Sublethal effects of pesticide mixtures on selected biomarkers of Carassius auratus

In the present study, the in vivo effects of commercial propoxur alone and in combination with isoprocarb and chlorpyrifos on acetylcholinesterase and glutathione S-transferases activities in goldfish (Carassius auratus) were investigated. Brain and muscle AChE activity was significantly inhibited (88% and 85% at most, respectively) by propoxur after 5 d of exposure at concentrations between 141 and 2263 μg/L, bell-shaped concentration–response curves were obtained. In most cases, time dependence was apparent. Under insecticide mixture exposure (binary mixture propoxur/isoprocarb and ternary mixture propoxur/isoprocarb/chlorpyrifos), the inhibition of AChE activity was significant in all cases and linear concentration–response relationships were observed. Propoxur (alone and in combination) significantly inhibited gill GST activity, and both mixtures also induced significant inhibition of liver GST activity. However, concentration and time dependence were not apparent. AChE is likely to be more informative than GST and seems to be a good biomarker to diagnose exposure to anticholinesterase pesticides in wild populations of this species.     

Responses of AChE and GST activities to insecticide coexposure in Carassius auratus

Organophosphates and carbamates are widely used pesticides and play an important role in global agriculture. The misuse of these compounds has caused environmental problems and has had a negative impact on wildlife. In this study, the in vivo effects of commercial chlorpyrifos and isoprocarb on acetylcholinesterase (AChE) and glutathione S‐transferase (GST) activities in goldfish (Carassius auratus) were investigated. Muscle and brain AChE activity was significantly inhibited by chlorpyrifos and isoprocarb (alone and in combination) after 2, 5, 10, and 15 days of exposure, and obvious concentration‐response and time‐response relationships were obtained. Gill GST activity was significantly inhibited by chlorpyrifos and isoprocarb (single compounds and in combination), however, concentration dependence and time dependence were not apparent. The joint effect of chlorpyrifos/isoprocarb was additive with regard to AChE activity inhibition and was antagonistic with regard to GST activity inhibition. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Single and combined effects of selected pharmaceuticals at sublethal concentrations on multiple biomarkers in Carassius auratus

In this study, the sublethal effects of caffeine, sulfamethoxazole and their mixture on goldfish (Carassius auratus) were investigated, the biomarkers including acetylcholinesterase (AChE) in brain, 7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST) and superoxide dismutase (SOD) in liver and vitellogenin (VTG) in serum were determined after 1, 2, 4, and 7 days of exposure. AChE activity was significantly inhibited by caffeine (≥0.4 mg/l), sulfamethoxazole (≥0.4 mg/l) and their mixtures (≥0.048 mg/l) during all exposure periods, and obvious concentration-response and time-response relationships were obtained. EROD, GST and SOD activities were significantly increased by individual compounds and mixtures in most cases. GST induction exhibited bell-shaped concentration-response curves. Serum VTG was significantly induced by 2 mg/l of caffeine, 10 mg/l of sulfamethoxazole and the mixtures at concentrations ≥1.2 mg/l. In general, the two pharmaceuticals induced similar biological responses. The joint effect of caffeine/sulfamethoxazole was additive with regard to AChE and GST activity variation and was antagonistic with regard to EROD and SOD induction. The results indicated that multiple biomarker response method might be a useful tool for describing an integrated toxicological effect of chemicals. VTG induction suggested that caffeine and sulfamethoxazole may cause a slightly feminization effect.     

Ultrastructural patterns of secretory activity in poison cutaneous glands of larval and juvenile Dendrobates auratus (Amphibia, Anura).

A transmission electron-microscope study has been performed on larval and juvenile skin of the Central American arrow-frog Dendrobates auratus to investigate early secretory processes and maturational changes in the serous (poison) glands. Poison biosynthesis involves the endoplasmic reticulum (both smooth and rough types), as well as Golgi stacks which release early serous product as secretory vesicles (or pre-granules). These vesicles contain fine-grained material, along with single electron-opaque bodies, spheroidal in shape, that accompany the grained product throughout its post-Gogian, maturational change. The first steps of this process involve condensation and lead to the formation of secretory granules with a glomerular-like substructure, resulting from a thick, random aggregation of rods (secretory granule subunits). Advanced maturational activity causes the loss of peculiar granule substructure: the dense bodies split into fragments, whereas the thick glomerular arrangement becomes looser, until the secretory product changes into a dispersed material.This ultrastructural study revealed biosynthesis and maturation processes in close sequence, suggesting the poison of D. auratus contains proteins and/or peptides as well as lipophilic compounds. Molecules of both these classes are known to perform several roles relevant to survival strategies in extant anurans. Furthermore, the ephemeral granules with a glomerular-like substructure detected in tadpoles and froglets exhibit the complex patterns of mature poisons in adult specimens of other anurans: Hylidae and related families. This agrees with current trends in the taxonomy of these advanced frogs and underlines the pertinence of an ontogenetic approach in investigating anuran phylogenesis.     

Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations

Acrylamide is an amide used in several industrial applications making it easily discharged to aquatic ecosystems. The toxicity of acrylamide to aquatic organisms is scarcely known, although previous studies with murine models provided evidence for deleterious effects. To assess the effects of acrylamide to freshwater fish, goldfish (Carassius auratus L.) were exposed to several concentrations of waterborne acrylamide and analysed for genotoxic damage, alterations to detoxifying enzymes and histopathology. Results revealed a dose‐dependent increase in total DNA strand breakage, the formation of erythrocytic nuclear abnormalities and in the levels of hepatic cytochrome P4501A (CYP1A) and glutathione S‐transferase (GST) activity. In addition, acrylamide induced more histopathological changes to pancreatic acini than to the hepatic parenchyma, regardless of exposure concentration, whereas hepatic tissue only endured significant alterations at higher concentrations of exposure. Thus, results confirm the genotoxic potential of acrylamide to fish and its ability to induce CYP1A, probably as a direct primary defence mechanism. This strongly suggests the substance's pro‐mutagenic potential in fish, similarly to what is known for rodents. However, the deleterious effects observed in the pancreatic acini, more severe than in the liver, could indicate a specific, albeit unknown toxic mechanism of acrylamide to fish that overran the organism's metabolic defences against a chemical agent rather than causing a general systemic failure. Copyright © 2013 John Wiley & Sons, Ltd.     

Biochemical biomarkers in liver and gill tissues of freshwater fish Carassius auratus following in vivo exposure to hexabromobenzene

Hexabromobenzene (HBB) is a novel brominated flame retardant (BFR) with ample evidence of its ubiquitous existence in the aquatic ecosystems. However, to date, the toxicological effects of this BFR on fish have been inadequately researched. The present study was conducted, based on an in vivo model, to investigate HBB‐induced biochemical changes in liver and gill tissues of Carassius auratus after medium‐term exposure to different concentrations (10, 150, and 300 mg/kg) for 7, 14, and 25 days. Oxidative stress was evoked evidently for the prolonged exposure, demonstrated by significant inhibition in antioxidant enzymes activities including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione S‐transferase, and a decrease in reduced glutathione level, as well as simultaneous elevation in malondialdehyde content. Moreover, Na⁺, K⁺‐ATPase activity, and protein level were remarkably reduced in fish tissues. Based on the integrated biomarker response, the toxic potency in each treatment was distinguished, and the more severe stress was mainly noted with the increasing concentrations and the extending durations. It was also observed that liver exhibited more pronounced alterations in biochemical parameters than gill, probably indicating the vulnerability of liver to HBB‐triggered oxidative stress. Taken together, the results of this study clearly showed that HBB was capable of inducing oxidative stress and inhibiting Na⁺, K⁺‐ATPase activity in different tissues of C. auratus after medium‐term exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1460–1470, 2014.     

Toxic effects induced by salt stress on selected freshwater prokaryotic and eukaryotic microalgal species

In order to determine the short-term impact induced by salt stress, cultures of Dictyosphaerium chlorelloides and Microcystis aeruginosa were grown in presence of increasing sea-salt concentrations. Growth rate and photosystem II activity in D. chlorelloides, and photosynthetic oxygen production (in both species) were analyzed. A concentration-dependent response was obtained with the presence of sea-salt in culture medium, being M. aeruginosa (EC₅₀₍₇₂₎ = 76.6 mM) more sensitive to salt stress than D. chlorelloides (EC₅₀₍₇₂₎ = 340.7 mM). However, comparative analysis between growth and Φ_PSII inhibition in D. chlorelloides shown that there are not significant differences among EC₅₀₍₇₂₎ values obtained. An immediate toxic response, induced by increase of sea-salt concentration, has been obtained applying the calculated EC₅₀₍₇₂₎ values in both species. These results shown that sea-salt acts as a sensitive and rapid toxic compound in algal cells, and that the sensitivity of M. aeruginosa to salinity stress is much higher than that of D. chlorelloides.     

Hematological effects of exposure to mixtures of selected ethylene glycol alkyl ethers in rats

Exposure to various ethylene glycol monoalkyl ethers (EGAEs) is known to result in hemolytic effect caused by their metabolites, appropriate alkoxyacetic acids, generated via both alcohol dehydrogenase and aldehyde dehydrogenase. It has been shown in many studies that administration of single doses of EGAEs to rats lead to dose- and time-dependent hemolytic anemia. The repeated exposure to isopropoxyethanol (IPE), and butoxyethanol (BE), contrary to methoxyethanol (ME) and ethoxyethanol (EE), resulted in significantly less pronounced hematological changes. While the majority of hematological effects were dramatic at the beginning of the exposure, later these changes clearly regressed despite continued weekly exposure to these ethers. The gradual recovery from the hemolytic anemia may be associated with tolerance development to the hemolytic effect of IPE and BE. ME demonstrated high hematotoxicity, which increased progressively and reached a maximum at the end of 4 week exposure, whereas EE revealed moderate hematological effects. It might be suspected that ME and EE may modified of IPE hemolytic activity in rats simultaneously treated with these compounds. In the rats co-exposed to IPE and ME subcutaneously at a relatively low doses of 0.75 mM + 0.75 mM for 4 weeks, a significantly less pronounced hematological changes at the beginning of the exposure in comparison with animals treated with IPE (0.75 mM) alone were observed. At the later period, i.e., at the end of 4 weeks exposure, the hematological alterations in the same animals were markedly pronounced and progressively elevated with exposure time, except for mean corpuscular volume (MCV) values, which were significantly lower in comparison with IPE group. ME at the higher dose of 1.25 mM/kg and EE at both doses of 0.75 and 1.25 mM/kg did not modify the hematotoxicity of IPE (at doses of 0.75 mM and 1.25 mM) at the beginning of the exposure, whereas increased its harmful effects at the end of the treatment. The amelioration in the majority of the hematological parameters at the beginning of the exposure may be caused by inhibitory effect of ME on IPE metabolism. On the contrary, an accumulation of the methoxyacetic acid and ethoxyacetic acid, toxic metabolites of ME and EE, respectively, and no tolerance development to the hemolytic effect of these two chemicals may be responsible for elevated hematological alterations at the end of the exposure.     

鹿药属植物化学成分及药理作用研究进展

鹿药Smilacina japonica是多年生百合科草本植物,有悠久的药用历史,尤其在民族药方面应用较多,作为药食同源植物近年来需求增加,因此对鹿药属植物化学成分和药理作用的研究增多。鹿药属植物含有甾体、黄酮、多糖、氨基酸、酚酸、酰胺等多种活性成分,具有抗菌、抗肿瘤、抗氧化等药理活性。根据国内外文献报道,总结并综述了鹿药、管花鹿药S.henryi、高大鹿药S.atropurpurea等鹿药属植物的化学成分及药理作用的研究进展,以期为鹿药属植物资源的开发及医药保健方面的合理应用提供参考。     

肾毒性小分子代谢标志物在5种中药毒性评价的适用性研究

目的考察前期发现的肾毒性小分子代谢标志物在中药毒性评价中的适用性。方法以已知具有肾毒性的5种中药雷公藤、马钱子、广防己、大黄和苍耳子提取液ig给予大鼠建立肾脏损伤模型,收集给药1和7 d后的血样,应用超高效液相色谱-质谱联用（UPLC/Q-TOF-MS）检测5种肾毒性生物标志物胸苷、溶血磷脂酰胆碱LPC（16：1）、LPC（18：4）、LPC（20：5）和LPC（22：5）水平,建立支持向量机（SVM）预测模型对其毒性进行判断;全自动生化仪测定血清中Cr和BUN的水平;各组大鼠于取血后处死,迅速取肾脏进行HE染色,光学显微镜下观察病理表现。结果对照组没有表现出毒性。5种中药在给药1 d后,生化检测没有发现肾脏损伤,肾毒性支持向量机预测模型发现异常;在给药7天后,SVM的预测结果与生化和病理检测结果一致,均出现肾毒性。结论代谢组学技术结合支持向量机模型可将肾毒性小分子代谢物更加灵敏、快速、准确地用于中药肾脏毒性评价,对于临床药源性肾损伤的防治具有重要意义。     

不同产地藤茶中二氢杨梅素含量及其与牛蒡子配伍的药效学研究

目的 探究不同产地、部位及加工工艺藤茶中二氢杨梅素含量的差异,及优选藤茶与牛蒡子配伍药效学研究。方法 二氢杨梅素高效液相方法学验证采用Agilent ZORBAX SB-C₁₈柱,流动相为甲醇-0.05 % 磷酸（30∶70）,流速为1 ml/min,检测波长为291 nm,柱温25 ℃。配伍药效验证方法采用大鼠棉球植入致炎及小鼠耳肿胀致炎模型,对大鼠棉球植入实验肉芽肿净量及小鼠耳肿胀率进行观察。结果 方法学验证二氢杨梅素在0.019 9～0.318 mg/ml范围内线性关系良好（r=0.999）,回收率在95.04 %～100.4 %之间,样品在24 h内稳定,该方法重复性较好。配伍药效学验证高剂量优选藤茶与牛蒡子配伍可致大鼠肉芽肿净量及小鼠耳肿胀率均显著低于空白对照组。结论 方法简便准确,二氢杨梅素在不同产地、部位及加工工艺中含量差异较大,其中以贵州省江口县自然晒干的芽尖部位藤茶含量最高。藤茶与牛蒡子配伍可显著改善咽部症状,减轻致炎程度,共同协同达到清咽效果。     

2种藏紫草体外抗氧化及抗菌活性比较

目的 比较长花滇紫草和团花滇紫草75％乙醇提取物的体外抗氧化及抗菌活性.方法 采用ABTS法、FRAP法、邻苯三酚自氧化法测定体外抗氧化能力,并采用肉汤连续稀释法初步考察了 2种藏紫草体提取物体外对15种不同菌株的抗菌作用.结果 不同浓度藏紫草的75％乙醇提取物对ABTS自由基和超氧阴离子都有较强的清除能力,与BHT阳...     

Evaluation of cytotoxicity and genotoxicity of pesticide mixtures on lymphocytes

The cytotoxicity and genotoxicity of pesticide mixtures viz. endosulfan?+?chlorpyrifos, chlorpyrifos?+?profenofos, and endosulfan?+?profenofos were evaluated on cultured human peripheral blood lymphocytes using assays for cell viability, and genotoxicity using chromosomal aberrations test and comet assay. The LC₅₀ values for cytotoxicity were 3.50?μM, 4.18?μM, and 10.5?μM for profenofos, endosulfan, and chlorpyrifos respectively. When combined in equimolar concentrations, the LC₅₀ values for cytotoxicity were 1.4?μM, 1.8?μM, and 2.0?μM for endosulfan?+?chlorpyrifos, chlorpyrifos?+?profenofos, and endosulfan?+?profenofos, respectively. Higher concentrations of individual pesticides (0.5–4.0?μM) but very low concentrations of pesticide mixtures caused significant DNA damage. Additive index values indicated a synergistic effect of toxicity for endosulfan?+?chlorpyrifos combination (1.12 TTU). The binary mixture of chlorpyrifos?+?profenofos showed an additive toxicity (0.46 TTU) while an antagonistic effect was observed for endosulfan?+?profenofos combination. Synergism could be due to these complementary pesticides simultaneously acting in different ways, magnifying their efficacy, whereas an additive interaction would imply that the chemicals are acting by the same mechanism and at the same target. Analysis of toxicity of pesticide mixtures may serve as important biomarker for occupational and household exposure to pesticides, with different modes of action.     

Insecticidal effects of canatoxin on the cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae).

Canatoxin (CNTX) is a variant form of urease isolated from Canavalia ensiformis (Leguminosaea) seeds. A possible role in the plant defense was proposed for CNTX, due to its toxicity upon feeding to the beetle Callosobruchus maculatus, and the hematophagous bug, Rhodnius prolixus. The toxic effect is caused by a canatoxin-derived peptide ( approximately 10kDa) formed by insect cathepsin-like digestive enzymes. In order to evaluate their potential as bioinsecticides, the effects of CNTX and its peptide were evaluated on a phytophagous hemipteran insect Dysdercus peruvianus, a pest of cotton culture. For the bioassays, the insects fed on gelatin capsules containing powdered cotton seeds, mixed with the freeze-dried protein and other test materials and were observed for survival rate, weight gain and molting. Ingestion of canatoxin, or a recombinant 10kDa peptide derived from it, severely affected young forms of the insects, delaying their development or leading to their death. In contrast, adults were insensitive to diets containing higher concentrations of canatoxin. Cathepsin-like proteinases predominated and showed distinct pattern of enzymatic activities in midguts homogenates according to the developmental stage of the insect, a fact which may explain the different susceptibility of nymphs as compared to adult D. peruvianus. The data presented confirm the potential use of canatoxin-like proteins and derived peptides as bioinsecticides.     

鱼腥草不同溶剂提取物的抗病毒活性研究

目的 研究鱼腥草不同溶剂提取物体外抗手足口病毒（enterovirus type 71,EV71）、单纯疱疹病毒1型（herpes simplex virus type 1,HSV-1）、呼吸道合胞病毒（respiratory syncytial virus,RSV）、柯萨奇病毒B3型（coxsackie virus type B3,CV-B3）、柯萨奇病毒B5型（coxsackie virus type B5,CV-B5）的活性。方法 采用细胞体外培养技术,建立不同病毒感染模型,用鱼腥草不同溶剂提取物进行治疗,通过细胞融合病变观察法以及MTT比色法检测得到治疗指数（therapeutic index,TI）,以此判定其体外抗病毒活性。结果 体外抗病毒试验结果证明,鱼腥草不同溶剂提取物对HSV-1、EV71抗病毒作用较强,对RSV、CV-B3、CV-B5的效果甚微或无明显作用。鱼腥草醇提物中黄酮类成分含量较高,抗病毒活性相对较高;30%,50%,75%乙醇提取物以及水提醇沉处理沉淀物对HSV-1的TI值分别是81.68,67.23,41.91,32.61,均高于阳性对照阿昔洛韦TI值23.43,显示鱼腥草水醇提取物抗HSV-1的活性较强;75%,50%,30%乙醇提取物对EV71的TI值分别是19.58,20.13,18.84,效果较为显著。结论 研究证实鱼腥草提取物对HSV-1、EV71的抗病毒活性较高,可一定程度为临床应用鱼腥草治疗这2种病毒感染引发的疾病提供参考依据。     

左金方的化学成分与药理作用研究进展

左金方为元朝医家朱震亨创制的经典中药复方,始载于《丹溪心法》,仅由黄连、吴茱萸以6∶1比例组方,治疗肝火犯胃所致胁肋胀痛、吞酸呕吐等,如今不仅用于多种消化系统病症（如胃溃疡、胃炎、胃癌、幽门螺杆菌感染等）,还在精神症状、非胃肠道肿瘤、高血压、糖尿病等非消化系统疾病的治疗中起到较好疗效,具有抗炎抗溃疡、抗肿瘤、抗菌等多种作用。就近5年来对左金方的化学成分与药理作用的研究进展进行归纳总结,同时对其未来可能的研究方向进行了展望,以期为其进一步综合开发利用奠定基础。     

二至丸及其组成药味保肝药理作用研究进展

二至丸由女贞子和墨旱莲两味中药组成,这两味药及其组方均有不同程度的保肝作用。二至丸中含有多种保肝有效成分,主要有三萜类、环烯醚萜类、苯乙醇-苷类、香豆素醚类等,它们能对抗多种化学性肝损伤、抗缺血再灌注性肝损伤以及抗肝纤维化等,其保肝作用机制包括抗氧化应激、抗炎、抑制肝星状细胞增殖和活化、促进肝星状细胞凋亡以及影响肝细胞代谢过程等。总结二至丸及其组成药味女贞子和墨旱莲的保肝作用,探讨其保肝作用机制,以期为进一步研究二至丸药效物质基础提供参考。     

丰肉结海绵相关青霉菌HLS-216次级代谢产物研究

目的 研究我国南海水域丰肉结海绵相关青霉菌Penicillium sp.HLS-216的次级代谢产物的提取分离方法 、结构鉴定及其抗肿瘤、抗炎活性.方法 采用大米固体发酵培养,乙酸乙酯提取后,经硅胶柱色谱、Sephadex LH-20凝胶柱色谱、高级液相色谱等手段进行分离,并对分离得到的单体化合物应用质谱、核磁共振等技术进行结构鉴定;采用噻唑蓝(MTT)法和Griess法对分离得到的单体化合物进行抗肿瘤、抗炎活性筛选.结果 分离得到7个化合物,分别鉴定为:黑麦酮酸F(secalonic acid F,1)、黑麦酮酸D(secalonic acid D,2)、meleagrin(3)、oxaline(4)、对羟基肉桂酰胺(4-hydroxycinnamamide,5)、对羟基苯乙酸甲酯(methyl 4-hydroxyphenylacetate,6)、对羟基苯乙醛(4-hydroxyphenylacetonitrile,7).结论 化合物5和7为首次从青霉菌中分离得到;化合物1和2显示出较强的抗肿瘤活性,化合物4表现出一定的抑制小鼠腹腔巨噬细胞一氧化氮生成的作用.