彗星试验检测环境致癌物的敏感性评价

目的：通过对彗星试验（又称单细胞凝胶电泳试验）检测致癌物的敏感性、检测谱、遗传毒性检出阈（检出阳性反应的最低剂量）的初步评价,为其在化学致癌性检测和环境致癌的生物监测中的应用提供依据。方法：选择１１类３７种已知致癌性化合物,利用彗星试验以悬浮暴露方式检测了各类物质诱发Ｖ７９细胞的ＤＮＡ损伤作用;根据结果评价了彗星试验的敏感性、检测谱与遗传毒性检测阈,并以这些评价指标,将彗星试验与Ａｍｅｓ试验进行了比较。结果：Ａ）苯并（α）芘等３７种致癌物中,彗星试验检出３３种具有ＤＮＡ损伤作用,检出的敏感性为８…     

柴油机车排出的颗粒提取物对V_(79)细胞DNA损伤的研究

目的：本试验是研究柴油机车排出的尾气颗粒,溶解在肺表面活性物质二棕榈酰卵磷脂（ＤＰＬ）溶液中,其提取物对中国仓鼠肺成纤维细胞（Ｖ７９细胞）ＤＮＡ的损伤和修复作用。并与溶解在二甲基亚砜（ＤＭＳＯ）中的柴油机尾气颗粒提取物对ＤＮＡ的损伤作一比较。方法：从柴油机车排气管抽取排出的尾气,经玻璃纤维滤膜阻留采样,干燥、取颗粒物,分别溶解在ＤＰＬ与ＤＭＳＯ中。设低、中、高三个剂量组和溶剂对照组,阳性对照组。将受试物分别加入培养的Ｖ７９细胞中,同时加入３Ｈ－ＴｄＲ,用放射自显影的方法显示掺入细胞核内的３Ｈ－Ｔ…     

应用蚕豆根尖细胞微核试验研究三氯甲烷与四氯化碳的遗传毒性

本文采用蚕豆根尖细胞微核测试法，三氯甲烷与四氯化碳的遗传毒性及其交互作用进行了研究。结果表明，三氯甲烷（０．５０３－５０．３０２μｍｏｌ／Ｌ）与四氯化碳（０．０１９５－１．９５０１μｍｏｌ／Ｌ）均分别显著诱发蚕豆根尖细胞微核的形成（Ｐ〈０．０１），并呈现明显的剂量效应，三氯甲烷与四氯化碳同时处理蚕豆根尖细胞时，诱发微核率仍然显著高于对照组，但表现一定的交互拮抗作用，结果提示，三氯甲烷与四氯化碳对生     

指状青霉提取物诱发大鼠肺及肝原代细胞DNA损伤作用

目的 探讨指状青霉（Ｐｅｎｉｃｉｌｉｕｍｄｉｇｉｔａｔｕｍ）提取物对大鼠肺及肝原代细胞的ＤＮＡ损伤作用，方法 用指状青霉素提取物诱发大鼠肺及肝原代细胞的程序外ＤＮＡ合成（ＵＤＳ），结果 指状青霉提取物均可诱导大鼠肺及肝原代细胞的ＵＤＳ，尤其是对肺原代细胞的作用更为明显（Ｐ〈０．０１）。结论 指状青霉素提取物对大鼠肺及肝原代细胞ＤＮＡ有损伤作用，从而证实其对哺乳细胞具有明显遗传毒性?     

α—干扰互和维甲酸对人卵巢癌细胞的联合抗增殖作用

采用台盼蓝拒染法及＾３Ｈ－脱氧胸苷和＾３Ｈ－亮氨酸掺入实验，观察－干扰素和维地人卵巢癌ＳＫＯＶ３细胞ＤＮＡ及蛋白质合成的影响。结果显示：１μｍ／Ｌ维甲 １０００＾／ｍｌα－干扰 联合可显著降低ＳＫＯＶ３细胞的增殖速度，抑制其ＤＮＡ和蛋白质的合成，联合作用强于二者单独应用之和，且细胞毒性并不增加。提示小剂量维甲酸和α干扰素联合可用于卵巢癌的治疗。     

d-筒箭毒碱对V79细胞和小鼠骨髓细胞有丝分裂的影响

目的：探讨烟碱型乙酰胆碱受体阻断剂ｄ－筒箭毒碱对Ｖ７９细胞和小鼠骨髓细胞有丝分裂的影响。方法：在离体情况下以受试物处理中国仓鼠Ｖ７９细胞，通过分析Ｖ７９细胞晚末期和早Ｇ１期细胞核与细胞质构型，双核细胞频率，探讨受试物对细胞质质裂的影响；研究同时探讨了活体情况下，该化合物对小鼠骨髓细胞的Ｃ－有丝分裂（Ｃ－Ｍ）效应，分析其对哺乳动物体细胞有丝分裂的影响。结果：ｄ－筒箭毒碱能使Ｖ７９细胞的晚末期一早Ｇ１     

^32P后标法分析二氯胺基酚—DNA加合物

本研究以核酸酶Ｐ１和正丁醇增强的＾３２Ｐ后标法分析了２，４－二氯－６－胺基酚（ＤＣＡＰ）染毒的Ｖ７９细胞ＤＮＡ加合物。试验结果：在核酸酶Ｐ１富集方式下，观察到４种ＤＣＡＰ诱发的ＤＮＡ加合物，其含量分别为３８．７、７１．５、８５．７和２２．４／１０＾８单核苷酸；而正丁醇萃取法，未检出加合物。该结果表明：ＤＣＡＰ具有与哺乳动物细胞ＤＮＡ产生共价结合的能力，与ＤＮＡ共价结合可能是ＤＣＡＰ产生遗传危害和致     

香烟烟雾对雄性小鼠生殖细胞DNA损伤的研究

为了研究香烟烟雾对生殖细胞的遗传毒性，本文采用目前检测ＤＮＡ损伤最敏感的方法—彗星试验，检测了香烟烟雾溶液对小鼠精原细胞的ＤＮＡ损伤作用。分别以二甲基亚枫（ＤＭＳＯ）和磷酸缓冲液（ＰＢＳ）作为吸收液，用大气收集器（Ｕ形多孔玻板吸收管）采集香烟主流烟雾，制得ＤＭＳＯ烟液和ＰＢＳ烟液。结果显示：ＤＭＳＯ烟液的原液至４倍稀释液（含烟量４×１０－３至１×１０－３支烟／ｍｌ）对精原细胞具有明显细胞毒性，孵育２ｈ后，细胞存活率小于８０％，而ＰＢＳ烟液的原液其细胞存活率也高于８０％。两种烟液的ＤＮＡ损伤作用也…     

B（a）P诱导的V79细胞HGPRT位点突变的检测

在加与不加Ｓ９两种情况下，用Ｖ７９中国仓鼠细胞检测了Ｂ（ａ）Ｐ诱ＨＧＰＲＴ突变频率和细胞毒性。结果表明，无论加与不加Ｓ９混合液，Ｂ（ａ）Ｐ对Ｖ７９细胞都具有明显毒作用，但在不加Ｓ９时，不会导致６－巯基嘌呤抗生细胞发生率的明显增加。在存在Ｓ９混合液时，Ｂ（ａ）Ｐ诱导的ＨＧＰＲＴ突变频率随Ｂ（ａ）Ｐ浓度的加大而明显增加。剂量在０．５－８μｇ／ｍｌ范围时，每１μｇ／ｍｌＢ（ａ）Ｐ诱发的Ｖ７９细胞ＨＧＰＲ     

彗星试验检测间接诱变剂对小鼠睾丸细胞的DNA损伤

目的：探讨组织原代细胞在检测间接诱变剂中的作用。方法：采用单细胞凝胶电泳试验（彗星试验）对３种典型的间接诱变剂（环磷酰胺、２－氨基芴和二甲基苯蒽）所致的离体小鼠睾丸细胞的ＤＮＡ损伤进行了研究。结果：在一定浓度范围内,３种受试物均可直接引起离体小鼠睾丸细胞ＤＮＡ单链断裂,出现拖尾的彗星细胞,其拖尾细胞百分率和ＤＮＡ迁移长度均随受试物浓度的增加而增加,且有明显的剂量反应关系。结论：原代小鼠睾丸细胞具有一定的代谢活化作用。可以用于彗星试验直接检测间接诱变剂所致的ＤＮＡ损伤研究。     

Structure-activity relationships of podophyllin congeners that inhibit topoisomerase II

Various analogs of etoposide have been studied and compared in different tests in order to identify which tests best correlate with antitumor activity. These tests included DNA breakage assays using standard alkaline elution procedures as a means of studying topoisomerase II inhibition in intact cells, cytotoxicity studies in naturally sensitive and resistant human carcinoma cell lines, in vitro assays of the effect of the different congeners on topoisomerase II activity, and a preliminary evaluation of the ability of etoposide and teniposide to induce resistance. As in previous studies, a direct correlation was seen between double strand DNA breakage and cytotoxicity but not between single strand DNA breakage and cytotoxicity. Analogs with blocked 4'-hydroxyl groups were poor antitumor agents but were still capable of inhibiting topoisomerase II as evidenced by the production of DNA breaks. However, this DNA breakage was qualitatively different from that produced by VP16. None of the analogs were able to overcome either naive or acquired drug resistance. The dihydroxy analog of VP16, a possible bioactivated analog, was much less potent and possibly less stable than VP16. A model is proposed for the inhibition of topoisomerase II by demethylepipodophyllotoxins that may explain the relationship between double strand DNA breakage and cytotoxicity.     

The antioxidant ascorbic acid mobilizes nuclear copper leading to a prooxidant breakage of cellular DNA: implications for chemotherapeutic action against cancer

Purpose

Ascorbic acid is an essential micronutrient and is considered to have an antioxidant function in living systems. For the past several decades, ascorbic acid has been the subject of considerable interest as an anticancer agent. Several studies have shown that ascorbic acid is cytotoxic to a variety of cancer cells, whereas normal cells are relatively resistant to such cytotoxic action. In this study, we propose a putative molecular mechanism that accounts for the preferential cytotoxicity of ascorbic acid against cancer cells.

Methods

Standard and lysed version of alkaline single-cell gel electrophoresis (Comet assay); ferrous oxidation?Cxylenol orange (FOX) assay.

Results

We show that ascorbic acid acts as a prooxidant and leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei. Scavengers of reactive oxygen species were able to inhibit ascorbic acid-induced DNA breakage, suggesting the involvement of reactive oxygen species in this reaction. We further show that such DNA breakage is inhibited by both iron and copper chelators in cells, whereas in nuclei, similar inhibition was achieved only by copper chelators, indicating an important role of chromatin-bound copper in the prooxidant cellular DNA breakage by ascorbic acid.

Conclusion

We propose that the copper-dependent cellular redox status is an important element in the cytotoxic action of ascorbic acid against cancer cells. It is well established that cellular copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper and ascorbate to generate reactive oxygen species. In light of these observations and those in literature, in this paper we explain that the preferential cytotoxicity of ascorbic acid against cancer cells is the result of elevated copper levels in such cells. Further, this study identifies nuclear copper as a novel molecular target for cytotoxic action of ascorbic acid, which has implications for its chemotherapeutic properties against cancer.     

Chlorinated ethylenes: their metabolism and effect on DNA repair in rat hepatocytes

The major initial metabolites of the chlorinated ethylenes inhepatocyte suspensions isolated from phenobarbital treated ratswere as follows (rates of metabolite production in nmol/l0⁶cells/min are given in brackets): vinylidene chloride, dichloroaceticacid (0.015); cis-1,2-dichloroethylene, 2,2-dichloroethanol(0.24); trans-1,2-dichloroethylene, dichloroacetic acid (0.005);trichloroethylene, chloral hydrate (2.7); tetrachloroethylene,trichloroacetic acid (0.08). Comparison of the metabolism ofthe chlorinated ethylenes by isolated hepatocyte suspensionsand hepatic microsomes indicates that the initial products ofthe three dichlorinated ethylenes from cytochrome P-450 in hepaticmicrosomes are rapidly and extensively metabolized in the hepatocyte,where the Phase II enzymes are present. In contrast, the initialmetabolites of trichloroethylene and of tetrachloroethylenein the two systems are identical. The abilities of the chlorinatedethylenes to induce unscheduled DNA synthesis was assessed inisolated hepatocytes using a method which does not require theblocking of semi-conservative DNA synthesis. Vinylidene chloride,cis-1,2-dichloroethylene and trichloroethylene induced unscheduledDNA synthesis, while trans-1,2-dichloroethylene and tetrachloroethylenedid not.     

Sensitivity of markers of DNA stability and DNA repair activity to folate supplementation in healthy volunteers

We have previously reported that supplementation with folic acid (1.2 mg day(-1) for 12 week) elicited a significant improvement in the folate status of 61 healthy volunteers. We have examined effects of this supplement on markers of genomic stability. Little is known about the effect of folate supplementation on DNA stability in a cohort, which is not folate deficient. Preintervention, there was a significant inverse association between uracil misincorporation in lymphocyte DNA and red cell folate (P < 0.05). In contrast, there were no associations between folate status and DNA strand breakage, global DNA methylation or DNA base excision repair (measured as the capacity of the lymphocyte extract to repair 8-oxoGua ex vivo). Folate supplementation elicited a significant reduction in uracil misincorporation (P < 0.05), while DNA strand breakage and global DNA methylation remained unchanged. Increasing folate status significantly decreased the base excision repair capacity in those volunteers with the lowest preintervention folate status (P < 0.05). Uracil misincorporation was more sensitive to changes in folate status than other measures of DNA stability and therefore could be considered a specific and functional marker of folate status, which may also be relevant to cancer risk in healthy people.     

卡铂对A_（549）肺癌细胞的抗癌作用及其机制的初步探讨

本文观察了卡铂对Ａ_（５４９）肺癌细胞ＤＮＡ合成、ＤＮＡ链间交联的影响及细胞超微结构的改变。依卡铂剂量不同将实验分为Ａ、Ｂ、Ｃ、Ｄ四组，剂量分别为２０μｇ／ｍｌ、４０μｇ／ｍｌ、６０μｇ／ｍｌ、８０μｇ／ｍｌ，另设空白对照Ｅ组。结果表明，卡铂可能是ＤＮＡ模板损伤型药物，通过引起ＤＮＡ交联而阻碍其复制，产生杀细胞效应。     

DNA damage in lung epithelial cells isolated from rats exposed to quartz: role of surface reactivity and neutrophilic inflammation

Respirable quartz has been classified as a human lung carcinogen (IARC, 1997). However, the mechanisms involved in quartz-induced carcinogenesis remain unclear. The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartz-exposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.     

Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro

The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy.     

The influence of Na+, K+-pump blockade on doxorubicin-mediated cytotoxicity and DNA strand breakage in human tumor cells

Summary We have previously shown that blockade of the Na⁺, K⁺-pump by the cardiac glycoside ouabain produces doxorubicin resistance and decreases topoisomerase II-mediated DNA strand breakage in hamster cells. To determine if this were a general phenomenon, the effect of pump blockade on doxorubicin resistance was assessed in three human tumor cell lines: A549 lung and HT29 colon adenocarcinomas and U1 melanoma. When cells were exposed to 1 μM ouabain prior to and during incubation with doxorubicin, cytotoxicity was markedly reduced. Ouabain had no effect on either the influx or the efflux of doxorubicin. However, all cell lines showed a ouabain-induced decrease in doxorubicin-induced topoisomerase-mediated DNA strand breakage (SSB). These data suggest that blockade of the Na⁺, K⁺ pump decreases doxorubicin cytotoxicity in human tumor cells by inhibiting topoisomerase-mediated SSB. Furthermore, they indicate that altered ionic gradients are a potential cause of resistance to drugs that use topoisomerase II as a target. This research was supported in part by a grant from the Rackham Foundation. Presented in part at the meeting of the American Association for Cancer Research in May, 1989     

DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide

Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells.     

Differential effects of all-trans-retinoic acid, docosahexaenoic acid, and hexadecylphosphocholine on cisplatin-induced cytotoxicity and apoptosis in a cisplantin-sensitive and resistant human embryonal carcinoma cell line

Apart from modulation of tumor-cell drug sensitivity, induction of differentiation might be an alternative in the treatment of tumors resistant to cytotoxic drugs. In this report the capacity to induce differentiation and to modulate the cis-diamminedichloroplatinum(II) (CDDP) sensitivity of all-trans-retinoic acid (RA), docosahexaenoic acid (DCHA), and hexadecylphosphocholine (HePC) is examined in human germ-cell tumor cell lines. In the embryonal carcinoma cell line Tera-2 and its 3.7-fold CDDP-resistant subline Tera2-CP, we evaluated the effects of 96 h of pretreatment with RA (0.1 μM ), DCHA (23 μM ), and HePC (25 μM ) on differenctiation induction and on CDDP-induced cytotoxicity, DNA platination (4-h incubation), and apoptosis (continuous incubation). Without drug treatment, Tera2-CP showed less apoptosis than Tera-2. Pretreatment with RA decreased the cytotoxicity and apoptosis induced by CDDP without resulting in decreased DNA platination and increased differentiation in both cell lines. DCHA enhanced CDDP-induced cytotoxicity and apoptosis and did not affect the embryonal character of either cell line. HePC did not affect CDDP cytotoxicity or differentiation in either cell lines. Effects of the modulators on differentiation and on CDDP-induced cytotoxicity, DNA platination, and apoptosis did not differ between Tera-2 and Tera2-CP. RA can be applied for differentiation induction in CDDP-resistant germ-cell tumor models. However, in this model, RA reduced the apoptotic susceptibility. DCHA potentiated CDDP cytotoxicity in vitro; its in vivo modulatory capacity in germ-cell tumor cells deserves further study. Received: 1 June 1997 / Accepted: 22 October 1997