围生期双酚A暴露对子代大鼠脾脏免疫功能的影响

为了研究围生期双酚A(BPA)暴露对子代脾脏免疫功能的影响,选择F344大鼠,按体重随机分为对照、低、中、高4个剂量组(BPA暴露剂量分别为0、4、40、400mg·kg-1·d-1),从雌鼠妊娠第0天开始至子鼠出生后30天,每天灌胃染毒1次,观察子鼠脾脏组织形态学的变化,并采用实时定量PCR检测IL-2、IL-12、IFN-γ、TNF-α4种细胞因子mRNA表达的改变.结果表明,1)与对照组相比,低剂量组子鼠脾重、脾脏指数均显著增加(p<0.05),体重无显著变化(p>0.05);中剂量组子鼠脾脏指数显著增加(p<0.05),体重和脾重无显著变化(p>0.05);高剂量组子鼠体重、脾重均显著降低(p<0.05),脾脏指数无显著变化(p>0.05).2)与对照组相比,各暴露组子鼠脾脏IL-2、IL-12、IFN-γ、TNF-α4种细胞因子mRNA表达均显著降低(p<0.01).以上结果提示,BPA可能能够透过胎盘屏障并通过乳汁传递给子代,降低子代的机体免疫功能.     

Effects of nonylphenol on endocrine regulation-associated gene expression profiles in whole brain of F1 generation male rats

In order to determine the effects of nonylphenol (NP) on endocrine regulation-associated gene expression profiles in whole brain of F1 generation rats, mRNA extracted from the brain of 2-day old F1 generation rats whose F0 male generation was treated with NP. The mRNA was then reversely transcribed to cDNA and labeled with cy5 and cy3 fluorescence. Subsequently, cDNA probes were hybridized to the Mouse40S cDNA microarray and the fluorescent signals of cy5 and cy3 were scanned and analyzed. Sixteen genes were identified which expressed differently, including 13 that were down-regulated in which 4 were related to brain regulation of endocrine function. Data suggest that NP mainly affects metabolism and synthesis of steroid hormone in various ways and disturbs the reproductive function of male rats when administered perinatally.     

Indium tin oxide nanoparticles-mediated DNA fragmentation and cell death by apoptosis in human lung epithelial cells

Indium tin oxide (ITO) nanoparticles (NP) have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and environmental impact have increased. Since exposure to ITO NP is mainly via skin and inhalation, this study was conducted utilizing human lung epithelial (A549) cell line. Cells were exposed to different concentrations of the ITO NP for 24 and 48 hr. A severe cytotoxic response of ITO NP was observed as evident by the (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red uptake assays after 48 hr exposure. ITO NP significantly reduced glutathione levels with a concomitant increase in lipid hydroperoxide levels, superoxide activity, and reactive oxygen species (ROS) generation after exposure. A significant induction in caspase activity and formation of condensed chromosomal bodies was also observed after ITO NP (10 or 25 µg/ml) exposure. Furthermore, a significant induction in DNA damage was observed by the Comet assay in cells exposed to ITO NP. Our data demonstrate that ITO NP display cytotoxic and genotoxic potential. However, increase in ROS levels and oxidative stress leading to oxidative DNA damage and condensed chromosomal bodies formation, suggests involvement of apotosis. Thus, ITO NP-mediated effects on cell viability indicate cytotoxicity, and therefore, exposures need to be carefully monitored in the industrial sector.     

Effects of nonylphenol on brain gene expression profiles in F1 generation rats

The effects of nonylphenol (NP) were examined on brain gene expression profiles of F1 generation rats by means of microarray techniques. The mRNA were extracted from the brain of two-day-old F1 generation rats whose F0 male generation were treated with NP, then reversely transcribed to cDNA and labeled with Cy5 and Cy3 fluorescence. Subsequently, the cDNA probes were hybridized to the mouse 40S cDNA microarray and the fluorescent signals of Cy5 and Cy3 were scanned and analyzed. Sixteen identified genes were expressed significantly differently from control, including 13 down-regulated, of which five were related to brain energy metabolism. Data suggest that it is possible that NP affects function of energy metabolism in male rats when administered perinatally.     

六溴环十二烷(HBCD)对H4细胞和SK-N-AS细胞脱碘酶2和3表达的影响

六溴环十二烷(hexabromocyclododecane,HBCD)与多溴联苯醚(polybrominated diethyl ethers,PBDEs)复合污染体系,对人类健康尤其神经系统所造成的潜在危害及其机制一直是笔者课题组的研究方向。HBCD是广泛使用的溴化阻燃剂,与PBDEs一样,会通过干扰内分泌系统、影响甲状腺激素分泌及损伤神经系统,对生物体产生发育神经毒性。作为系列研究之一,本研究以H4人脑神经胶质瘤细胞和SK-N-AS人神经母细胞瘤细胞为体外生物模型,通过观察HBCD对H4细胞Ⅱ型脱碘酶(Dio2)和SK-N-AS细胞Ⅲ型脱碘酶(Dio3)表达的调控,初步探讨了HBCD对神经系统局部甲状腺激素水平的潜在影响。H4细胞和SK-N-AS细胞分别暴露于0、1、3和9μmol·L-1HBCD 24 h后,采用MTT法检测细胞活力,Western Blot和RT-PCR法分别分析Dio2和Dio3蛋白和基因的表达,酶联免疫吸附测定(ELISA)法检测H4细胞脑源性神经细胞营养因子(BDNF)的分泌。结果表明,HBCD以剂量依赖方式降低H4细胞和SK-N-AS细胞生存率,引起H4细胞Dio2蛋白和基因表达下调,而致SK-N-AS细胞Dio3蛋白和基因表达上调。此外,HBCD还降低H4细胞BDNF的分泌。这表明,HBCD很可能通过影响神经和胶质细胞脱碘酶的表达,影响脑局部甲状腺激素水平,从而引起神经系统损伤及发育神经毒性。     

纳米氧化锌对羊角月牙藻毒性效应及其在藻细胞内外的分布

为研究纳米氧化锌(ZnO NP)的毒性效应及其在细胞内外分布,以羊角月牙藻(Selenastrum capricornutum)为模型藻类,研究了不同浓度ZnO NP对羊角月牙藻生长、叶绿素含量、可溶性蛋白含量、超氧化物岐化酶(SOD)及过氧化物酶(POD)活性、丙二醛(MDA)含量及细胞内外ZnO NP含量变化。结果表明,ZnO NP对羊角月牙藻的生长抑制与处理浓度呈现正相关。在45 mg·L~(-1)ZnO NP暴露24 h后,其生长抑制率已达到95%。当ZnO NP处理藻细胞72 h后,羊角月牙藻细胞的叶绿素含量与处理浓度之间存在剂量-效应关系。低浓度(0.5 mg·L~(-1))ZnO NP处理后藻细胞可溶性蛋白质含量、SOD和POD活性明显下降,MDA含量升高,其产生的毒性效应高于高浓度组(5 mg·L~(-1)、45 mg·L~(-1))。细胞培养液溶出Zn2+量及藻细胞外吸附的ZnO NP量与ZnO NP处理浓度成正比,但是藻细胞内ZnO NP量与ZnO NP浓度没有相关性,胞内积累量基本维持不变。研究表明,各浓度组对藻细胞毒性的差异,不仅与细胞内Zn2+量有关,还与细胞外粘附的ZnO NP有关。     

三(1,3-二氯-2-丙基)磷酸酯诱发肝脏损害及病理改变研究

本研究观测有机磷酯阻燃剂(OPFRs)污染是否可以诱发肝脏损害,考察其发生及发展程度,并探讨其发生机理,为有机磷阻燃剂污染的防治和相关疾病的有效治疗提供基础数据和科学依据。实验以大鼠为动物模型,将60只SPF级SD雄性大鼠分为5组,每组12只,选取典型的氯代有机磷阻燃剂三(1,3-二氯-2-丙基)磷酸酯(TDCPP)对大鼠进行染毒,空白对照组不做任何处理,溶剂对照组以相同体积的橄榄油灌胃,染毒组以不同剂量的TDCPP进行灌胃(125 mg·kg~(-1)·d~(-1)、250 mg·kg~(-1)·d~(-1)和500 mg·kg~(-1)·d~(-1)),每周测量体重,于第4周和第8周取血检测肝功及其他生化指标,在第8周每组抽取3只大鼠取肝脏组织做HE染色,并用透射电镜观察分析肝组织病理学改变。TDCPP对大鼠染毒8周后,结果表明:(1)体重指标在灌胃1周后开始发生差异,TDCPP处理组大鼠的体重有下降的趋势,染毒组与空白对照组和溶剂对照组相比较,差异显著(*P0.05,**P0.01),其中高剂量灌胃组的体重下降最为明显(**P0.01);(2)血清肝功指标表现出显著变化,血清谷丙转氨酶、谷草转氨酶、胆固醇和甘油三脂水平在第8周呈现明显下降趋势,染毒组与空白对照组和溶剂对照组比较,差异明显(*P0.05,**P0.01);(3) TDCPP暴露组生理生化指标变化明显,血清乙酰胆碱酯酶活性显著降低,MDA含量显著升高,SOD活力显著性降低,造成氧化损伤,与空白对照组和溶剂对照组比较,差异显著(*P0.05,**P0.01);(4)病理切片结果显示染毒组与对照组比较,细胞坏死现象明显,且高剂量组坏死更为严重。研究结果显示:TDCPP可引起大鼠体重明显下降,大鼠肝脏细胞损伤、合成功能下降,造成肝脏代谢功能紊乱,造成较为严重的肝损伤。     

Purification of metallothionein from thymus and spleen,induced by zinc administration

Zinc is an essential trace element to maintain human and generally mammal homeostasis; its biological signification could be explained through the participation or as structural element of more than 200 protein molecules in mammals.

The synthesis of proteins in spleen and thymus of rats is induced by zinc, a fact which moved us to purify and isolate such proteins, before proceeding to its identification or characterization. Rats are treated with zinc during 4 weeks and then sacrificed; blood is extracted under light ether anesthesia. The extracts of thymus and spleen were prepared by homogenization with Tris‐HC1 buffer 1.0 × 10^‐2M pH 7.4 and submitted to Sephadex G‐75 column (40 × 1.6 cm) after centrifugation and filtration through 0.45 μ filters. The elution profile of proteins were established at 253 and 280 nm, and zinc concentration was measured by atomic absorption spectrophotometry. The peak containing zinc was submitted to a second column of Sephadex G‐25 (40×1.6 cm) obtaining a peak of purified protein with physico‐chemical characteristics similar to metallothionein.     

Hepatoprotective activity of 1-(4-(Dimethylamino)Benzylidene)-5-(2-Oxoindolin-3-ylidene) Thiocarbohydrazone in rats

The aim of the present study was to evaluate the hepatoprotective activity of 1-(4-(dimethylamino)benzylidene)-5-(2-oxoindolin-3-ylidene) thiocarbohydrazone, a novel isatin derivative against carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats. Hepatic damage was induced by administration of CCl₄ (1 ml/kg, b.w., p.o.) in combination with liquid paraffin (1:1) as a single dose. The hepatotoxic rats were treated with test compound at doses of 50 or 100 mg/kg for three days and liver damage biomarkers, including activities of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), serum alkaline phosphatase (ALP), and levels of total serum bilirubin (TB) measured in blood samples. Results demonstrated that treatment with test compound at doses of 50 or 100 mg/kg to hepatotoxic rats produced a significant dose-dependent reduction of elevated SGOT, SGPT, ALP activities and TB levels indicating a hepatoprotective effect that was confirmed by histopathological examination of liver tissues. The study results confirmed the hepatoprotective activity of 1-(4-(dimethylamino)benzylidene)-5-(2-oxoindolin-3-ylidene) thiocarbohydrazone in rats.     

Determination of 4-nonylphenol in carp blood samples by solid phase extraction and chromatographic techniques

In this study, selective methods were developed for isolation, purification, separation and determination of 4-nonylphenol (4NP) in biological samples. Several methods such as high-performance liquid chromatography (HPLC) with fluorescence and diode array detection and gas chromatography coupled to mass spectromotry (GC-MS) are described for the simultaneous determination of 4NP. The nonylphenols in the plasma of fish were extracted through solid phase process by using octadecyl, polymeric and octadecyl endcapping sorbents. The average recoveries in plasma samples spiked with 4NP of levels 1?µg?mL^?1 were between 80–95% using added surrogate standards. The aim of this study was to determine whether trace amounts of 4NP in fish plasma samples could be detected by solid phase extraction and chromatographic methods (HPLC, GC-MS). This technique of monitoring the levels of endocrine-disruptors in plasma samples is consistent, reliable as well as inexpensive.     

Toxicological effects of mouse diet contaminated with tributyltin on the immune and neurological systems of C57BL/6 mice

Organotin compounds, widely used as antifouling agents, are known to bioaccumulate in the food chain. Among organotin compounds, tributyltin (TBT), a toxic and widespread contaminant, has become a serious factor in environmental pollution and is suspected of being immunotoxic in animals. The purpose of this study was to assess the effects of 80 µg of TBT per kilogram of body weight (kg bw), provided in food, on the immune and neurological systems of C57Bl/6 mice. Data showed that TBT increased the proliferation of T and B lymphocytes in both adult and juvenile female and juvenile male mice, but that it decreased the proliferation of both T and B lymphocytes in adult male mice. The macrophages activity in female and male juvenile mice was higher than in adults of both sexes. The natural killer cytotoxic activity was also increased in juvenile and adult males and females compared to the control groups. In the brain, we observed the presence of TBT in the hippocampus, the striatum, the cortex, and the cerebellum in both the male and female groups. The highest levels were observed in the cortex of females and males, while the lowest levels were found in the cerebellum. TBT also induced an increase in the levels of the antioxidant glutathione (GSH) in the striatum, the hippocampus and in cortical structures but not in the cerebellum where the levels of TBT are lower. Our findings indicate that TBT modulated the immune and nervous systems causing endocrine and nervous perturbations.     

Cellular response of mouse splenocytes to heavy metals exposure

Among the environmental contaminants recognised for their toxicity and their global presence, heavy metals are certainly a major concern. They can elicit a number of immunomodulatory effects leading ultimately to an enhanced susceptibility (sensitivity) of immune cells to microbial agents and the appearance of neoplastic diseases and autoimmune phenomena. Heavy metals also provoke changes in the function(s) of immune cells. A striking biological effect of heavy metals is the induction of intracellular thiols (cysteine, glutathione, metallothioneins). Thiols are involved in many physiological processes, including protection from free radical damage and detoxification of chemicals. The purpose of this study was to assess the differences of susceptibility (sensitivity) in both pre-activated (concanavalin A was used) and non-pre-activated cells in the presence of heavy metals. Five were evaluated on murine splenocytes. The lymphoblastic proliferation test was performed for lymphocytes and a phagocytosis test for macrophages. Data showed that the levels of thiols in the pre-activated cells are greater than non-pre-activated cells following exposure to various heavy metals; macrophages were more resistant than lymphocytes to the toxic effects of heavy metals, and pre-activated cells were more resistant than cells at rest. One possible explanation is that macrophages produce more thiols than lymphocytes and this provides an increased protection from the deleterious effects of heavy metals.     

Impacts of UV-B radiation on short-term cultures of sea urchin coelomocytes

Three specialized cell types constitute the heterogeneous population present in the coelomic cavity fluid of sea urchins. The list includes: phagocytes, which undergo a stress-induced petaloid-filopodial transition, white or red amoebocytes and vibratile cells. As a whole, they act as the immune defense system of the sea urchin and respond to environmental and experimental challenge triggering specific stress markers. Here we extended our studies on coelomocytes short-term cultures by describing the morphology and occurrence of each cell type and analyzing their response to UV-B radiation at the biochemical level and with respect to DNA damage. The effects of different doses, ranging from 500 to 2,000 J/m², on cells, which were then cultured in vitro for 1–6 h were tested. As early as 1 h after irradiation we found an increase in the levels of the heat shock protein70 (hsp) stress marker. A peak at 1,000 J/m² corresponding to a 3.4-fold hsp70 increase over the levels of control coelomocytes was observed. Concurrently, we found an increase in the number of apoptotic nuclei detected by Hoechst staining, which varied from 7.1 to 15.6% in cells that were exposed to 500 and 2,000 J/m², respectively. On the basis of our findings we confirm sea urchin coelomocytes as a sensitive cell culture system for environmental studies and propose their use for the analysis on the effects of UV-B radiation. Physical and Chemical Impacts on Marine Organisms, a Bilateral Seminar Italy-Japan held in November 2004. This paper is dedicated to the memory of Aurelia Koros, Ph. D., University of Pittsburg, who committed her scientific life to the study of natural killer cell antigens expressed in small lung carcinoma cells and sea urchin coelomocytes.     

Fate and effects of nonylphenol in the presence of the cyanobacterium Microcystis aeruginosa

Interactions of nonylphenol (NP), a toxic and oestrogenic degradation product of widely used non-ionic surfactants, with the cyanobacterium Microcystis aeruginosa were studied. Batch cultures were incubated for 10 days with NP concentrations between 10 and 570 nM. NP was removed more quickly in the presence of M. aeruginosa (half-life 2.7–5.2 days) than in its absence (half-life 6.7–10.2 days) at all concentrations tested. At the end of the experiment, NP could not be found in the biomass, so the biotic removal is due to uptake and chemical transformation, and not to physical binding on the cells. The observed effective concentrations, EC₅₀ and EC₂₀, were 0.45 and 0.25 μ M, respectively. Therefore, NP is expected to have toxic effects on M. aeruginosa only in very contaminated surface waters. However, for concentrations that go far beyond environmental levels, cyanobacteria are able to cope with NP toxicity by internalising the compound in a less toxic form. Therefore, the presence of cyanobacteria may increase the rate of NP removal from the aquatic environment.     

Oxidative stress in the blood of pregnant rats and reduction of organ weights caused by the polychlorinated biphenyl congener 77

3,3′,4,4′-Tetrachlorobiphenyl (PCB 77), one of the environmentally persistent polychlorinated biphenyls that have been used for industrial purposes, was repeatedly administered to pregnant rats by gavage at a dose of minimal, non-lethal toxicity in order to study its effects on routine blood variables, parameters of oxidative stress and hematopoietic organs in pregnant female rats. Of the routine blood parameters, PCB 77 reduced the mean corpuscular hemoglobin concentration and platelet counts; the levels of malondialdehyde and glutathione peroxidase in the blood were higher during the first days of gestation (1–7), as compared to the respective controls. In the subsequent period between days 8 and 18, these parameters did not show any significant change after PCB 77 treatment. Routine blood parameters and oxidative stress parameters indicated a moderate degree of oxidative stress which alone could not bring about the serious weight reduction of the thymus, pituitary, and kidneys. The extent of oxidative stress did not correlate with the weight reduction of some of the blood-forming organs.     

Time-dependent effects of pentabrominated diphenyl ether (PBDE) on thyroid hormones and histology in rats

In order to examine the time-dependent effects of pentabrominated diphenyl ether (BDE-99) on thyroid hormones levels and histological structure in rats, 4-week-old SD rats were divided into 10 groups randomly, according to their body weight. Rats in treatment groups were orally gavaged with BDE-99 at the dose of 60 mg kg^?1 for 10, 15, 20, 30 or 40 days, while rats in control group received equal volume of corn oil. Serum levels of thyroxine (FT4), triiodothyronine (FT3), and thyroid stimulating hormone (TSH) were measured by radioimmunoassay. Sections of thyroids were dyed with hematoxylin-eosin (HE) to detect any pathologically alterations. In BDE-99 treated groups, on day 10, changes in serum levels of FT4, FT3 and TSH were not found. On days 15 and 20, serum levels of FT4 and FT3 decreased, accompanied by increased levels of TSH. On days 30 and 40, serum levels of these hormones returned to control values. As for alterations in histology, on day 15, follicular epithelium of the thyroid were found to proliferate into two layers in BDE-99 treated rats. On day 20, in focal areas the two-layer follicular epithelia were observed to continuously proliferate into 3–4 layers of follicular epithelium, called hyperplasia plaques. On days 30 and 40, solid buds and secondary follicles to solid buds were detected. The earliest alteration of thyroid hormones in treated SD rats occurred at the duration of 10–15 days, with corresponding histopathologic changes. As the exposure time increased, the serum level of hormones and histopathology altered accordingly in a time-dependent manner.     

Effects of nonylphenol on the brain cytochrome P450 gene expression in F1 generation rats

In order to explore the effects of nonylphenol (NP) on brain cytochrome P450 gene expression in F1 generation rats microarray analysis techniques were used. mRNA were extracted from the brain of 2-day-old F1 generation rats whose F0 male generation were treated with NP, then reversely transcribed to cDNA and labeled with cy5 and cy3 for fluorescence. Subsequently, the cDNA probes were hybridized to the mouse 40S cDNA microarray; and fluorescent signals produced by cy5 and cy3 were scanned and analyzed. Sixteen identified genes were found to be expressed differently from control, including three cytochrome P450 genes, in which two were up-regulated and one down-regulated. Data suggest that NP affects the expression of some cytochrome P450 genes in rat brain when administered perinatally.     

应激大鼠脾脏内诱导型一氧化氮合酶活性变化及其意义

在捕食压力下,大鼠脾脏诱导型一氧化氮合酶(iNOS)明显表达,说明iNOS起到了重要的生理功能,然而,NOS的表达及在亚细胞水平的定位以及相应的差异变化等都很不明确,本试验采用捕食应激模型,利用免疫组织化学方法和生化测定,检测心理性应激对大鼠脾脏iNOS活性的影响及探讨这种影响与应激动物免疫力下降之间的关系.结果表明iNOS免疫阳性细胞表达部位集中于脾脏的红髓和边缘区,白髓中几乎没有.与对照组相比,在应激1~12d的过程中,应激组大鼠脾脏内iNOS的活性逐渐升高,至d12最为显著(P<0.01).但在后期应激13~30d,脾内iNOS的表达又逐渐减少.生化测定结果表明,NOS和NO的含量变化与iNOS的活性变化相似,在心理性应激早中期较高,至d12最为显著(P<0.01),而到中后期则逐渐降低.结果提示,iNOS活性程度与动物心理应激程度密切相关;应激过程中大鼠脾脏内由于iNOS的活性增加导致产生过量的NO,这可能与应激导致大鼠免疫力下降有关.图1表2参10     

Effect of formaldehyde exposure on structure and function of epididymis in adult rats: a histological and biochemical study

Formaldehyde (FA), a ubiquitous environmental pollutant, is extensively used in hospitals, laboratories, and industrial settings. Previous studies showed that FA exerts adverse effects on testicular function and as epididymis is known to play an important role in the maturation and storage of sperm, the effects of FA were examined on epididymis. In particular, this study was designed to investigate the influence of FA on structure and function of epididymis in adult male rats using histological and biochemical methods. Sprague-Dawley adult rats were randomly allotted to three groups and exposed to FA at a 0 (control), 0.5, or 10 mg m^?3 by inhalation for 28 days. The results indicated that epididymal toxicity of FA was concentration dependent. Epdididymal structure and function in rats of 0.5 mg m^?3 FA exposure group showed no apparent difference from control. However, epididymal weight, sperm count and motility, the activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were significantly decreased, whereas the levels of malondialdehyde (MDA) was significantly increased in epididymis of rats exposed to 10 mg m^?3 FA. Moreover, microscopy with hematoxylin and eosin (H&;E) staining showed atrophy of epididymal tubules, disintegration of epididymal epithelium, disorganization, and even vacuolar denaturalization of epididymal epithelial cells. There was hyperemia in interstitial vasculature and lumina were oligozoospermic in rats of 10 mg m^?3 FA exposure group. In conclusion, FA exposure alters the epididymal structure and function by inducing oxidative stress in epididymis of adult rats.