiO₂ (NRs, NSs, or NWs), as determined by the Comet assay and ROS analysis, respectively. A concentration–effect relationship was observed in hemocytes exposed to the NMs. Data demonstrated that selected forms of TiO₂.-induced genotoxicity in D. melanogaster larvae hemocytes indicating this organism is susceptible for use as a model to examine in vivo NMs-mediated effects.     

荜茇提取物对地鼠脂质代谢紊乱的影响

目的研究荜茇提取物对高血脂症地鼠血脂的影响。方法给予高脂饲料建立金黄地鼠高血脂模型,将动物随机分为正常对照组、模型组、辛伐他汀组、荜茇提取物组、去胡椒碱荜茇提取物组,除正常对照组外其余各组均给予高脂饲料喂养2或4周,每周测量体质量,分别在第2周末和第4周末用全自动生化分析仪检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、肝脏指数、肝脂质TC和TG。结果与正常对照组比较,模型组TC,TG和LDL显著增加(P<0.01),HDL显著降低;与模型组比较,荜茇提取物组在第2周和第4周均能显著降低TC,TG和LDL,显著升高HDL(P<0.05或P<0.01);去胡椒碱荜茇提取物组在第2周能显著降低LDL,升高HDL,在第4周有降低TC,TG,LDL的趋势,但差异无统计学意义,能显著升高HDL(P<0.05);各受试物组动物在前两周体质量显著低于模型组,而后2周差异无统计学意义;4周时荜茇提取物组能显著降低肝脂质TC,TG;各受试物组均能显著降低肝指数。结论荜茇提取物可显著改善高血脂症地鼠血脂水平,其中胡椒碱为其主要有效成分,且与其他各成分有协同降血脂作用。     

Effects of guggulsterone isolated from Commiphora mukul in high fat diet induced diabetic rats

Sedentary lifestyle, consumption of energy-rich diet, obesity and longer lifespan are some of the major reasons for the rise of metabolic disorders like type II diabetes, obesity, hypertension and dyslipidemia among people of various age groups. High fat diet induced diabetic rodent models resembling type II diabetic condition in human population were used to assess the anti-diabetic and hypolipidemic activity of guggulsterone (isolated from Commiphora mukul resin). Four groups of rats were fed high fat diet, for 16 weeks. On feeding the normal rats with fat rich diet they showed increased serum glucose, cholesterol and triglyceride levels along with increase in insulin resistance significantly (p < 0.05) in comparison to control animals. Different biochemical parameters like GTT, glycogen content, glucose homeostatic enzymes (like glucose-6-phosphatase, hexokinase), insulin release in vivo and expression profiles of various genes involved in carbohydrate and lipid metabolism clearly demonstrated the hypoglycemic effect of this extract. Guggulsterone demonstrated a differential effect with a significantly improved PPARγ expression and activity in in vivo and in vitro conditions, respectively. However, it inhibited 3T3-L1 preadipocytes differentiation in vitro. The results presented here suggest that the guggulsterone has both hypoglycemic and hypolipidemic effect which can help to cure type II diabetes.     

京尼平苷体内外微透析回收率的研究

目的 探究京尼平苷在自制线性微透析探针上的体内外回收率与灌流速度、药物浓度、透析膜长之间的关系。方法 采用HPLC测定微透析样品浓度,考察不同灌流速度、不同药物浓度和不同透析膜长对体外正向和体外反向回收率、体内反向回收率的影响。结果 自制探针性质稳定;探针回收率与京尼平苷的药物浓度无关,与灌流速度成反比,随透析膜长增加而增加;体外的正向与反向探针回收率有显著性差异（P<0.01）,而体内反向回收率低于体外反向回收率,与体外正向回收率无显著性差异;新探针体内回收率大于使用过1次的探针体内回收率（P<0.05）。结论 在京尼平苷的皮肤药动学研究中,宜采用体内反向回收率或体外正向回收率作为药物的探针回收率来校正。     

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs < 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC > 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.     

刺老苞根皮总提物对去势大鼠骨质疏松的影响

目的 研究刺老苞Araliae Echinocaulis Radicis et Cortex根皮总提取物对去势雌性大鼠骨质疏松症的影响。方法 75只SD雌性大鼠随机分成对照组、假手术组、模型组、仙灵骨葆胶囊组和刺老苞根皮总提取物组,每组15只。经去势手术2周后,仙灵骨葆胶囊组、刺老苞根皮总提取物组ig相应药物悬液(20 mg/kg),每天1次,连续12周。观察大鼠给药前后体质量变化;试剂盒法检测血清总胆固醇(TC)、高密度脂蛋白-胆固醇(HDL-C)、低密度脂蛋白-胆固醇(LDL-C)、碱性磷酸酶(ALP)水平;取右侧股骨检测骨密度、骨微量元素和羟脯氨酸(Hyp)水平;取左侧股骨进行骨力学测定。结果 与对照组比较,模型组大鼠血清TC、LDL-C水平均显著升高(P<0.01),血清HDL-C、ALP、骨微量元素(钙、磷、镁、锰)、Hyp水平,骨密度、骨力学指标均显著下降(P<0.01);与模型组比较,刺老苞根皮总提取物组与仙灵骨葆胶囊组TC、LDL-C水平均显著降低(P<0.05),血清HDL-C、ALP、骨微量元素(钙、磷、镁、锰)、Hyp水平,骨密度、骨力学指标均显著升高(P<0.05)。结论 刺老苞根皮总提取物有效改善去势雌性大鼠骨质疏松症的发生。     

Genotoxic effects of glyphosate on Physalaemus tadpoles

Genotoxicity studies have revealed that pesticides bind to genetic material in non-target vertebrates, thereby impairing the genetic integrity of these animals. The main objective of this study was to determine the genotoxic damage in erythrocytes of two native South American amphibian Physalaemus cuvieri and Physalaemus gracilis, both species exposed to a glyphosate-based herbicide. We evaluated the presence of micronuclei (MN) and erythrocyte nuclear abnormalities (ENA) as biomarkers for potential genotoxic compounds. Tadpoles were exposed to doses permitted by Brazilian legislation and concentrations found naturally in Brazilian and Argentinian waters (500, 700 and 1000 μg/L). Glyphosate-based herbicide caused micronuclei formation and several types of erythrocyte nuclear abnormalities in both Physalaemus species. The total frequency of MN and ENA demonstrated the occurrence of cell damage at all tested concentrations. Glyphosate herbicide can be considered a genotoxic that may impact the genetic integrity of native populations of P. cuvieri and P. gracilis.     

中药葛根及其同属植物的资源利用和评价

对我国葛属(Pueraria DC.)9种和1变种植物进行了调查研究,认为目前国内市售中药葛根的主流品种为野葛P.lobata(Wild.)Ohwi和粉葛P.thomsoni Benth.。野葛在我国大部份省区都有分布,资源最为丰富;粉葛在广西载培历史悠久;其他种主要分布在西南地区,并有少数种在当地作中药葛根用。从成分分析结果看,野葛的葛根素(puerarin)及总黄酮含量显著高于本属其它植物,粉葛次之。除野葛和粉葛外其他种均不宜作葛根药用。峨嵋葛P.omeiensis Wang et Tang是个有争议的种,我们比较了化学成分的含量,其结果与粉葛相近,而与山葛藤有所区别,有待进一步研究。     

Induction of micronuclei and nuclear lesions in Channa punctatus following exposure to carbosulfan,glyphosate and atrazine

Abstract

The genotoxic effects of commonly used agricultural pesticides viz., carbosulfan, glyphosate, and atrazine, were evaluated in Channa punctatus (Pisces, Perciformes) using micronucleus (MN) test and induction of nuclear lesions (NL). The 96?h LC₅₀ value were estimated by probit analysis as 0.27, 32.0 and 42.0?mg?L^?1, respectively, for carbosulfan, glyphosate, and atrazine using semi-static bioassays. Based on these values, three sublethal test concentrations of carbosulfan (0.07, 0.13, 0.20?mg?L^?1), glyphosate (8.1, 16.3, 24.4?mg?L^?1) and atrazine (10.6, 21.2, 31.8?mg?L^?1) corresponding to ¼, ½ and ¾ of the LC₅₀ of the pesticides respectively, were selected for exposure for 96?h. Peripheral blood samplings were taken at intervals of 24?h for assessment of MN and NL frequencies. Considerably higher genotoxic damage was induced by carbosulfan as compared to glyphosate and atrazine. There were significant effects (p?<?0.01) of concentrations in all the treated groups. The induction of MN and NL was highest at 96?h pesticide exposure at all test concentrations. The nuclear abnormalities recorded in this study, such as blebbed-, lobed-, notched- and bi-nuclei, other than micronuclei, are indicators of genotoxic damage.