胆汁酸遗传毒性和细胞间隙连接通讯抑制作用的研究

胆汁酸遗传毒性和细胞间隙连接通讯抑制作用的研究梁立新，毕洁，刘建，印木泉（上海军医大学卫生毒理学教研室２００４３３）本文应用Ａｍｅｓ试验，ＣＨＬ细胞株染色体畸变试验检测了脱氧胆酸和石胆酸的遗传毒性，用ＮＩＨ／３Ｔ３细胞恶性转化试验观察了脱氧胆酸和石胆...     

两种胆汁酸诱发MW／3T3细胞恶性转化的作用

两种胆汁酸诱发ＭＷ／３Ｔ３细胞恶性转化的作用陈耀富，印木泉（上海军医大学卫生毒理学教研室２００４３３）众所周知，胆汁酸是一种促癌物。但仍有认为胆汁酸可能是一种遗传毒性致癌物的报道。本文观察了两种胆汁酸（石胆酸和脱氧胆酸）诱发ＭＷ／３Ｔ３培养细胞的恶性...     

环磷酰胺、噻替哌诱发人支气管上皮细胞的染色体畸变

目的与方法：以永生化人支气管上皮细胞（ＢＥＡＳ－２Ｂ）受环磷酰胺、噻替哌诱导并发生癌性转化的细胞为模型,运用染色体Ｇ－显带技术,观察环磷酰胺、噻替哌的遗传病毒作用引起的细胞转化过程中的染色体动态畸变。结果：ＢＥＡＳ－２Ｂ细胞染色体众数４６条,近二倍体,核型稳定,携带有Ｍ１,Ｍ２,Ｍ３三个标志染色体。环磷酰胺转化细胞（ＢＥＡＳ－ＣＰ）为二倍体核型,丢失了１个１４号染色体,增加了Ｍ４异常染色体,该畸变可能与细胞转化的始动,促进和进展有关。噻替哌转化细胞（ＢＥＡＳ－Ｔ）在培养过程中渐趋多倍体细胞,１５代以后部分细胞的１４和２１号染色体各丢失１条,ＢＥＡＳ－Ｔ ２３代在软琼脂上形成克隆的细胞（ＢＥＡＳ－ＳＴ）是多倍体细胞,并具有高频率的非稳定性畸变,ＢＥＡＳ－Ｔ ２５代时为３％,ＢＥＡＳ－Ｓ为３４％,多倍体背景上出现２     

海狗油脂肪乳的致突变试验研究

目的 目的观察海狗油脂肪乳是否存在遗传毒性.方法 应用Ames试验、中国仓鼠肺细胞体外培养染色体畸变试验和小鼠骨髓嗜多染红细胞微核试验,研究海狗油脂肪乳的致突变作用.结果 Ames试验在P《5.0mg/皿浓度下未见回复突变菌落数显著增加;中国仓鼠肺细胞体外培养染色体畸变试验在P《6.6mg/ml浓度下未出现细胞染色体畸变率显著增高;小鼠骨髓嗜多染红细胞微核试验在P《6.0g/kg剂量下未诱发微核细胞率的增高.结论 海狗油脂肪乳在试验剂量范围内,无致突变作用.     

生殖细胞是否对辐照食品敏感？─辐射鱼对雄性小鼠生殖细胞的影响

以往辐照食品的遗传毒性研究多数采用体细胞为指标,并认为辐照食品是安全的,仅有少数学者提出辐照食品对生殖细胞有“辐射毒”,本研究是以３ＫＧＹ辐照鱼为饲料（占饲料的３０％左右）长期喂养Ｓｗｉｓｓ雄性小鼠,并以睾丸生殖细胞中期Ⅰ的染色体畸变、精子畸变及生育能力为指标,观察辐照鱼对小鼠遗传物质的影响。结果表明,在此条件下,可诱发睾丸生殖细胞染色体单价体的明显增高（５９．１％）,并可诱发结构畸变;精子畸变率也明显增高（５４．４‰）,但对亲代小鼠生育能力无影响,放生殖细胞遗传物质的畸变是不是辐照食品的一个敏感指标值得进一步探讨。     

钆喷酸二葡甲胺的致突性研究

本文应用鼠伤寒沙门菌／徽粒体试验，小鼠骨髓细胞微核试验和体外哺乳动物细胞（ＣＨＬ）染色体畸变试验对钆喷酸二葡甲胺进行了致突性研究．结果表明：钆喷酸二葡甲胺各剂量ＴＡ９７，ＴＡ９８，ＴＡ１００，ＴＡ１０２在加和不加Ｓ９条件下均无致突性。在徽核试验中，各剂量诱发小鼠骨髓多染红细胞的微核率与阴性对照相比，无显著差异（Ｐ＞０．０５）染色体畸变试验也未观察到钆喷酸二葡甲胺对体外培养细胞的损伤作用．研究表明，钆喷酸二葡甲胺对原核生物和哺乳动物细胞无致突变作用．     

草苔虫内酯的遗传毒性评价

目的：检测草苔虫内酯的遗传毒性。方法：采用Ames试验、体外培养中国仓鼠卵巢（chinese hamsterovary,crto）细胞染色体畸变试验和小鼠骨髓微核试验检测草苔虫内酯的遗传毒性。结果：Ames试验显示在每皿100、10、1、0．1g受试剂量下,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102及TAl535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示,在3．75、1．88和0．94g／ml 3个剂量组,在加S9条件下培养24h和不加S9培养24、48h的CHO细胞染色体畸变率与溶剂对照组相比差异有统计学意义（P〈0．05）。小鼠骨髓微核试验,在12．5、25、50g／kg3个剂量下对ICR小鼠的微核诱发率呈剂量反应关系,与溶剂对照组相比差异有统计学意义（P〈0．01）。结论：在本实验条件下,草苔虫内酯对鼠伤寒沙门氏菌无致突变性,对哺乳动物培养细胞染色体的致畸变作用为可疑阳性,对ICR／b鼠有诱发骨髓嗜多染红细胞微核的效应,提示草苔虫内酯对人体具有潜在的遗传毒性。     

桑椹对小鼠骨髓细胞诱发突变的抑制作用

本文用小鼠骨髓细胞微核试验方法和小鼠骨髓细胞染色体畸变试验方法观察了新鲜桑椹汁对环磷酰胺（ＣＰ）诱发小鼠骨髓嗜多染红细胞微核和染色体畸变的抑制作用。发现新鲜桑椹汁具有抑制环磷酰胺诱发骨髓微核率和染色体畸变率升高的作用，且有明显的剂量反应关系，相关系数分别为－０．９４和－０．９８。说明新鲜桑椹汁具有一定的抗诱变作用。     

荧光原位杂交技术检测外周T细胞淋巴瘤的染色体畸变

运用荧光原位杂交技术，在４１例外周Ｔ细胞淋巴瘤（简称ＰＴＬ）中发现３２例（７８％）和１４例（３４％）病人分别有常见的第３号染色体三体（＋３）以及一个额外Ｘ染色体（＋Ｘ）畸变，含有＋３和＋Ｘ的畸变细胞率分别为４．５％和８．２％。研究表明，＋３和＋Ｘ是ＰＴＬ常见的、且可能是特异性的染色体畸变，恶性肿瘤细胞在受累淋巴结中只占少数，荧光原位杂交技术在检测染色体畸变中比传统的细胞遗传学方法敏感。     

锂离子照射人气管上皮细胞诱发的以体畸变初步观察

本研究用中国原子能科学院物理研究所的ＨＩ－１３串列加速器产生的Ｌｉ（Ｚ＝３，ＬＥＴ＝１００ｋｅＶ／μｍ）离子束以０．５－６．０Ｇｙ剂量照射人支气管上皮细胞系（ＢＥＡＳ－２Ｂ），来观察重离子辐射诱发的细胞染色体畸变。分析经Ｌｉ离子照射细胞的染色体，除了如同对照组那样的发现５号、１５号、２２号的Ｘ染色体丢失、出现４条标记染色体及染色体数目仍以４６条为主以外，在早期细胞中有非稳定性畸变，对照组细胞丢失的     

Okadaic Acid and Phorbol Esters: Comparative Effects of These Tumor Promoters on Cell Transformation, Intercellular Communication and Differentiation in vitro

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.     

Okadaic acid and phorbol esters: comparative effects of these tumor promoters on cell transformation, intercellular communication and differentiation in vitro

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.     

Inhibition of tumor promoter activity toward mouse fibroblasts and their in vitro transformation by tissue inhibitor of metalloproteinases- 1 (TIMP-1)

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.      

Possible mechanism for the cocarcinogenic effect of bile acids: increased intracellular uptake of methylcholanthrene by C3H/10T1/2 fibroblasts in vitro

The cocarcinogenic effect of bile acids on the chemical transformation of C3H/10T1/2 fibroblasts was examined in vitro. The assimilation of 3H-methylcholanthrene (3H-MCA) by C3H/10T1/2 cells pretreated with bile acids was examined by the measurement of uptake and by autoradiography. Cells that were pretreated for 48 h with bile acids (100 microM lithocholic acid, 500 microM cholic acid) and then maintained in medium that contained 3H-MCA showed an increase in radioactivity compared to control cells. These results indicate that the transfer of carcinogens into cells is enhanced by pretreatment with bile acids.     

In vitro transformation of C3H/10T1/2 and NIH/3T3 cells by acrylonitrile and acrylamide

Acrylonitrile (AN) and acrylamide (AM) are carcinogenic in a number of rodent organs and AN is a suspected human carcinogen. We sought to determine whether AN and/or AM could produce morphological transformation in vitro in C3H/10T1/2 and NIH/3T3 mouse fibroblast cells. Both AN and AM induced a dose-dependent cytotoxic effect in C3H/10T1/2 and NIH/3T3 cells and readily transformed both cell lines. Our conclusions are based on the appearance of cells exhibiting a transformed phenotype and growth in soft agar. AN and AM transformed NIH/3T3 cells to a greater extent than C3H/10T1/2 cells. This is the first reported transformation of cells in vitro by AM.     

Inhibition of Balb/c 3T3 cell transformation by synthetic acyclic retinoid NIK-333; possible involvement of enhanced gap junctional intercellular communication

BACKGROUND: In an attempt to develop effective and non-cytotoxic cancer chemopreventive derivatives of retinoids, a novel acyclic retinoid has previously been synthesized. This acyclic retinoid, NIK-333, had been reported to suppress recurrence of primary hepatocellular carcinomas. We explored the molecular mechanisms by which NIK-333 exerts anti-proliferative effects. METHODS: We employed Balb/c 3T3 cells, since they can be used as a quantitative transformation assay and since we can study a possible involvement of gap-junctional intercellular communication (GJIC) in their growth control. We included all-trans-retinoic acid (ATRA) for comparison. RESULTS: We first confirmed that these cells express the retinoid receptors, RARalpha, gamma and RXRalpha, suggesting that they respond to NIK-333 and ATRA. When NIK-333 or ATRA was added to Balb/c 3T3 cells during the induction of cell transformation by a standard (3-methylcholanthrene (MCA) alone) or two-stage (low dose of MCA plus 12-O-tetradecanoylphorbol-13-acetate (TPA)) protocol, there was a significant decrease in the number of transformed foci. The extent of inhibition of transformation by NIK-333 was similar to that exerted by ATRA. Employing the microinjection dye-transfer assay, we found that both retinoids increase GJIC level when measured 24h after treatment. The extent of GJIC increase by NIK-333 was similar to that of ATRA. These retinoids also increased the amount of connexin 43 (Cx43) on the plasma membrane as revealed by immunostaining. CONCLUSION: These data indicate that NIK-333 suppresses chemical carcinogenesis in vitro and support the hypothesis that enhancement of GJIC is involved in this process.     

Inhibition of gap junctional intercellular communication and enhancement of growth in BALB/c 3t3 cells treated with connexin43 antisense oligonucleotides

Many studies have correlated reductions in gap junctional intercellular communication (a) with altered cellular growth, tumor promotion, and neoplastic transformation. To test directly whether reduced GJIC affects cellular growth, GJIC was inhibited in murine BALB/c 3T3 fibroblasts by treatment with a phosphorothioatemodified antisense oligonucleotide targeted against the connexin43 translation start codon, and in vitro cell growth was monitored. The cells were incubated with the oligonucleotide (0.1-0.5 μM) in liposomes in serumless culture medium for 16 h; washed and refed with serum-containing medium; and analyzed for dye-coupling, connexin43 protein and mRNA levels, and cell growth over the next 5 d. The antisense oligonucleotide inhibited dye-coupling and reduced connexin43 protein levels in a concentration-dependent manner but had no effect on connexin43 mRNA levels. Cell growth rate was not affected, but saturation density was increased approximately threefold by the oligonucleotide. These data support a role for GJIC in the establishment of contact inhibition of in vitro cell growth. © 1995 Wiley- Liss, Inc.     

A mutant cell line derived from NIH/3T3 cells: two oncogenes required for in vitro transformation

EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.     

The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation

The AGR2 gene encodes a secretory protein that is highly expressed in adenocarcinomas of the esophagus, pancreas, breast, and prostate. This study explores the effect of AGR2 expression with well-established in vitro and in vivo assays that screen for cellular transformation and tumor growth. AGR2 expression in SEG-1 esophageal adenocarcinoma cells was reduced with RNA interference. Cellular transformation was examined using NIH3T3 cells that express AGR2 after stable transfection. The cell lines were studied in vitro with assays for density-dependent and anchorage-independent growth, and in vivo as tumor xenografts in nude mice. SEG-1 cells with reduced AGR2 expression showed an 82% decrease in anchorage-independent colony growth and a 60% reduction in tumor xenograft size. In vitro assays of AGR2-expressing NIH3T3 cells displayed enhanced foci formation and anchorage-independent growth. In vivo, AGR2-expressing NIH3T3 cells established tumors in nude mice. Thus, AGR2 expression promotes tumor growth in esophageal adenocarcinoma cells and is able to transform NIH3T3 cells. Immunohistochemistry of the normal mouse intestine detected AGR2 expression in proliferating and differentiated intestinal cells of secretory lineage. AGR2 may be important for the growth and development of the intestine as well as esophageal adenocarcinomas.