Embryotoxicity of Arsenite and Arsenate: Distribution in Pregnant Mice and Monkeys and Effects on Embryonic Cells in Vitro

Abstract: The distribution of ⁷⁴As-labelled arsenate and arsenite in pregnant mice and a monkey has been studied by autoradiography and gamma counting of isolated tissues, and their in vitro toxicity to a chondro-genic system has been investigated. With both arsenic forms, given as single intravenous injections to the mother, the ⁷⁴As-arsenic appeared to pass the mouse placenta relatively freely and approximately to the same extent. The retention time in maternal tissues including the placenta was, however, around three times longer with arsenite than with arsenate. In early gestation, high activity was registered in the embryonic neuroepithelium, which correlates well with reported CNS malformations in rodents. In late gestation, the distribution pattern was more like that in the adults. Accumulation in skin and squamous epithelia of the upper gastrointestinal tract (oral cavity, oesophagus and oesophageal region of stomach) dominated the distribution picture, especially at a long survival interval. Arsenate, but not arsenite, showed affinity for the calcified areas of the skeleton. A marmoset monkey in late gestation receiving arsenite showed a somewhat lower rate of placental transfer than the mice. Skin and liver had the highest concentrations (at 8 hrs), both in mother and foetuses. This species is known not to methylate arsenic, resulting in stronger binding and longer retention times of arsenic as compared with other species. The stronger binding in maternal tissues may possibly explain the lower rate of placental transfer. Arsenite was shown to inhibit cartilage formation in a chick limb bud mesenchymal spot culture system (ED50~ 5-10 μM), while arsenate seemed to be without effect at concentrations up to 200 μM (highest tested). Arsenate, however, showed a potentiation of the arsenite toxicity.     

不同的培养方法对鼠胚体外发育的影响

目的比较鼠胚在不同的培养体系中的体外发育情况,探讨培养液的体积对体外培养的影响。方法把收集的小鼠2-细胞胚胎分成两组,A组于20μl培养液的微滴中培养,B组与50μl培养液的微滴中培养,观察胚胎发育情况。结果 96h后A、B组的囊胚形成率分别为83.64%和81.63%,差异无统计学意义(P>0.05)。结论适当体积的培养滴可促进胚胎的体外发育。     

口服避孕药预处理在体外受精-胚胎移植中的利弊研究

目的 评估口服避孕药预处理在体外受精-胚胎移植周期中的利弊,为生殖中心临床合理应用口服避孕药(Oral contraceptive,OCP)提供依据.方法 回顾性分析341例行IVF/ICSI的患者,按促排卵周期前是否使用过OCP药物分为OCP治疗组182例和对照组159例,对入选患者的病历资料进行统计分析,评估口服避孕药对患者促排卵的效果以及对助孕结局的利弊影响.结果 OCP预处理明显减少卵巢功能性囊肿形成(P＜0.0001).OCP处理组比对照组Gn天数平均增加0.34d(P=0.042),但剂量总量差异无统计学意义(P=0.55),且两组获卵数、成熟卵数、总受精数、正常受精数、卵裂数、卵巢过度刺激发生率、流产率、临床妊娠率等差异均无统计学意义.结论 OCP预处理能较好地控制促排卵的时间,有利于医务人员安排不孕患者进周期的时间,同时也减轻了患者往返医院监测排卵的不便,又能有效减少卵巢功能性囊肿形成,但对助孕结局无明显改善.     

体外受精与胚胎移植周期血清性激素水平与妊娠率的关系

目的　探讨IVF -ET治疗周期中血清性激素水平与妊娠率的关系。方法　对 134个采用促性腺激素释放激素激动剂 促卵泡生成素 绒毛膜促性腺激素 (GnRH -a FSH hCG)方案促排卵的IVF和单精子胞浆内注射 (ICSI)周期 ,采用时间分辨荧光读数法 ,在注射hCG日测定促卵泡生成激素 (FSH)、黄体生成素 (LH)、雌二醇 (E2 )、孕酮 (P)、泌乳素(PRL) ,在胚胎移植 (ET)日测定E2、P。结果　注射hCG日FSH、LH、E2、P、PRL均与妊娠无关 ;但P E2与妊娠有关。当P E2 <0 5ng ml时 ,妊娠率最高 (P =0 0 2 4)。ET日 ,雌激素水平与妊娠无关 ,孕激素水平与妊娠率有显著性差异 ,P≥ 6 0ng ml时临床妊娠率达 44 4% (P =0 0 0 3)。结论　在IVF和ICSI周期中 ,注射hCG日的P E与ET日的P值均与妊娠结局有关。该两项是预测IVF -ET临床妊娠率的一个较好的指标。若P值偏低 ,可在黄体期加强黄体支持以提高妊娠率。     

Effect of hexavalent chromium on histone biotinylation in human bronchial epithelial cells

Chromium is a potent human mutagen and carcinogen. The capability of chromium to cause cancers has been known for more than a century, and numerous epidemiological studies have been performed to determine its carcinogenicity. In the post-genome era, cancer has been found to relate to epigenetic mutations. However, very few researches have focused on hexavalent chromium (Cr(VI))-induced epigenetic alterations. The present study was designed to investigate whether Cr(VI) would affect the level of a newfound epigenetic modification: histone biotinylation. Histone acetylation and histone biotinylation were studied in detail using human bronchial epithelial (16HBE) cells as an in vitro model after Cr(VI) treatment. Our study showed that Cr(VI) treatment decreased histone acetylation level in 16HBE cells. In addition, low doses of Cr(VI) (≤0.6 μM) elevated the level of histone biotinylation. Furthermore, immunoblot analysis of biotinidase (BTD), a major protein which maintains homeostasis of histone biotinylation, showed that the distribution of BTD became less even and more concentrated at the nuclear periphery in cells exposed to Cr(VI). Moreover, Cr(VI)-induced histone deacetylation may take part in the regulation of histone biotinylation. Together, our study provides new insight into the mechanisms of Cr(VI)-induced epigenetic regulation that may contribute to the chemoprevention of Cr(VI)-induced cancers and may have important implications for epigenetic therapy.     

硝苯地平对小白鼠离体小肠收缩活动的影响

目的探讨硝苯地平对小白鼠消化道平滑肌收缩的影响。方法以1%丙酮溶液0.2ml作对照组,采用离体器官灌流的实验方法,分别滴加0.02%硝苯地平0.05ml、0.10ml、0.15ml、0.20ml,观察小白鼠离体小肠收缩曲线下面积和自发收缩活动频率的变化。结果滴加0.02%硝苯地平0.05ml后对离体小肠收缩活动的影响不显著。0.02%硝苯地平在0.10～0.20ml后均能引起离体小肠收缩曲线下面积下降(P<0.05,P<0.01),自发性收缩频率下降(P<0.01),与0.02%硝苯地平0.05ml、1%丙酮溶液0.2ml比较,差异均有统计学意义(P<0.05,P<0.01)。但0.02%硝苯地平在0.10～0.20ml范围内引起离体小肠收缩活动变化差异无统计学意义。结论硝苯地平用量达一定程度对小白鼠离体小肠平滑肌收缩具有抑制作用。     

Influence of 13-cis and all- trans retinoic acid on rat embryonic development in vitro: correlation with isomerisation and drug transfer to the embryo

In vitro experiments using whole rat embryo cultures show that all -trans retinoic acid (all- trans RA) administered at low concentrations (30 ng/ml culture medium) is 10 times more active than 13-cis retinoic acid (13-cis RA) and 3 times more active when administered at high concentrations (1000 ng/ml culture medium). Morphological investigation of the embryos shows that both substances directly influence embryonic development in an identical manner. Isomerisation products of the administered compounds (all-trans RA from 13-cis RA and vice versa) were detected by HPLC both in the culture medium and the embryo. Correlation of embryonic retinoid concentration with the observed effects led us to suggest that the isomerisation to all- trans RA is crucial in regard to 13-cis RA-induced abnormal embryonic development. A 100% effect can be induced in vitro with very low amounts of all- trans RA (7.2 ng/g) in the embryo.     

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25 μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.     

Developmental cardiotoxicity effects of four commonly used antiepileptic drugs in embryonic chick heart micromass culture and embryonic stem cell culture systems

Antiepileptic drugs (AEDs) are commonly used drugs in pregnant women with epilepsy. Prenatal exposure to AEDs increases the risk of major or minor congenital malformation during embryonic development. The precise mode of action and intracellular mechanisms of these AEDs during embryonic development remains unclear. To determine relative teratogenic risk of AEDs, four AED drugs including valproic acid (VPA), phenytoin (PHT), phenobarbital (PB), and trimethadione (TMD) were tested using two in vitro systems (the embryonic chick heart micromass (MM) culture and the in vitro differentiating mouse embryonic stem cells into cardiomyocytes culture systems). Cardiomyocyte cultures (the heart MM and ES cell-derived cardiomyocytes) were treated with or without different concentrations of PHT, PB, TMD (10–100 μM), and VPA (100–2000 μM). 5-Fluorouracil (5-FU) (1–10 μM) and l-ascorbic acid (10–1000 μM) were used as positive and negative controls. It was found that these four commonly used AEDs and 5-FU tested have the potential to inhibit embryonic heart cell differentiation (p < 0.05) without inducing any cytotoxicity. VPA at higher concentrations (⩾800 μM), and 5-FU at all doses proved to be cytotoxic in the differentiating ES cell culture system. The results demonstrated in this study suggest that the use of these four commonly prescribed AEDs during pregnancy may have an effect on embryonic heart development, and may be associated with congenital cardiovascular defects.     

Fast uptake kinetics in vitro of 51Cr (VI) by red blood cells of man and rat

Fast transport kinetics of ⁵¹Cr (VI) into red blood cells (RBCs) in vitro were studied. No significant species differences were found between RBCs of man and rat. The uptake of ⁵¹Cr (VI) by RBCs in whole blood was composed of two different first order processes of different velocities (apparent t_1/2 of 22.7 s and 10.4 min for man and 6.9 s and 10.1 min for rat, respectively). However, even after longer time periods a fixed portion of approximately 15% of the administered dose remained in the plasma and did not penetrate into RBCs Over the entire concentration range studied (10 M–50 mM), the fast initial uptake followed Michaelis-Menten kinetics. The maximal capacity of this Cr(VI) transport into RBCs of man and rat was 3.1×10⁸ CrO₄ ^2– ions × cell^–1 × min^–1 and 2.5×10⁸ CrO₄ ^–2 ions × cell^–1 × min^–1, respectively. It is likely that Cr(VI) is transported into RBCs via a physiological anion carrier (band-3-protein).     

人表皮干细胞体外分离和培养的研究

目的建立人表皮干细胞体外分离和培养的技术方法。方法用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞,并以人成纤维细胞条件培养基为基础制备表皮干细胞培养液,用以人表皮干细胞(Ⅳ型胶原快速粘附法富集分离)的体外培养,通过检测β1整合素和角蛋白19的表达水平及其克隆形成率和克隆维持时间对其进行鉴定,角朊细胞作对照。结果表皮干细胞体外培养,可见细胞呈克隆样生长、β1整合素及角蛋白19免疫组织化学染色呈阳性,且其克隆形成率和克隆维持时间分别为(17.04±1.01%)和15～18d,明显高于对照组的(8.72±0.73%)和9～10d(P<0.01)。结论应用Ⅳ型胶原快速粘附法及人成纤维细胞条件培养基,对人表皮干细胞成功进行了体外分离和培养,为表皮干细胞体外的大量扩增奠定了基础。     

Dynorphin-(1-13): the effect of in vivo treatment on opiate bindings in vitro

Mice were injected intracerebroventricularly with different doses of dynorphin-(1-13) in vivo and decapitated after different intervals of time; crude P2 fractions were then prepared and used for in vitro binding with [3H]dihydromorphine (DHM) and [3H][D-Ala2,D-Leu5]enkephalin. Dynorphin pretreatment was found to decrease DHM binding in vitro in a both time-dependent and dose-dependent manner. Its maximal effect lasted from 30 min to 3 h and recovery took place in 6-12 h. Analysis of the Scatchard plots showed that dynorphin decreased the number of high affinity-binding site for DHM, while KD of this site was essentially unaltered. Neither Bmax nor KD of low affinity site were much affected. A similar decrease in high affinity Bmax for DADLE was also obtained; however, the recovery was even more rapid. Extra washes of tissue did not significantly increase the binding, but preincubation of tissue in the presence of morphine reversed the dynorphin effect and increased DHM binding significantly to almost control levels. The probable mechanism of dynorphin in decreasing opiate receptor binding is discussed.     

The effects of tricentric chromium(III) propionate complex supplementation on pregnancy outcome and maternal and foetal mineral status in rat

Chromium(III) is an essential element for carbohydrate and lipid metabolism, and various chemical forms of this element are widely used as dietary supplements. Of particular interest is [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺ cation (CrProp), that has been proposed as an alternative source of Cr. However, its safety has not been studied completely. In this study we investigated the effects of CrProp supplementation on pregnancy outcome and maternal and foetal mineral status in the rat. Female Wistar rats (n = 20, 14 weeks old) were mated with males and, after successful conception were fed either AIN-93G diet supplemented with CrProp (100 mg Cr/kg diet, equals to 7.2 mg Cr/kg body mass/day) or non-supplemented diet (0.27 mg Cr/kg diet, equals to 0.02 mg Cr/kg body mass/day) for 21 days. Dams were sacrificed on 21 day of gestation, and their foetuses were examined for adverse effects. Maternal and foetal organs were analysed for minerals contents (Fe, Cu, Zn, Cr) using the AAS-method. Supplemental Cr given did not affect pregnancy outcome, litter size, body and inner organ masses, maternal blood biochemical indices. No abnormalities in gross organ morphology of foetuses were detected. Supplemental CrProp increased maternal liver and kidney Cr levels by 177% and 455%, decreased liver Cu and Zn concentrations by 9% and 12%, increased foetal liver Zn by 181%, and decreased kidney Cu level by 34%.     

Low level chromium (VI) inhalation effects on alveolar macrophages and immune functions in Wistar rats

In inhalation chambers, 5-week-old male Wistar rats of the strain TNO-W-74 were continuously exposed to submicron aerosols of sodium dichromate in concentrations from 25 (low level) to 200 g/m³ Cr (high level). Subacute exposure (28 days) to 25 and 50 g/m³ Cr resulted in activated alveolar macrophages with stimulated phagocytic activities, and significantly elevated antibody responses to injected SRBC's. After subchronic (90 days) low level exposure there was a more pronounced effect on activation of the alveolar macrophages, with increased phagocytic activities. However, at high Cr (VI) exposure level (200 g/m³), inhibited phagocytic function of the alveolar macrophages was seen. In rats which were exposed to this chromium aerosol concentration for 42 days, the lung clearance of inert iron oxide was reduced significantly. The humoral immune system was still stimulated at subchronic low chromium aerosol concentrations of 100 g/m³, but significantly depressed at 200 g/m³ Cr. These results show that respiratory defence and immunologic functions were stimulated or inhibited depending on dose and time of chromium (VI) inhalation.This paper was presented in part at the International Seminar on the Immunological System as a Target for Toxic Damage, November 6–9, 1984, Luxembourg     

安心康滴丸体外抗柯萨奇病毒的实验

目的 评价安心康滴丸的体外抗柯萨奇病毒（CVB3）活性。方法 在大鼠心肌细胞上进行抗病毒实验，观察安心康滴丸对细胞病变的影响、对病毒繁殖抑制作用和对肌钙蛋白含量的影响。结果 安心康滴丸能有效的修复病变细胞，抑制病毒繁殖和降低肌钙蛋白含量。结论 安心康滴丸在体外具有显著的抗柯萨奇病毒作用。     

Antiproliferative effects of aspirin and diclofenac against the growth of cancer and fibroblast cells: In vitro comparative study

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of several cancer cell lines. The aim of this study is to compare the cytotoxic effect of aspirin with diclofenac on the growth of HeLa cell, mammary cell carcinoma, rhabdomyosarcoma and fibroblast cell lines in the culture media. The cells are cultured in RPMI-1640 culture media supplemented with 5% fetal calf serum and antibiotics. Aspirin (5 mg/well) and diclofenac (0.625 mg/well) significantly inhibit the growth of HeLa, rhabdomyosarcoma and fibroblast cells. The cytotoxic effect of aspirin against rhabdomyosarcoma is significantly (p < 0.001) higher than that of diclofenac with a potency approximated 2.6. It concludes that aspirin and diclofenac inhibit the growth of fibroblast and cancer cell by inhibiting the up-regulation of cyclooxygenases enzymes in cancer cells. Aspirin is more effective than diclofenac against the growth of rhabdomyosarcoma cell line.     

Effects of culture conditions on estrogen-mediated hepatic in vitro gene expression and correlation to in vivo responses

Refinement of in vitro systems for predictive toxicology is important in order to develop high-throughput early toxicity screening assays and to minimize animal testing studies. This study assesses the ability of mouse Hepa-1c1c7 hepatoma cell model under differing culture conditions to predict in vivo estrogen-induced hepatic gene expression changes. Custom mouse cDNA microarrays were used to compare Hepa-1c1c7 temporal gene expression profiles treated with 10 nM 17beta-estradiol (E2) in serum-free and charcoal-stripped serum supplemented media at 1, 2, 4, 8, 12, and 24 h. Stripped serum supplemented media increased the number gene expression changes and overall responsiveness likely due to the presence of serum factors supporting proliferation and mitochondrial activity. Data from both experiments were compared to a gene expression time course study examining the hepatic effects of 100 microg/kg 17alpha-ethynyl estradiol (EE) in C57BL/6 mice at 2, 4, 8, 12, 18, and 24 h. Only 18 genes overlapped between the serum-free and in vivo studies, whereas 238 genes were in common between Hepa-1c1c7 cells in stripped serum data and C57BL/6 liver samples. Stripped serum cultured cells exhibited E2-elicited gene expression changes associated with proliferation, cytoskeletal re-organization, cholesterol uptake and synthesis, increased fatty acid beta-oxidation, and oxidative stress, which correlated with in vivo hepatic responses. These results demonstrate that E2 treatment of Hepa-1c1c7 cells in serum supplemented media modulate responses in selected pathways which appropriately model estrogen-elicited in vivo hepatic responses.     

环孢素A微球的体外释放影响因素

目的以环孢素A为模型药,使用生物可降解材料乳酸/羟基乙酸PLGA制备微球。并评价微球的体外释放。方法使用乳化溶剂挥发法制备环孢素微球,并对微球的外观,粒径和载药量进行评价。通过衡量温度,pH值和表面活性剂等因素筛选体外释放介质。结果制得的微球外观圆整,粒径统一,平均粒径50μm左右,载药量为13%。DSC结果表明环孢素A和PLGA有结合作用。体外释放实验表明微球的释放受温度,pH值和表面活性剂的影响,加入30%的异丙醇可使微球在2周内释放完全。结论乳化溶剂挥发法可制备得到质量符合要求的环孢素微球,含30%异丙醇的释放介质是微球体外释放评价的理想介质。     

多抗甲素对脐血树突状细胞体外扩增、成熟的影响

目的观察多抗甲素(polyactin A,PA)对脐血树突状细胞(dendritic cell,DC)体外扩增、成熟的影响。方法分别用加有PA、GM-CSF+TNF-α+IL-4(GTI)及GTI+PA(GTIP)的RPMI-1640培养液体外诱导培养脐血单核细胞,培养d7,Elivision免疫组化方法检测各组细胞CD1a、CD83、HLA-DR、CD34抗原表达情况,透射电镜观察细胞形态。结果PA组、GTI组及GTIP组均有一定数量的典型DC,电镜观察该细胞表面有大量粗细不等的树枝状突起;PA组CD1a+、CD83+细胞比例分别达19·63%±3·61%、14·52%±5·79%,高于对照组(即单纯培养液组);GTIP组CD1a+细胞比例升高最明显,高于GTI组。结论PA不仅能促进脐血DC体外扩增、成熟,还能协同GM-CSF、TNF-α、IL-4促进DC生成,PA是一种实用的DC活化剂。