1、肿瘤是一类严重影响人类健康和生命的疾病 Age-adjusted Cancer Death Rates,by Site,US,1930-201919571957、19841984、20192019、20192019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1957198420192019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21.6
2、6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6.56WORLD HEALTH ORGANIZATIONINTERNATIONAL AGENCY FOR RESEARCH ON CANCERIARC Monograph EvaluationsLYON,FRANCESlide courtesy of V.Cogliano(IARC)IAR
3、C(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
4、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
5、 21st CenturyNew perceptions of previously known carcinogens:Combined effects of multiple exposuresExamples:o Alcohol drinking and aflatoxinso Alcohol drinking and HBV/HBCo Alcohol drinking and tobacco smokingo Tobacco smoking and asbestos/arsenic/radon 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
6、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
7、 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,The false positive
8、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 genot
9、oxic tests in detecting rodent carcinogens as analyzed by Kirkland et al.(2019).Sensitivity(%)58.873.178.7Specificity(%)73.939.030.8Sensitivity(%)81.085.987.090.7Specificity(%)32.412.010.05.0Performance of simultaneous testing batteries of genotoxic tests in detecting rodent carcinogens as analyzedI
10、n vitro genotox testing:the problem Good!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 vi
11、vo 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
12、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.2019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
13、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 2019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
14、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
15、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 化学致癌化学致癌(chemical carcinogenesis)化学
16、物质引起化学物质引起或增进正常细胞发生恶性转化并发展成为肿瘤的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过程。具有这类作用的化学物质称为化学致癌物化学致癌物(chemical carcinogen).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 首次报道从事苯胺染料生产的工人发生膀胱癌首次报道从事苯胺染料生产的工人发生膀胱癌1914,T Boveri 提出恶性肿瘤起源于存在染色体异常的单个细提出恶性肿瘤起源
17、于存在染色体异常的单个细胞（这就是著名的癌症胞（这就是著名的癌症体细胞突变理论体细胞突变理论和和肿瘤单细胞克隆肿瘤单细胞克隆起源学说起源学说）1915,Yamagiwa,K Ichikawa 通过长期给兔耳涂煤焦油成功地诱发通过长期给兔耳涂煤焦油成功地诱发皮肤癌（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，先后给雄性小鼠注射雌激素诱发乳腺癌、通过慢性先后给雄性小鼠注射雌激素诱发乳腺癌、通过慢性饲喂偶氮染料饲喂偶氮染料O氨基偶氮甲苯诱发出大鼠肝癌、使用氨基偶氮甲苯诱发出大鼠肝癌、使用萘胺萘胺诱发狗膀胱癌成功诱发狗膀胱癌成功 1941-1944,首次提出首次提出启
18、动启动(Initation)和和促进促进(Promotion)的概念的概念,根据根据多次使用巴豆油能促进苯并芘诱发小鼠皮肤癌提出多次使用巴豆油能促进苯并芘诱发小鼠皮肤癌提出小鼠皮肤癌小鼠皮肤癌两阶段致癌模型两阶段致癌模型;1949 Foulds提出提出肿瘤演进肿瘤演进(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样适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瘤的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
19、癌物诱发的小鼠皮肤乳头状首次报道在化学致癌物诱发的小鼠皮肤乳头状瘤中的瘤中的c-Ha-ras基因被激活；随后发现多种致癌物可使不同的基因被激活；随后发现多种致癌物可使不同的癌癌基因活化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 PromotionCellular and Molecula
20、r Mechanisms in Multistage Cellular and Molecular Mechanisms in Multistage Carcinogenesis:Carcinogenesis:INITIATIONINITIATIONInitiating event involves cellular genome MUTATIONSTarget genes:-oncogenes/tumor suppressor genes -signal transduction -cell cycle/apoptosis regulators“Simple”genetic changesG
21、entic and Epigenetic Models of The Cancer InitiationEpigenetically reprogrammed cellsMutator phenotype cellsALTERATIONS IN CELLULAR EPIGENOMENormal cellsCancer cellsClonal selection and expression of initiated cellsMutator phenotype cellsACQUISITION OF ADDITIONAL RANDOM MUTATIONSNormal cellsCancer c
22、ellsCellular and Molecular Mechanisms in Multistage Cellular and Molecular Mechanisms in Multistage Carcinogenesis:Carcinogenesis:PROMOTIONPROMOTIONReversible enhancement/repression of gene expression:-increased cell proliferation-inhibition of apoptosisNo direct structural alteration in DNA by agen
23、t or its metabolitesCellular and Molecular Mechanisms in Multistage Cellular and Molecular Mechanisms in Multistage Carcinogenesis:Carcinogenesis:PROGRESSIONPROGRESSION Irreversible enhancement/repression of gene expression Complex genetic alterations(chromosomal translocations,deletions,gene amplif
24、ications,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 eno
25、ugh 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(apoptosi
26、s)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 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
27、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。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 conversi
28、on,”which leads to true a carcinoma This set of processes often takes YEARSGenotoxic carcinogens increase tumour frequency in animal cancer bioassay positive results from in vitro and in vivo genotoxicity tests either direct-acting or indirectly acting genotoxic carcinogensNon-genotoxic carcinogens
29、usually act as tumor promoters positive in cancer bioassay in animals,but negative in genotoxicity tests The mechanism of carcinogenicity may include the chronic injury and regeneration hormonal mechanisms increase in the cell proliferation or decrease in the cell death in target organ When one fore
30、ign chemical is suffient to cause cancer,either as a direct or indirect carcinogen,it is said to be complete When it requires a tumor promoter to cause cancer,it is an incomplete carcinogen Promotors are compounds that induce cells,like the mutated cancer initiator cell,to grow and divide,making mor
31、e DNA 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
32、 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
33、 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
34、 of in vivo models providing insight into carcinogenic endpoints such as and models of carcinogenesis using or 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 huma
35、ns,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
36、 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
37、 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/bone
38、Eyes 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 In
39、cidence 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 approa
40、ches for assessing carcinogenic potentialTumorigenicity in humans,nonhuman primates and rodentsSpontaneous tumor rates in the breeder and control animalsPietra et al.(1959).The neonatal mouse is one of the alternative in vivo models,for detecting the carcinogenic potential of pharmaceuticals.This is
41、 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 di
42、fferent 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.(2019)considered this model to have high sensit
43、ivity 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.McClain et al.(2019)reported that neonatal mice have been shown to have a reduced time for tumor induction
44、,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
45、 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.Fe
46、males 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
47、,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,
48、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 v
49、ehicles 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/
50、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)
