药物的致癌性课件

药物致癌性的研究现状和动态第二军医大学药物安全性评价中心第二军医大学药物安全性评价中心第二军医大学卫生毒理学教研室第二军医大学卫生毒理学教研室张天宝张天宝药物致癌性研究的必要性药物致癌性研究的必要性肿瘤是一类严重影响人类健康和生命的疾病 肿瘤已成为人类死亡的第肿瘤已成为人类死亡的第肿瘤已成为人类死亡的第肿瘤已成为人类死亡的第 1 1 1 1或或或或2 2 2 2位原因，每年位原因，每年位原因，每年位原因，每年约有约有约有约有700700700700万人死于癌症。据万人死于癌症。据万人死于癌症。据万人死于癌症。据2003200320032003年年年年WHOWHOWHOWHO资料资料资料资料,目前每目前每目前每目前每年新增肿瘤年新增肿瘤年新增肿瘤年新增肿瘤1000100010001000万人，其中男性万人，其中男性万人，其中男性万人，其中男性530530530530万人，与万人，与万人，与万人，与1990199019901990年相比，全球癌症患者发病率增长年相比，全球癌症患者发病率增长年相比，全球癌症患者发病率增长年相比，全球癌症患者发病率增长19191919，死亡率死亡率死亡率死亡率增长了增长了增长了增长了18181818。我国恶性肿瘤我国恶性肿瘤我国恶性肿瘤我国恶性肿瘤(2000(2000(2000(2000年年年年)在各种死因中排在第二在各种死因中排在第二在各种死因中排在第二在各种死因中排在第二位，城市已排在首位。每年新增肿瘤位，城市已排在首位。每年新增肿瘤位，城市已排在首位。每年新增肿瘤位，城市已排在首位。每年新增肿瘤200200200200万人。据万人。据万人。据万人。据预测我国预测我国预测我国预测我国2010201020102010年年发病数年年发病数年年发病数年年发病数220220220220万，万，万，万，2020202020202020年为年为年为年为300300300300万。万。万。万。Age-adjusted Cancer Death Rates,by Site,US,1930-200519571957、19841984、19991999、20042004年我国城市主要疾病死因年我国城市主要疾病死因构成比及死因顺位构成比及死因顺位顺顺位位1957198419992004死死 因因比例比例(%)死死 因因比例比例(%)死死 因因比例比例(%)死因死因比例比例（%）1呼吸系统呼吸系统16.86心脏病心脏病22.65脑血管病脑血管病22.28恶性肿瘤恶性肿瘤27.232传染病传染病7.93脑血管病脑血管病21.13恶性肿瘤恶性肿瘤21.66脑血管病脑血管病18.473肺结核肺结核7.51恶性肿瘤恶性肿瘤21.11心脏病心脏病16.37心脏病心脏病17.234消化系统消化系统7.31呼吸系统呼吸系统8.79呼吸系统呼吸系统15.28呼吸系统呼吸系统12.775心脏病心脏病6.61消化系统消化系统4.32意外伤害意外伤害6.52损伤中毒损伤中毒6.56特别严重的是特别严重的是特别严重的是特别严重的是,肿瘤是我国最佳劳动人口的首要死因，在肿瘤是我国最佳劳动人口的首要死因，在肿瘤是我国最佳劳动人口的首要死因，在肿瘤是我国最佳劳动人口的首要死因，在3535353559595959岁年龄人口中，所有年龄组的第一位死因都是肿瘤，只有岁年龄人口中，所有年龄组的第一位死因都是肿瘤，只有岁年龄人口中，所有年龄组的第一位死因都是肿瘤，只有岁年龄人口中，所有年龄组的第一位死因都是肿瘤，只有到了到了到了到了60606060岁以后脑血管或心血管疾病才上升为第岁以后脑血管或心血管疾病才上升为第岁以后脑血管或心血管疾病才上升为第岁以后脑血管或心血管疾病才上升为第1 1 1 1位死因。位死因。位死因。位死因。What may cause cancer?Hereditary disorders Hereditary disorders ChemicalsChemicals Viruses Viruses Chronic inflammation Chronic inflammation?WORLD HEALTH ORGANIZATIONINTERNATIONAL AGENCY FOR RESEARCH ON CANCERIARC Monograph EvaluationsLYON,FRANCESlide courtesy of V.Cogliano(IARC)IARC(2009)-monographs.iarc.frCarcinogenic to humans (group 1)108 agentsProbably carcinogenic to humans(group 2A)66Possibly carcinogenic to humans(group 2B)248Not classifiable as to its carcinogenicity to humans (group 3)515 Probably not carcinogenic to humans(group 4)1IARC:IARC Group 1 Carcinogenic to humansMedical drugs and treatments24Industrial processes13Infectious agents or processes10Physical agents10Industrial chemicals 7Inhaled particulates 5Metals and inorganic salts 5Lifestyle factors(incl.herbal remedies)7Other 8Group 2A 66 Medical drugs and treatments 12 Chemical Carcinogenesis in the 21st CenturyNew perceptions of previously known carcinogens:Combined effects of multiple exposuresExamples:oAlcohol drinking and aflatoxinsoAlcohol drinking and HBV/HBCoAlcohol drinking and tobacco smokingoTobacco smoking and asbestos/arsenic/radon 在研究药物的潜在致癌作用中，致癌试验比现在研究药物的潜在致癌作用中，致癌试验比现在研究药物的潜在致癌作用中，致癌试验比现在研究药物的潜在致癌作用中，致癌试验比现有遗传毒性试验和系统暴露评价技术更有意义。有遗传毒性试验和系统暴露评价技术更有意义。有遗传毒性试验和系统暴露评价技术更有意义。有遗传毒性试验和系统暴露评价技术更有意义。致癌试验仍是目前评价药物致癌作用最可靠和最有意义的方法 已评价的致癌物中有已评价的致癌物中有93%(515/554)93%(515/554)至少在三项至少在三项标准遗传毒性试验中有一项呈阳性标准遗传毒性试验中有一项呈阳性,表明在检测表明在检测致癌物（敏感性）是成功的；然而鉴定非致癌物致癌物（敏感性）是成功的；然而鉴定非致癌物的能力（特异性）较差的能力（特异性）较差 ，183183种在大、小鼠致癌种在大、小鼠致癌试验中为阴性的物质试验中为阴性的物质80%80%以上有体外遗传毒性阳以上有体外遗传毒性阳性的资料。性的资料。The European Centre for the Validation of Alternative Methods(ECVAM)A recent analysis of nearly 1000 chemicals for which data have been published has highlighted the strikingly imprecise nature of in vitro genetic toxicology tests in discriminating non-carcinogens from carcinogens.When the standard battery of two or three in vitro genotoxicity tests was performed,at least 80%of at least 80%of the 177 non-carcinogenic compounds tested the 177 non-carcinogenic compounds tested gave a false positive result in at least one test.gave a false positive result in at least one test.The false positive rate was highest in mammalian cell tests such as those to detect chromosomal Aberrations or micronucleus in Chinese hamster cells,or Mutations in the mouse lymphoma assay.A similar outcome was obtained in analysis by the U.S.FDA of an even larger database of chemicals.Performance of individual genotoxic tests in detecting rodent carcinogens as analyzed by Kirkland et al.(2005).AmesAmesMLNMLNMNMNSensitivity(%)58.873.178.7Specificity(%)73.939.030.8Ames+Ames+MLAMLAAmes+Ames+MNMNMLA+MLA+MNMNAmes+Ames+MLA+MNMLA+MNSensitivity(%)81.085.987.090.7Specificity(%)32.412.010.05.0Performance of simultaneous testing batteries of genotoxic tests in detecting rodent carcinogens as analyzedIn vitro genotox testing:the problem poor specificitypoor specificity大多数致癌物在组合试验大多数致癌物在组合试验大多数致癌物在组合试验大多数致癌物在组合试验中呈阳性中呈阳性中呈阳性中呈阳性Good!大多数非致癌物在组合试大多数非致癌物在组合试大多数非致癌物在组合试大多数非致癌物在组合试验中也呈阳性验中也呈阳性验中也呈阳性验中也呈阳性Bad!特特异异性性敏敏感感性性AmesMLA AmesMN MNIndomethacin(吲哚美锌吲哚美锌)tested negative for in vivo cytogenetic assays in the regulatory tests,but was reported positive for the induction of DNA adducts in the literature.Halothane(氟烷氟烷)and pyrazinamide(吡嗪酰胺吡嗪酰胺)were also in vivo positive for comet test in human lymphocytes and induction of sperm head abnormalities in mice,respectively,which are considered non-regulatory tests.某些药物是非遗传毒性的致癌物某些药物是非遗传毒性的致癌物-用遗传毒性用遗传毒性试验无法检出试验无法检出很多管理机构都提出了致癌试验的要求很多管理机构都提出了致癌试验的要求日本日本(1990)，如果临床预期连续用药如果临床预期连续用药6 6个月或更长时间，个月或更长时间，则需要进行致癌试验。尽管连续用药少于则需要进行致癌试验。尽管连续用药少于6 6个月，如果个月，如果存在潜在致癌性因素，也可能需要进行致癌试验。存在潜在致癌性因素，也可能需要进行致癌试验。美国，美国，一般药物使用一般药物使用3 3个月或更长时间，需要进行致癌个月或更长时间，需要进行致癌试验。试验。欧洲，欧洲，规定长期应用的药物，即至少规定长期应用的药物，即至少6 6个月的连续用药，个月的连续用药，或频繁的间歇性用药以致总的暴露量与前者相似的药物或频繁的间歇性用药以致总的暴露量与前者相似的药物需要进行致癌试验需要进行致癌试验.2010年年04月月01日我国日我国SFDA制定发布了制定发布了药物致癌试药物致癌试验必要性的技术指导原则验必要性的技术指导原则药物致癌试验药物致癌试验 2010年年04月月01日日SFDA制定发布了制定发布了药物致癌试药物致癌试验必要性的技术指导原则验必要性的技术指导原则 预期临床用药期至少连续预期临床用药期至少连续6 6个月的药物一般应进行。个月的药物一般应进行。连续用药没有连续用药没有6 6个月，但以间歇的方式重复使用，个月，但以间歇的方式重复使用，如治疗慢性和复发性疾病如治疗慢性和复发性疾病(包括过敏性鼻炎、抑郁症包括过敏性鼻炎、抑郁症和焦虑症和焦虑症)，而需经常间歇使用的药物，一般也需进，而需经常间歇使用的药物，一般也需进行。行。某些可能导致暴露时间延长的释药系统，也应考虑某些可能导致暴露时间延长的释药系统，也应考虑进行致癌试验。进行致癌试验。存在潜在致癌的担忧因素存在潜在致癌的担忧因素 1 1）已有证据显示此类药物具有与人类相关的潜在致癌性；）已有证据显示此类药物具有与人类相关的潜在致癌性；2 2）其构效关系提示致癌的风险；）其构效关系提示致癌的风险；3 3）重复给药毒性试验中有癌前病变的证据；）重复给药毒性试验中有癌前病变的证据；4 4）导致局部组织反应或其它病理生理变化的化合物或其代）导致局部组织反应或其它病理生理变化的化合物或其代谢产物在组织内长期滞留谢产物在组织内长期滞留 内源性肽类、蛋白类物质及其类似物内源性肽类、蛋白类物质及其类似物 对于替代治疗的内源性物质（浓度在生理水平），尤其是当同类产品对于替代治疗的内源性物质（浓度在生理水平），尤其是当同类产品（如动物胰岛素、垂体来源的生长激素和降钙素）已有临床使用经验时，（如动物胰岛素、垂体来源的生长激素和降钙素）已有临床使用经验时，通常不需要进行致癌试验通常不需要进行致癌试验 1 1）其生物活性与天然物质明显不同；）其生物活性与天然物质明显不同；2 2）与天然物质比较显示修饰后结构发生明显改变）与天然物质比较显示修饰后结构发生明显改变 3 3）药物的暴露量超过了血液或组织中的正常水平）药物的暴露量超过了血液或组织中的正常水平 化学致癌的过程及其机制化学致癌的过程及其机制化学致癌化学致癌(chemical carcinogenesis)化学物化学物质引起或增进正常细胞发生恶性转化并发展成为质引起或增进正常细胞发生恶性转化并发展成为肿瘤的过程。具有这类作用的化学物质称为肿瘤的过程。具有这类作用的化学物质称为化学化学致癌物致癌物(chemical carcinogen).化学致癌研究的重要历史事件化学致癌研究的重要历史事件 1761,J Hill 提出使用鼻烟可能会诱发鼻咽癌提出使用鼻烟可能会诱发鼻咽癌1775,P Pott 提出扫烟囱男童阴囊癌与煤烟过度暴露有关提出扫烟囱男童阴囊癌与煤烟过度暴露有关1895,L Rehn 首首次次报报道道从从事事苯苯胺胺染染料料生生产产的的工工人人发发生生膀膀胱胱癌癌1914,T Boveri 提提出出恶恶性性肿肿瘤瘤起起源源于于存存在在染染色色体体异异常常的的单单个个细细胞胞（这这就就是是著著名名的的癌癌症症体体细细胞胞突突变变理理论论和和肿肿瘤瘤单单细细胞胞克隆起源学说克隆起源学说）1915,Yamagiwa,K Ichikawa 通通过过长长期期给给兔兔耳耳涂涂煤煤焦焦油油成成功功地诱发皮肤癌（地诱发皮肤癌（实验性化学致癌研究的开端实验性化学致癌研究的开端）1932-1938，先先后后给给雄雄性性小小鼠鼠注注射射雌雌激激素素诱诱发发乳乳腺腺癌癌、通通过过慢慢性性饲饲喂喂偶偶氮氮染染料料O氨氨基基偶偶氮氮甲甲苯苯诱诱发发出出大大鼠鼠肝肝癌癌、使使用用萘萘胺诱发狗膀胱癌成功胺诱发狗膀胱癌成功 1941-1944,首首次次提提出出启启动动(Initation)和和促促进进(Promotion)的的概概念念,根根据据多多次次使使用用巴巴豆豆油油能能促促进进苯苯并并芘芘诱诱发发小小鼠鼠皮皮肤肤癌癌提提出出小小鼠鼠皮皮肤肤 癌癌 两两 阶阶 段段 致致 癌癌 模模 型型;1949 Foulds提提 出出 肿肿 瘤瘤 演演 进进(Progression)概念概念 1971-1981,C Peraino等等,发现小鼠皮肤癌两阶段致癌理论同发现小鼠皮肤癌两阶段致癌理论同样适用于大鼠肝癌的发生情况；随后建立了适用于各种脏器肿样适用于大鼠肝癌的发生情况；随后建立了适用于各种脏器肿瘤的瘤的多阶段癌变理论多阶段癌变理论1984-,A Balmain,首次报道在化学致癌物诱发的小鼠皮肤乳首次报道在化学致癌物诱发的小鼠皮肤乳头状瘤中的头状瘤中的c-Ha-ras基因被激活；随后发现多种致癌物可使不基因被激活；随后发现多种致癌物可使不同的同的癌基因活化癌基因活化和和抑癌基因失活抑癌基因失活 Initiating EventCell Proliferation(clonal expansion)ProgressionCell ProliferationCell Proliferation MalignancySecond Mutating EventN Mutating Event Initiation PromotionStages of CarcinogenesisCellular and Molecular Mechanisms in Multistage Carcinogenesis:INITIATIONInitiating event involves cellular genome MUTATIONSTarget genes:-oncogenes/tumor suppressor genes -signal transduction -cell cycle/apoptosis regulators“Simple”genetic changesGentic and Epigenetic Models of The Cancer InitiationEpigenetically reprogrammed cellsMutator phenotype cellsEndogenousEndogenousEnvironmentalEnvironmentalALTERATIONS IN CELLULAR EPIGENOMENormal cellsCancer cellsClonal selection and expression of initiated cellsMutator phenotype cellsEndogenousEndogenousEnvironmentalEnvironmentalACQUISITION OF ADDITIONAL RANDOM MUTATIONSNormal cellsCancer cellsCellular and Molecular Mechanisms in Multistage Carcinogenesis:PROMOTIONReversible enhancement/repression of gene expression:-increased cell proliferation-inhibition of apoptosisNo direct structural alteration in DNA by agent or its metabolitesCellular and Molecular Mechanisms in Multistage Carcinogenesis:PROGRESSION Irreversible enhancement/repression of gene expression Complex genetic alterations(chromosomal translocations,deletions,gene amplifications,recombinations,etc.)Selection of neoplastic cells for optimal growth genotype/phenotype in response to the cellular environment“Complex”genetic changes致癌过程不同阶段的特征致癌过程不同阶段的特征Initiation DNA modification,Mutation,Genotoxic One cell division necessary to lock in mutation Modification is not enough to produce cancer Nonreversible,Single treatment can induce mutationPromotion No direct DNA modification,Nongenotoxic,No direct mutation Multiple cell divisions necessary,Threshold Clonal expansion of the initiated cell population Increase in cell proliferation or decrease in cell death(apoptosis)Reversible,Multiple treatments(prolonged treatment)necessaryProgression DNA modification,Genotoxic event?Mutation,chromosome disarrangement,Irreversible Changes from preneoplasia to neoplasia benign/malignant Number of treatments needed with compound unknown遗传毒物对遗传毒物对遗传毒物对遗传毒物对DNADNADNADNA和非和非和非和非DNADNADNADNA相互作用的机制相互作用的机制相互作用的机制相互作用的机制正常细胞正常细胞正常细胞正常细胞遗传改变遗传改变遗传改变遗传改变（突变）（突变）（突变）（突变）遗传改变遗传改变遗传改变遗传改变的细胞的细胞的细胞的细胞癌细胞癌细胞癌细胞癌细胞异倍体异倍体异倍体异倍体细胞细胞细胞细胞表遗传表遗传表遗传表遗传干扰干扰干扰干扰遗传毒性致癌物遗传毒性致癌物遗传毒性致癌物遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物遗传毒性和非遗传毒性致癌遗传毒性和非遗传毒性致癌物在多阶段致癌中的区别物在多阶段致癌中的区别遗传毒性和非遗传毒性致癌物作用机制的比较遗传毒性和非遗传毒性致癌物作用机制的比较 遗传毒性致癌物遗传毒性致癌物遗传毒性致癌物遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物非遗传毒性致癌物遗传毒性测试遗传毒性测试遗传毒性测试遗传毒性测试阳性阳性阳性阳性阴性阴性阴性阴性多阶段致癌作用多阶段致癌作用多阶段致癌作用多阶段致癌作用 遗传改变遗传改变遗传改变遗传改变表遗传改变表遗传改变表遗传改变表遗传改变 第一阶段第一阶段第一阶段第一阶段 基因突变基因突变基因突变基因突变细胞增殖细胞增殖细胞增殖细胞增殖 染色体畸变染色体畸变染色体畸变染色体畸变 再生再生再生再生 生长因子刺激生长因子刺激生长因子刺激生长因子刺激过氧化物酶体增殖过氧化物酶体增殖过氧化物酶体增殖过氧化物酶体增殖细胞间间隙连接通讯细胞间间隙连接通讯细胞间间隙连接通讯细胞间间隙连接通讯基因组印记基因组印记基因组印记基因组印记 以后阶段以后阶段以后阶段以后阶段 遗传或表遗传改变遗传或表遗传改变遗传或表遗传改变遗传或表遗传改变 遗传或表遗传改变遗传或表遗传改变遗传或表遗传改变遗传或表遗传改变 低剂量的剂量低剂量的剂量低剂量的剂量低剂量的剂量反应关系反应关系反应关系反应关系无致癌性阈值无致癌性阈值无致癌性阈值无致癌性阈值线性剂量反应曲线线性剂量反应曲线线性剂量反应曲线线性剂量反应曲线 致癌性阈值致癌性阈值致癌性阈值致癌性阈值S S S S型剂量反应曲线型剂量反应曲线型剂量反应曲线型剂量反应曲线 化学致癌的生物学特征化学致癌的生物学特征致癌物多数具有遗传毒性，遗传毒性致癌物尽管致癌物多数具有遗传毒性，遗传毒性致癌物尽管致癌物多数具有遗传毒性，遗传毒性致癌物尽管致癌物多数具有遗传毒性，遗传毒性致癌物尽管化学结构和性质不尽相同，但有一共同的特点，化学结构和性质不尽相同，但有一共同的特点，化学结构和性质不尽相同，但有一共同的特点，化学结构和性质不尽相同，但有一共同的特点，即皆为亲电子剂。即皆为亲电子剂。即皆为亲电子剂。即皆为亲电子剂。致癌作用依赖于化学致癌物的剂量致癌作用依赖于化学致癌物的剂量,大剂量的致大剂量的致癌物可增强肿瘤的发生癌物可增强肿瘤的发生,缩短潜伏期缩短潜伏期.肿瘤的产肿瘤的产生取决于化学致癌物的总剂量。同时暴露于几种生取决于化学致癌物的总剂量。同时暴露于几种致癌物，可发生联合作用。致癌物，可发生联合作用。致癌作用的充分表达需相当长的时间致癌作用的充分表达需相当长的时间致癌作用的充分表达需相当长的时间致癌作用的充分表达需相当长的时间,无论致癌无论致癌无论致癌无论致癌物的剂量和性质如何，在肿瘤形成前，总有一个物的剂量和性质如何，在肿瘤形成前，总有一个物的剂量和性质如何，在肿瘤形成前，总有一个物的剂量和性质如何，在肿瘤形成前，总有一个潜伏期。在细胞恶变以前，细胞存在着多阶段的潜伏期。在细胞恶变以前，细胞存在着多阶段的潜伏期。在细胞恶变以前，细胞存在着多阶段的潜伏期。在细胞恶变以前，细胞存在着多阶段的癌前病变。癌前病变。癌前病变。癌前病变。致癌作用所引起的细胞变化可传到子细胞致癌作用所引起的细胞变化可传到子细胞 致癌作用可被非致癌因子所修饰致癌作用可被非致癌因子所修饰 细胞增生是细胞癌变过程的重要阶段细胞增生是细胞癌变过程的重要阶段 The development of cancer is a multi-step process“Initiation”forms an early adenoma“Promotion”leads to a late adenoma“Progression”leads first to a cancer in situ,then on to “Malignant conversion,”which leads to true a carcinomaThis set of processes often takes YEARSGenotoxic carcinogensincrease tumour frequency in animal cancer bioassaypositive results from in vitro and in vivo genotoxicity testseither direct-acting or indirectly acting genotoxic carcinogensNon-genotoxic carcinogensusually act as tumor promoterspositive in cancer bioassay in animals,but negative in genotoxicity tests The mechanism of carcinogenicity may includethe chronic injury and regenerationhormonal mechanismsincrease in the cell proliferation or decrease in the cell death in target organThe concept of a“complete”vs.an“incomplete”carcinogenWhen one foreign chemical is suffient to cause cancer,either as a direct or indirect carcinogen,it is said to be completeWhen it requires a tumor promoter to cause cancer,it is an incomplete carcinogenPromotors are compounds that induce cells,like the mutated cancer initiator cell,to grow and divide,making more Fifteen example key events representing diverse Fifteen example key events representing diverse carcinogenic modes of actioncarcinogenic modes of actionDNA reactivity(covalent binding)Gene mutationChromosomal breakageAneuploidyEnzyme-mediated effects on DNA damage or repairEpigenetic effectsCell signaling:nuclear receptor-mediatedCell signaling:other than nuclear receptor-mediatedImmune response modulationInflammationCytotoxicity and compensatory cell proliferationMitogenicityChronic metabolic or physiologic overloadNutrient deficiency relatedInterference with intercellular communicationICH Guideline S1B onTesting for Carcinogenicity of Pharmaceuticalschoosing one 2-year rodent carcinogenicity study(rat)plus one other study that supplements the 2-year study and providing additional information that is not readily available from the 2-year study:either(1)a short-or medium-term in vivo rodent test system or(2)a 2-year carcinogenicity study in a second rodent species(mouse).the short-or medium-term models was intended to focus on the use of in vivo models providing insight into carcinogenic endpoints such as initiationinitiationpromotion rodent models promotion rodent models and models of carcinogenesis using transgenictransgenic or neonatal neonatal rodentsrodents药物致癌性评价方法药物致癌性评价方法Stipulated Rationale for Choosing a Short-or Medium-Term Test System as Supplement to One 2-Year Bioassay The mechanism of carcinogenesis in the model should most likely be relevant to humans,and therefore the use of the model should be applicable to human risk assessment.The use of the model should supplement the 2-yearcarcinogenicity study and it should provide additional information that is not readily available from the 2-yearstudy.Animal welfare,animal numbers,and overall economy of the carcinogenic evaluation process should be considered.Two-year Carcinogenesis“Bioassay”Protocol Current Global Carcinogenicity Study RequirementsStandard Tissue ListKidney Urinary bladder AortaHeart Trachea LungsLiver Gallbladder PancreasFat Salivary gland SpleenCervical lymph node Mesenteric lymph node ThymusTongue Esophagus StomachDuodenum Jejunum IleumCecum Colon Mammary glandSkin Skeletal muscle Sciatic nerveParathyroid Thyroid Adrenal glandPituitary Prostate Seminal vesiclesTestes Epididymides OvariesOviducts Uterine horns Uterine bodyCervix Vagina BrainSpinal cord Sternum Rib/boneEyes Harderian glands BM smearNares Clitoral/preputial gland Zymbal s glandGross lesions美国毒性病理学会（美国毒性病理学会（STP）建议致癌试验进行组织）建议致癌试验进行组织病理学检查的最基本的受检内容目录病理学检查的最基本的受检内容目录Tumor-Bearing Animals in Control Groups from Rodent StudiesSource:J.K.Haseman(unpublished summary of U.S.NTP data).Comparative Percent Incidence of Pertinent Neoplasia in Different Strains of Rats and Mice(104 Weeks Old)Note:F344,Fischer 244 rats;S-D,Sprague Dawley rats;B6C3F1,mice,(C57BL/6N+C3H/HeN)F1;CD-1,1CRCr:CD-1 mice;NA,nonapplicable;the average number used by species/strain/gender was in excess of 750 animalsPreclinical approaches for assessing carcinogenic potentialTumorigenicity in humans,nonhuman primates and rodentsSpontaneous tumor rates in the breeder and control animalsThe Neonatal MouseThe Neonatal MousePietra et al.(1959).The neonatal mouse is one of the alternative in vivo models,for detecting the carcinogenic potential of pharmaceuticals.This is in agreement with the suggestions of ICH,which allows the use of one alternative study in place of one of the 2-year carcinogenicity studies.When treatment begins within the first 24 hours of life,the study design is described as“newbornmouse”.“neonatal mouse”includes test item administration at different timepoints from birth to three weeks of age.Fujii(1991)reported that the neonatal mouse assay showed a sensitivity of 85%and a positive prediction rate of 96%compared to the results of the adult mouse 2-year carcinogenicity study.Flammang et al.(1997)considered this model to have high sensitivity and specificity to detect genotoxic carcinogens as well as presenting advantages such as reduced test article requirements,decreased animal numbers and costs and a reduced completion time.It does not respond to chemicals It does not respond to chemicals acting via epigenetic mechanisms.acting via epigenetic mechanisms.McClain et al.(2001)reported that neonatal mice have been shown to have a reduced time for tumor induction,a higher multiplicity of induced tumors,a lower spontaneous tumor rate and an equivalent or higher sensitivity to carcinogens when compared to adult mice.This model also responds to a wide range of structurally dissimilar genotoxic compounds.Additionally,the neonatal mouse possesses the majority of the phase I and II biotransformation liver enzymes involved in the processes of activation and detoxification of carcinogens from different chemical classes.CD-1 mice 10 to 12 weeks of age.Mice were caged with 5 females per male and examined each day for the presence of a vaginal copulation plug.Females were isolated until delivery,6 litters with 4 neonates/sex/litter were assigned to each group during the first week after birth.Three or four dose levels,a vehicle and a positive control were used.Groups consisted of 24 animals/sex/group.They were dosed on the basis of their average bodyweight,on days 8 and 15 of age,using dose volumes of up to 100 and 200 l,respectively.Dose levels were selected on the basis of the results obtained in dose range finding studies,in which the MTD or the MFD(Maximum Feasible Dose)for neonatal mice,were determined.The pups were weaned around 22 days of age,housed 4/sex/cage and then maintained until 1 year of age,when they were sacrificed.DEN(diethylnitrosamine)at a dosage of 2 mg/kg dissolved in water was used as the positive control.Tg.AC(v-Ha-ras)Transgenic Mouse89 weeks oldGroups of 15 mice/sex/dose were randomly assigned to the study groups.The vehicles used for drugs and positive control agents were acetone,ethanol or DMSO.TPA(12-o-tetradecanoylphorbol-13-acetate)was the positive control compound26 weeksHemizygous p53+/Knockout Mouse6 to 10 weeks old,Genotype analysis was recommended prior to assignment to dose groupsGroups of 15 mice/sex/group,Three dose levels,a vehicle and a positive control were used.Two additional groups of 15 wild-type mice/sex/group received the vehicle and the high dosage of the test compound.p-Cresidine at 400 mg/kg/day by gavage in corn oil(10ml/kg),or benzene at 100 mg/kg/day by gavage in corn oil(5 ml/kg)were recommended as positive controls.18 to 24 month bioassaycarcinoGENOMICSDevelopment of a high throughput genomics-based test for assessing genotoxic and carcinogenic properties of chemical compounds in vitroCombining pathway-associated gene expression with metabolic proiles generated in vitro,as is foreseen in carcinoGENOMICS,represents a highly innovative approach possibly leading to in silico models that may be used to predict the carcinogenic potential of a compound in vivo.Furthermore,toxicogenomics-based assays mayoutperform currently available tests for genotoxicity,as well as for genotoxic and non-genotoxic carcinogenicity,without using animals.These in vitro methods may therefore play a major role under the new system of the Community regarding the REACH initiative.研究动向研究动向An in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expressionMaeshima et al.established an in vitro real-time PCR screening assay with BALB/c 3T3 cells for the assessment of the tumor promoting potential of chemicals.based on 22 marker genes,enables earlier assessment,and is easier to con