肝细胞癌临床病理学特征与p53蛋白表达的关系

应用免疫组织化学技术检测29例肝细胞癌（肝癌）和23例肝硬化标本中突变型p53蛋白的表达，并探讨后者与肝癌临床病理学特征之间的关系。结果：肝癌突变型p53蛋白表达阳性率为70％（20／29），癌旁组织阳性率为60％（18／29），肝硬化组织阳性率为30％（7／23），有血管侵犯的12例肝癌变变型p53蛋白表达均为阳性，而无血管侵犯的17例中仅8例为阳性（P＜0．05）；突变型p53蛋白阳性组血清甲胎蛋白（AFP）明显高于阴性组（8370±4764ng／ml比98．7±64．3ng／ml，P＜0．05）。提示：P53蛋白表达在肝硬化时即发生了异常；p53蛋白异常可能是AFP基因被激活的原因之一；p53蛋白异常有利于肝癌向门静脉转移。     

MDM2和p53在反应性及肿瘤性星形胶质细胞中的表达

目的：检测MDM2和p53蛋白在星形胶质细胞反应性增生与星形胶质细胞瘤中的表达，探讨二者在胶质瘤形成和发展中的作用及其相关性。方法：应用组织芯片和免疫组化染色技术检测正常脑组织、星形胶质细胞反应性增生、低级别（Ⅰ-Ⅱ级）和高级别（Ⅲ-Ⅳ级）星形胶质细胞瘤中MDM2和p53蛋白的表达情况。结果：正常脑组织中MDM2和p53蛋白均呈阴性表达；反应性增生组、低级别肿瘤组及高级别肿瘤组中MDM2蛋白的阳性率分别为32.7％（16／49）、59．2％（29／49）、80．0％（40／50）；p53蛋白的阳性率分别为27．3％（12／49）、57．1％（28／49）、82．0％（41／50）。二者阳性表达率均随着病变恶性程度的增加而升高，MDM2和p53在各实验组间的比较差异均有统计学意义（P〈0．05）；且MDM2和p53表达密切相关（P〈0．05）。结论：MDM2和p53在星形胶质细胞反应性增生及星形胶质细胞瘤中均呈不同程度的过度表达，且随着病变恶性程度的增加表达水平增高，MDM2扩增和p53突变是胶质瘤发生的早期事件，二者的联合检测可能会对星形胶质细胞反应性增生与低级别胶质细胞瘤的鉴别诊断以及星形胶质细胞瘤的早期诊断提供一定的依据。     

碳酸锂对化学诱导大鼠肝癌的保护作用

目的采用黄曲霉毒素B1（AFB1）诱导的大鼠肝癌模型，以肝组织形态学变化，胎盘型谷胱甘肽硫转移酶（GST-P）及增殖细胞核抗原（PCNA）为观察指标，探讨碳酸锂（Li2CO3）对化学诱导肝癌的保护作用。方法Wistar大鼠144只，随机分为A、B、C、D四组（A组：阴性对照组；B组：阳性对照组；C组：Li2CO3同时给药组；D组：Li2CO3先期给药组），于实验第6、9、10周分批断头处死动物，取动物肝脏进行肝组织学检查，GST-P及PCNA的免疫组化染色。结果C、D两组动物健康状况明显改善，肝癌前期病变程度明显减轻，肝癌标志酶GST-P表达减弱（P〈0．01），PCNA阳性率显著降低；D组动物一般状况明显好于C组，GST-P阳性灶及PCNA阳性率的下降趋势优于C组。结论Li2CO3对化学诱导肝癌有抑制和预防作用。     

凋亡相关蛋白Survivin及突变型p53在膀胱移行细胞癌中的临床意义

目的：Survivin、突变型p53在膀胱移行细胞癌（BTCC）中表达的临床意义。方法：应用SP免疫组织化学法检测60例行TUR—BT术切除BTCC标本组织石蜡切片中Survivin,突变型p53表达,结合临床资料进行分析,20例正常膀胱黏膜为对照。结果：Survivin在BTCC标本中的表达阳性率为70．0％（42／60）,而正常对照组中无1例呈阳性表达;Survivin表达与BTCC的初发、复发、病理分级有显著相关（P〈0．05）,但与肿瘤数目,临床分期无关（P〉0．05）;突变型p53在BTCC标本中表达阳性率为65．0％（39／60）,与对照组阳性率35．0％（7／20）相比差异有统计学意义（P〈0．05）,与BTCC的初发、复发、病理分级,临床分期有显著相关（P〈0．05）,它与肿瘤数目无统计学意义（P〉0．05）,BTCC中Survivin和突变型p53相比差异无统计学意义（P〉0．05）。结论：Survivin在膀胱移形细胞癌中高表达提示该基因在BTCC发生发展中有着重要的作用,Sur—vivin和突变型p53对临床医生判断BTCC的预后有着一定的临床指导意义。     

68例膀胱癌p53的检测及其预后意义

用抗p53单克隆抗体Do-7河检测68例膀胱移行上皮细胞癌．总阳性率44．1％16（30／68）。病理分级G_1p53阳性事28．6％（8／28）．G_248％（12／25），G_366．7％（10／15）（P＜0．05）。浸润型膀胱癌（T_2～T_4）的p53阳性率61．3％（19／31）．显著高于钱表型膀胱癌（Ta～T1）的29．7％（11／37）（P＜0．01）。随访材料（中位时间79个同）提示；p53阳性病例有很高的复发（P＜0．05〕、转移（P＜0．01）比例和较低的存活概率（P＜0．01）、本研究表明：膀恍癌p53过度表达与肿瘤恶性生物学行为密切相关，p53异常蛋白的检测可能是临床上有实用价值的客观预后指标。     

p33ING1、p53在结直肠癌中的表达及其相互关系

目的：探讨p33^ING1、p53在结直肠癌中的表达及其相互关系。方法：应用免疫组化SP法检测60例结直肠癌组织及相应正常黏膜组织中p33^ING1、p53的表达。结果：结直肠癌组织、相应正常黏膜组织中p33^ING1蛋白的阳性表达率分别为43．3％（26／60）、100％（60／60）（P〈O．01），p53蛋白分别为51．6％（31／60）、0％（0／60）。p33^ING1在无淋巴结转移组及淋巴结转移组癌组织中的阳性表达率分别为57．6％（19／33）、25．9％（7／27）（P〈0．05）；在DukesA、B期、Dukes C、D期病例中分别为56．7％（17／30）、30．0％（9／30）（P〈0．05）。在p53表达阴性的29例中有12例（41．4％，12／29）p33^ING1表达缺失，而p53阳性的31例病例中有22例（71．0％，22／31）p33^ING1表达阴性（P〈0．05），在p53蛋白表达阳性的病例中p33^ING1蛋白表达明显缺失，两者表达呈负相关。结论：p33^ING1在结直肠癌组织中低表达，与p53互相协同，在结直肠癌的发生、发展中可能起重要作用。     

非小细胞肺癌中甲胎蛋白受体表达的临床意义

目的：研究非小细胞肺癌（NSCLC）中甲胎蛋白受体（alpha—fetoprotein receptor，AFPR）表达的临床意义。方法：用免疫组织化学方法分析57例NSCLC组织、癌旁正常肺组织AFPR表达情况。结果：①NSCLC患者肿瘤组织中AFPR阳性率为40．4％（23／57）较正常肺组织15．8％（9／57）明显增高（P〈0．05）。②腺癌及腺鳞癌AFPR阳性率59．4％（19／32）显著高于非腺癌16％（4／25）（P〈0．05），腺癌AFPR表达与p53呈负相关（P〈0．05）。③在不同年龄、不同性别、不同分期、有无淋巴结转移的不同肺癌组织中，AFPR表达阳性率均无显著性差异（P〉0．05）。结论：NSCLC组织中AFPR表达明显高于癌旁正常肺组织，AFPR表达与肿瘤病理类型有一定关系。     

NF-κBp50、p53、Bcl-2在子宫颈癌组织中的表达及其与人乳头瘤病毒感染的关系

目的探讨子宫颈癌组织中NF-κBp50、p53、Bcl-2的表达及其与人乳头瘤病毒（HPV）感染的关系。方法采用免疫组织化学S—P法对46例子宫颈癌、26例正常子宫颈组织进行NF-κBp50、p53、Bcl-2蛋白检测；用PCR技术对各组织标本的HPV—DNA进行了检测。结果NF-κBp50、p53、Bcl-2蛋白在子宫颈癌组织中的表达率显著高于正常对照组（．P〈O．01）。NF-κBp50与p53或Bcl-2之间的表达具有相关性（P〈0．05），p53与Bcl-2之间无相关意义（P〉0．05）。在HPV—DNA阳性子宫颈癌组织中NF-κBp50的阳性率显著高于阴性组（P〈0．05）。p53、Bcl-2的阳性率在HPV—DNA阳性和阴性组之间差异无显著性（P〉0．05）。结论NF-κBp50、p53、Bcl-2的表达与子宫颈癌的发病有密切关系，HPV感染与NF-κBp50活化有关。     

启东肝癌高发区肝癌患者血清中p53突变研究

目的：研究启东地区肝癌患者血清中p53基因突变，并确定其对肝癌诊断的意义。方法：收集25例肝癌，20例肝硬化，30例健康对照者的血标本，提取DNA，应用限制性酶切及直接序列分析方法测定p53基因第七外显子突变。结果：显示p53基因第七外显子的249密码子产生突变，ARC变为SER，肝癌，肝硬化，健康者中发生率分别为44％（11／25），20％（4／20），7％（2／30）（P＜0．01），结论：启东地区肝癌患者血清中p53突变与肝癌发生密切相关，其可作为新的肝癌早期诊断标志。     

增殖细胞核抗原和p53蛋白在胆囊癌中的表达

本文应用PCNA和p53抑癌基因蛋白的免疫络化方法，对11例胆囊上皮良性病变和33例胆囊癌进行了研究，结果发现胆囊良性病变和胆囊癌的平均PCNA指数分别为：良性病变为24．1％（n＝11．x±24．6），高分化癌为48．0％（n＝13，x±12．8），中分化癌为49．5％（n＝11，x±15．4）．低分化癌为76．4％（n＝9，x±18．1）；高分化胆囊癌平均PCNA指数明显高于良性病变（P＜0．05）；低分化癌的平均PCNA指数明显高于高分化癌（P＜0．05）；11例胆囊上皮良性病变均无p53蛋白阳性表达；33例胆囊癌p53阳性表达12例（36．4％），高分化癌p53阳性率为15．4％（2／13）．中分化癌45．5％（5／11）．低分化癌55．6％（5／9）：各组阳性率比较无显著性差异（P＞0．05）；PCNA指数≥50％的胆囊癌其p53蛋白阳性率明显高于PCNA指数＜50％的胆囊癌（P＜0．05）。结果提示：PCNA指数与胆囊癌的分化程度有关．PCNA及p53蛋白的免疫组化染色对胆囊上皮良、恶性病变的鉴别论断有帮助。本文还就胆囊癌p53蛋白表达与PCNA指数的关系进行了讨论。     

DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long-Evans Cinnamon rats

The mutant strain Long-Evans Cinnamon (LEC) rat accumulates copper, resulting in spontaneous hepatitis and subsequent development of hepatocellular carcinomas (HCCs) in the liver, providing a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis. We examined DNA strand breaks in peripheral blood cells and p53 expression in livers during acute and chronic hepatitis in LEC rats, along with preneoplastic lesions, and cell proliferation and apoptosis in non-cancerous portions of livers from LEC rats aged 7-115 weeks. Immunohistochemistry using antibodies against glutathione S-transferase placental-form (GST-P), proliferating cell nuclear antigen (PCNA), and in situ DNA nick labeling (TUNEL) were used. Long-Evans Agouti (LEA) rats, a sibling line of the LEC strain, were used as controls. In the LEC rats, DNA strand breaks and expression of p53 were significantly higher than that of LEA rats at 24 weeks of age. The number of GST-P-positive (GST-P+) foci/cm2 increased and peaked at 48 weeks old, and the areas rapidly expanded thereafter. The level of cell proliferation increased with the development of hepatitis and was highest at about 48 weeks old. The induction of apoptosis in LEC rats was transiently higher than that in LEA rats during the period from 24 to 34 weeks of age. However, the ratio of PCNA-positive cells to the apoptotic index showed a growth imbalance in favor of cell proliferation, supporting sustained net growth in LEC rats. These findings suggest that DNA damage, reflected in DNA strand breaks, plays a critical role in the development of hepatocellular preneoplastic foci, with an imbalance between high proliferation and relatively low apoptosis.     

Increased expression of c-jun gene during spontaneous hepatocarcinogenesis in LEC rats

We have studied the expressions of nine proto-oncogenes (c-myc, N-myc, c-fos, C-jun, p53, H-ras, N-ras, c-raf, hst) and two other genes (PCNA, GST-P) during the spontaneous development of hepatocellular carcinomas (HCCs) in LEC rats. Expression of c-myc, H-ras, N-ras, C-raf, p53 and PCNA genes was detected, but this did not significantly change during the development of HCCs in LEC rats. Expression of N-myc and hst genes was not detectable. Expression of c-fos gene was detected in one HCC case out of four. Significantly increased expression of c-jun gene was observed in the liver tissues of LEC rats aged 8 months. This high expression was decreased with the development of HCCs. On the other hand, the expression of GST-P gene increased in parallel with the clinical course of the development of HCCs in LEC rats. The pattern of c-jun mRNA augmentation was different from that of GST-P mRNA. These observations suggest that c-jun gene may play a role in the spontaneous development of HCCs in LEC rats.     

Gene expression of placental glutathione S-transferase in hereditary hepatitis and spontaneous hepatocarcinogenesis of LEC strain rats

Liver tissues of LEC rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S-transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

Gene Expression of Placental Glutathione S-Transferase in Hereditary Hepatitis and Spontaneous Hepatocarcinogenesis of LEC Strain Rats

Liver tissues of LEG rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S -transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

3′-Me-DAB诱导大鼠肝癌发生过程中p53及相关基因mRNA的定量分析

背景与目的:有关肝癌中 p53基因突变及 p53蛋白表达异常已有报道 , 但其在 mRNA水平上的变化尚不清楚 . 为了解在肝癌发生、发展和预后过程中 p53、谷胱甘肽转硫酶 P( glutathione S-transferase P,GST-P) 、甲胎蛋白 ( α -fetoprotein,AFP) 和白蛋白 mRNA水平的变化 , 本研究定量分析癌前病变和癌灶中 GST-P、 AFP和白蛋白 mRNA的量 . 方法:在 3′ -甲基 -4-二甲胺偶氮苯 (3′ -methyl-4-dimethylaminoazobenzene,3′ -Me-DAB)诱发 F344大鼠肝癌过程中 , 用激光捕获显微取样仪 (LCM)准确获得大鼠肝脏中微小癌灶或癌前病变组织后 , 采用 LightCyclerTM V3 System 实时 (real-time)RT-PCR定量分析这些病灶组织中 mRNA水平 . 结果:在实验的第 6、 12和24周,癌前病变组织中p53mRNA量均显著高于正常组织(P≤0.001)。从第6周到第24周,癌前病变组织中p53mRNA逐渐降低(P<0.01)。癌组织中p53mRNA含量高于正常组织,低于同期癌前病变组织(P=0.028和0.0136)。第24周的癌前病变组织或癌组织中细胞核呈p53强染色。各实验期,癌前病变中GST-PmRNA量明显高于正常组织和癌组织(P<0.001)。癌组织中AFPmRNA的表达量显著高于癌前病变组织和正常组织(P<0.001),白蛋白mRNA的表达量显著低于这两种组织(P<0.01)。GST-P和AFP分别在癌前病变组织和癌组织中呈灶状强表达。结论：GST-P和AFPmRNA分别在癌前病灶组织和癌组织中强表达,是这些病变组织的重要标志物。p53mRNA在早期肝癌前病变和癌灶中显示高水平的表达,而较晚期p53蛋白升高。     

p53基因在甲状腺癌中的突变研究

目的：为探讨p53基因突变与甲状腺癌的发生、发展及预后的关系。方法：应用PCR单链构象多态性（SSCP）分析技术,对p53基因第7,8外显子突变进行了检测和分析。结果：在37例甲状腺癌中有11例在第7.8外显子发生突变,突变率为29.9％。p53基因突变在复发的患者中显著高于未复发的患者;p53基因突变与转移、组织学类型和分化状况无显著差异。结论：p53基因的突变可能与甲状腺癌患者预后有关。     

High Sensitivity of LEC Rats with Chronic Hepatitis to Hepatocarcinogenesis: Decreases in Unscheduled and Replicative DNA Synthesis of the Hepatocytes

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age-matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione-S-transferase placental-form (GST-P)-positive foci in the 33-week-old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25-week-old LEC rats with chronic hepatitis showed about one-third the level of UDS induced by UV irradiation, as compared to that of age-matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8-week-old LEC rats and age-matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8-week-old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25-week-old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non-initiated hepatocytes.     

珍珠梅提取物对二乙基亚硝胺所致大鼠肝脏癌前病变的抑制作用及抗氧化活力的影响

目的研究珍珠梅对二乙基亚硝胺致大鼠肝脏癌前病变灶及抗氧化活力的抑制作用.方法75只大鼠随机分为A、B、C 3组对照组、模型组和治疗组,每组25只.按略改良的Solt-Farber氏方法制作大鼠肝脏癌前病变模型,用珍珠梅提取物饲养大鼠6周后处死,通过免疫组织化学检测癌前病变组织谷胱甘肽S-转移酶(GST-P)和p53蛋白的表达,放射免疫法检测血清肿瘤坏死因子(TNF-α)的含量,比色分析法检测血清、肝线粒体超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)的含量.结果B和C组GST-P的表达率为86.7%和28.6%,p53的表达率为56%和36%,差异均有显著意义,P<0.05;B组血清TNF-α的含量与C组比较,差异有显著意义,P<0.05;B组血清、肝线粒体SOD、GSHPx活性及MDA含量与C组比较,差异均有显著意义,P<0.05.结论珍珠梅提取物对DEN所致大鼠癌前病变灶有抑制作用,可诱发TNF-α的生成,并有抗氧化作用.     

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.     

Tumor promoting potential in male F344 rats and mutagenicity in Salmonella typhimurium of dipyrone

For assessment of the carcinogenic potential and the mutagenicity of dipyrone, an antipyretic anodyne, -[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino]-methanesulfonic acid sodium salt monohydrate, three experiments were conducted using dipyrone A produced in Japan and/or dipyrone B obtained from the Federal Republic of Germany. (i) Carcinogenic potential of dipyrone A for rat liver: 8 week old male F344 rats were pretreated with 0.01% diethylnitrosamine (DEN) in drinking water for 2 weeks and, after 1 week of resting, administered 0.4% dipyrone in drinking water, 5 days a week, for 72 weeks. After an 8 week recovery period, all surviving rats were killed at 83 weeks. Hepatocellular carcinomas developed at a higher incidence in the DEN + dipyrone group (18 of 29 rats, 62%) than in the DEN alone group (9 of 29 rats, 31%), the difference being statistically significant (P less than 0.05). No carcinogenic activity of dipyrone was demonstrated in the groups given 0.4% dipyrone for 72 weeks or 0.4% dipyrone for 25 weeks, followed by 0.05% phenobarbital (PB) for 50 weeks. However, glutathione S-transferase P positive (GST-P+) preneoplastic hepatic foci in these groups were observed at a higher incidence than in the untreated control group (P less than 0.01). (ii) Effect of dipyrone A and dipyrone B on induction of DEN-initiated GST-P+ hepatic foci in a medium-term bioassay system: 0.4% dipyrone A in drinking water and 0.57% dipyrone A or dipyrone B in powdered diet after DEN initiation had similar enhancing effects on the development of GST-P+ foci (P less than 0.001). (iii) The Ames mutation test in Salmonella: both dipyrone A and dipyrone B proved weakly mutagenic for strain TA100 in the presence or absence of S9 fraction.