Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals

Increased levels of oxidatively damaged DNA have been documented in studies of metal, metal oxide, carbon‐based and ceramic engineered nanomaterials (ENMs). In particular, 8‐oxo‐7,8‐dihydroguanine‐2'‐deoxyguanosine (8‐oxodG) is widely assessed as a DNA nucleobase oxidation product, measured by chromatographic assays, antibody‐based methods or the comet assay with DNA repair enzymes. However, spurious oxidation of DNA has been a problem in certain studies applying chromatographic assays, yielding high baseline levels of 8‐oxodG. Antibody‐based assays detect high 8‐oxodG baseline levels, related to cross‐reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations between exposure to ENMs and oxidized DNA in tissue than studies showing acceptable baseline levels (odds ratio = 12.1, 95% confidence interval: 1.2–124). Nevertheless, reliable studies indicate that intratracheal instillation of nanosized carbon black is associated with increased levels of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO₂, carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration‐dependent associations between ENM exposure and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential. Environ. Mol. Mutagen. 56:97–110, 2015. © 2014 Wiley Periodicals, Inc.     

The unique uterine structure of the basal monogenean Chimaericola leptogaster (Monogenea: Polyopisthocotylea), an ectoparasite of the relictual holocephalan fish Chimaera monstrosa

Ultrastructural studies of the monogenean uterus are few in number and no non-polystomatid polyopisthocotyleans have been investigated. The uterus of Chimaericola leptogaster, a basal polyopisthocotylean monogenean, has several unusual features, including six reflexed loops comprising four ascending and three descending, longitudinally oriented, linear sections. At the ultrastructural level, three readily distinguishable uterine regions and other distinctive characteristics are apparent. One novel feature occurs in the proximal uterus, where the lining forms a so-called ‘single-layered multi-rowed cellular epithelium’, which includes two types of cells, tall (ca. 14–19 μm in height) and short (ca. 6–9 μm in height) cells, both lying on the basement membrane. Although known from other bilaterian groups, this is the first record of this type of epithelium in the Neodermata. The lining of the middle uterine region comprises a single regular layer of columnar glandular epithelial cells, which produce numerous rounded, electron-dense granules associated with Golgi complexes. The presence of the uterine glands in the middle region of the uterus is an unusual feature for a monogenean, having previously been described only for basal orders of the Cestoda, i.e. the Gyrocotylidea, Caryophyllidea and Spathebothriidea. Seen in cross-section, the epithelium of the distal uterus contains three areas of tall single-layered columnar epithelium (ca. 30 μm deep) interspersed by three areas of flattened epithelium (ca. 0.2–0.9 μm deep). Such a pattern is quite different from those reported for other monogeneans and, indeed, other neodermatan groups. The investigation has shown that the outer layer of the fully formed eggshell is assembled from epithelial secretions in the middle uterine lumen, but is modified in terms of its shape in the distal uterus. Possible phylogenetic implications arising from the unusual features described are discussed in relation to other neodermatan groups and recent molecular phylogenies of the Bilateria.

     

体外纯化后和诱导后骨髓间充质干细胞与骨髓单个核细胞移植治疗心肌梗死的效果比较

目的:将骨髓单个核细胞、体外培养纯化后及诱导后的骨髓间充质干细胞注射至大鼠心肌梗死区域,观察移植细胞的增殖及分化情况,分析其对缺血心肌细胞修复重建能力与心功能改善的影响。方法:实验于2005-07/2006-04在安徽省干细胞研究和治疗中心完成。取清洁级健康雄性新西兰白兔40只,按随机数字表法分为4组:骨髓间充质干细胞组、5-杂氮胞苷诱导组、骨髓单个核细胞组、对照组,10只/组。均于左心耳下缘结扎冠状动脉左前降支建立心肌梗死模型,心电图出现S-T段弓背样抬高认为结扎成功。2周后骨髓间充质干细胞组注射第3代自体骨髓间充质干细胞1×106个,5-杂氮胞苷诱导组注射经5-杂氮胞苷诱导24h的第3代自体骨髓间充质干细胞1×106个,骨髓单个核细胞组注射骨髓单个核细胞2.5×107个/400μL,对照组仅注射400μL L-DMEM培养基。分别在术前、移植前、移植后2,4周应用超声心动图评价心脏功能,同时利用心肌声学造影观察缺血区的血流灌注情况。细胞移植后8周采用免疫荧光检测移植细胞在梗死区的生长状况,苏木精-伊红染色于普通显微镜下记数梗死区毛细血管密度。结果:骨髓间充质干细胞组9只、5-杂氮胞苷诱导组9只、骨髓单个核细胞组10只、对照组6只进入结果分析,其余动物因造模死亡未能完成整个实验。①超声心动检查发现术前、细胞移植前4组左室射血分数、左室短轴短缩率组间比较差异不显著;与对照组相比,细胞移植后2,4周骨髓间充质干细胞组和5-杂氮胞苷诱导组左室射血分数、左室短轴短缩率明显升高[对照组:(60.4±5.1)%,(62.4±7.8)%;(28.8±1.4)%,(27.2±5.3)%;骨髓间充质干细胞组:(70.8±4.6)%,(70.4±6.1)%;(33.8±3.4)%,(33.9±3.5)%;5-杂氮胞苷诱导组:(71.7±6.8)%,(70.3±5.8)%;(34.7±5.7)%,(35.5±6.3)%,P<0.05];左室收缩末内径、左室舒张末内径细胞移植组与对照组相比,差异不显著;心肌声学造影见细胞移植后心肌梗死区血流灌注改善明显。②细胞移植术后8周,所有细胞移植组均见Dil阳性移植细胞存活,并表达α横纹肌肌动蛋白和结蛋白。5-杂氮胞苷诱导组Dil阳性细胞出现具有典型横纹和肌小节样结构的心肌样细胞,各细胞移植组毛细血管密度均较对照组明显增多[(38.6±7.6)/mm2,(32.9±5.7)/mm2,(38.5±2.0)/mm2,(21.4±3.9)/mm2,P<0.05],各移植组间差异不显著。结论:①体外纯化培养与经5-杂氮胞苷诱导24h的骨髓间充质干细胞,以及新鲜分离的骨髓单个核细胞自体移植8周后均存活于梗死心肌中,并表达心肌细胞特异性标志α横纹肌肌动蛋白和结蛋白,移植后可明显改善左室收缩功能,增加毛细血管密度,改善局部血流灌注。②5-杂氮胞苷有助于促进骨髓间充质干细胞在体内向心肌细胞的分化成熟。     

Role of oxidative stress in carbon nanotube-generated health effects

The development of products containing carbon nanotubes (CNTs) is a major achievement of nanotechnology, although concerns regarding risk of toxic effects linger if the hazards associated with these materials are not thoroughly investigated. Exposure to CNTs has been associated with depletion of antioxidants, increased intracellular production of reactive oxygen species and pro-inflammatory signaling in cultured cells with primary function in the immune system as well as epithelial, endothelial and stromal cells. Pre-treatment with antioxidants has been shown to attenuate these effects, indicating a dependency of oxidative stress on cellular responses to CNT exposure. CNT-mediated oxidative stress in cell cultures has been associated with elevated levels of lipid peroxidation products and oxidatively damaged DNA. Investigations of oxidative stress endpoints in animal studies have utilized pulmonary, gastrointestinal, intravenous and intraperitoneal exposure routes, documenting elevated levels of lipid peroxidation products and oxidatively damaged DNA nucleobases especially in the lungs and liver, which to some extent occur concomitantly with altered levels of components in the antioxidant defense system (glutathione, superoxide dismutase or catalase). CNTs are biopersistent high aspect ratio materials, and some are rigid with lengths that lead to frustrated phagocytosis and pleural accumulation. There is accumulating evidence showing that pulmonary exposure to CNTs is associated with fibrosis and neoplastic changes in the lungs, and cardiovascular disease. As oxidative stress and inflammation responses are implicated in the development of these diseases, converging lines of evidence indicate that exposure to CNTs is associated with increased risk of cardiopulmonary diseases through generation of a pro-inflammatory and pro-oxidant milieu in the lungs.     

Growth and development following marrow transplantation for leukemia

One hundred forty-two patients between the ages of 1 and 17 years who survived disease-free more than 1 year after marrow transplantation for hematologic malignancy had growth and development evaluations from one to 14 years posttransplant (median 4 years). Prior to transplant all children received multiagent chemotherapy and 55 also received central nervous system irradiation, but none had growth and development evaluations. Marrow transplant preparation included high-dose chemotherapy and total body irradiation (TBI) given as a single dose of 9.2 to 10.0 Gy (79 patients) or as fractionated doses of 2.0 to 2.25 Gy/d for six to seven days (63 patients). After transplant abnormal thyroid function was present in 39%. Stimulated 11-desoxycortisol levels were subnormal in 24% of patients evaluated. Growth hormone (GH) deficiency was present in 17 of 25 children who received previous cranial irradiation. Partial GH deficiency was present in 4 of 25 who received previous cranial irradiation and in 6 of 18 who had not received cranial irradiation. Height velocity was decreased in all patients. After transplant, height was significantly influenced by chronic graft-v-host disease and single-dose TBI. Sixty-eight percent had delayed development of secondary sexual characteristics. Gonadal failure occurred in nearly all who were postpubertal at transplant. While it is not possible to determine how many of these endocrine abnormalities occurred as a result of treatment administered prior to transplantation, these data do demonstrate that children who become long-term survivors after marrow transplantation for hematologic malignancy have endocrine abnormalities that adversely affect growth and development.     

Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.