叶绿酸对7,12—二甲基苯蒽诱发大鼠乳腺癌的影响

本文研究了叶绿酸（ＣＨＬ）对７，１２－二甲基苯蕙（ＤＭＢＡ）诱发大鼠乳腺癌的影响。４３天龄雌性Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠随机分为３组，每组２５只。给ＤＭＢＡ前Ｉ组给自来水，Ⅱ、Ⅲ组在饮水中加ＣＨＬ１．５ｍｍｏｌ，１ｗｋ后每只大鼠一次灌胃给ＤＭＢＡ １０ｍｇ。给ＤＭＢＡ后Ⅰ、Ⅱ组给自来水，Ⅲ组饮水中继续加ＣＨＬ，持续２４ｗｋ，结果３组大鼠乳癌发生率分别为５０．０％、３３．３％和２３．８％。给Ｃ     

叶绿酸对7，2—二甲基苯蒽诱发大鼠乳腺癌的影响

本文研究了叶绿酸（ＣＨＬ）对７，１２—二甲基苯蒽（ＤＭＢＡ）诱发大鼠乳腺癌的影响。４３天龄雌性Ｓｐｒａｇｕｅ—Ｄａｗｌｅｙ大鼠随机分为３组，每组２５只。给ＤＭＢＡ前Ⅰ组给自来水，Ⅱ、Ⅲ组在饮水中加ＣＨＬ１．５ｍｍｏｌ，ｌｗｋ后每只大鼠一次灌胃给ＤＭＢＡ１０ｍｇ．给ＤＭＢＡ后Ⅰ、Ⅱ组给自来水，Ⅲ组饮水中继续加ＣＨＬ，持续２４ｗｋ。结果３组大鼠乳癌发生率分别为５０．０％、３３．３％和２３．８％．给ＣＨＬ的两组与对照组比较有下降趋势，但差异无显著性，说明ＣＨＬ的抗癌作用还有待进一步证实。实验中未发现ＣＨＬ有促癌作用。     

二甲基苯并蒽诱导金黄地鼠颊囊白斑癌变过程中血液流变特性的初步观察

人类口腔鳞状细胞癌约占整个口腔区域恶性肿瘤的90-95％，大多数学者认为．其发生多是由癌前病变发展而来．口腔白斑是最常见的口腔癌前病变，其发生及癌变与烟草等刺激密切相关．因此。采用烟草的主要有害成分之一的二甲基苯并蒽(DMBA)成功地在地鼠颊囊造成了白斑损害模型．近来．人们注意到，恶性肿瘤患者体内常存在血液流变特性的改变。     

有氧运动对二甲基苯蒽诱导大鼠乳腺癌发生及其机制探讨

目的：研究中等强度有氧运动对二甲基苯蒽（7,12-dimethyl-benz{a}anthracene,DMBA）诱导大鼠乳腺癌发生的影响。方法：120只7周龄无特定病原体级（SPF）雌性Sprague—Dawley（sD）大鼠按2×2析因设计随机数字表法分为空白对照组（CON）、运动对照组（ECON）、药物诱导组（DD和运动＋药物诱导组（EDI）,每组30只。ECON组和EDI组进行有氧运动训练,每周5d,25mL／min,30min／d。药物诱导组和运动＋药物诱导组给予1mL的10mg／mLDMBA麻油溶液灌胃2次。每2周观察1次大鼠的体质量、乳腺肿瘤体积和荷瘤数。18周后处死所有大鼠,对所有乳房肿瘤进行病理诊断,并测定脂肪湿重、脂肪指数、Lee’S指数、血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL—C）、低密度脂蛋白胆固醇（LDL—C）和血清雌二醇（E2）水平。结果：实验终末CON组、ECON组、DI组和EDI组的体质量分别为（352．67±32．93）、（322．30±29．18）、（296．50±28．60）和（289．30±27．05）g。析因分析显示,有氧运动（F=12．149,P=0．001）和DMBA（F=68．442,P〈0．001）都显著影响大鼠的体质量增长,两者有交互作用,F=4．620,P=0．034。DI组和EDI组的潜伏期分别为（13．86士2．88）和（15．65土2．23）d,差异有统计学意义,t=～2．456,P=0．018;荷瘤数分别为（3．24±1．12）和（2．30土1．02）个,差异有统计学意义,t=3．111,P=0．003;肿瘤体积为（2107．07±526．76）和（1793．78±541．08）差异有统计学意义,t=2．105,P=0．040。有氧运动和DMBA可以显著影响大鼠的脂肪湿重、脂肪指数和Lee’S指数,P〈0．01,且两者之间无明显交互作用,P〉0．05。有氧运动可以显著影响大鼠血清TG、TC、HDL—c和LDc和雌激素水平,P〈O．01,且与DMBA无明显交互作用,P〉0．05。结论：有氧运动可以抑制二甲基苯蒽诱导的大鼠乳腺癌的发生,其机制可能与有氧运动通过影响大鼠体脂水平而降低体内E2水平有关。     

二苯并（a,h）蒽诱发小鼠肺腺癌超微结构的研究

用二苯并蒽诱发了高发病率(90％以上)小鼠肺肿瘤,其中80％以上为腺癌。电镜观察发现有的腺癌具有肺泡Ⅱ型上皮细胞的特点,有的具有Clara细胞的特点,多数腺癌则兼具有Ⅱ型上皮细胞及clara细胞的特点。     

DMBA诱发的地鼠口腔癌发病机制研究

目的：用DMBA诱发的地鼠口腔癌模型，探讨口腔癌的发病机制。方法：0.5%DMBA涂于地鼠左侧颊囊，每周3次，共涂6周，观察至24周；用免疫组化方法检测口腔不同病变包括正常粘膜，单纯增生，异常增生，乳头状瘤和癌组织中Brdu标记指数和血管密度，并以形态学方法记数凋亡细胞，计算凋亡指数。结果：Brdu标记指数和血管密度均随着口腔癌病变的发展而增高，与病变呈正相关。细胞凋亡指数从正常粘膜，单纯增生到异常增生，乳头状瘤和癌组织增高，但异常增生，乳头状瘤和癌组织之间无显著性差异。结论：细胞增殖和新生血管形成是口腔癌发病的重要机制。     

PCR和探针自我杂交──Northern Blot方法对二甲基苯蒽诱导的P_（450）Cyp~（1b－1）mRNA的定量

通过ＰＣＲ和探针自我杂交──ＮｏｒｔｈｅｒｎＢｌｏｔ方法对０．３μＭ二甲基苯蒽（ＤＭＢＡ）诱导的２×１０６个小鼠胚胎成纤维细胞１０Ｔ１／２产生的Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ进行了定量。在这个方法中，与Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ相同的特定的单链ＣＤＮＡ探针片断通过ＰＣＲ合成。而双链探针中的一条链与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的特定片断是相同的。双链探针上的另一条链（Ｈ链）则与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的特定片断是互补的。用３２Ｐ－ｄＣＴＰ标记Ｈ链后，它就能够与单链探针和Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ同时进行杂交。这样，就能用已知浓度的单链探针作为标准物制做标准工作曲线和对Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ的含量进行定量。经测定，在０．３μＭＤＭＢＡ分别于１、２、３、４、６小时对２×１０６个细胞进行诱导后，２×１０６个细胞在不同时间产生的Ｐ４５０Ｃｙｐ１ｂ－１ｍＲＮＡ分别为０．００９８、０．０１３５、０．０１７４、０．０１９０和０．０１２０μＭ。     

β—胡萝卜素和叶绿酸对DMBA诱发大鼠乳腺癌的影响

５０天龄雌性ＳＤ大鼠随机分为４组，每组２５只，Ｉ组每只大鼠一次罐胃给予ＤＭＢＡ１０ｍｇ；Ⅱ组结ＤＭＢＡ前饮水中加ＣＨＬ１．５ｍｍｏｌ／Ｌ持续１ｗｋ，Ⅲ组给ＤＭＢＡ后饲料中加ＢＣ９０ｍｇ／ｋｇ饲料，持续到实验结束；Ⅳ组给ＤＭＢＡ前后分别给ＣＨＬ和ＢＣ。结果各组的肿瘤发生率分别为５０．０％，３３．３％，１８．３％和１７．４％，给ＢＣ组肿瘤发生率低于对照组（Ｐ〈０．０５），而单给ＣＨＬ组的肿瘤发生率与对     

Loss of heterozygosity at the N-ras locus in 7,12-dimethylbenz[a]anthracene-induced rat leukemia

The 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia model enables scientists to analyze cell altered by carcinogens at various stages of leukemogenesis. We have reported that a consistent type of point mutation, A→T transversion at the second base in codon 61 of the N-ras gene, was present in this leukemia and that this mutation appeared in bone marrow cells as early as 48 h after a single dose of DMBA. In addition, two leukemia cell lines with the N-ras mutation had no wild-type N-ras allele. Therefore, we examined whether these alterations were essential to the DMBA-induced leukemias. In the study reported here, we confirmed the occurrence of this N-ras mutation in 18 (86%) of 21 primary leukemias and loss of the N-ras wild-type allele in 12 (67%) of 18 leukemias with the mutated N-ras. By using microsatellite markers on chromosome 2, loss of heterozygosity (LOH) at the N-ras locus was observed in eight leukemias, all of which were shown to have lost the wild-type N-ras allele by mutant-allele-specific amplification. These results suggest that LOH related to loss of the wild-type N-ras allele reproducibly occurs in leukemias with the N-ras mutation. Considering the timing of the N-ras mutation and LOH, it is likely that the N-ras mutation is induced early, and cells that have lost the wild-type N-ras allele seem to develop into leukemia. We believe that this system provides a suitable model for studying a series of genetic alterations from the earliest stage of carcinogenesis that cannot be approached in human malignancies. Mol. Carcinog. 18:206–212, 1997. © 1997 Wiley-Liss, Inc.     

Optimization of 7,12-dimethylbenz(a)anthracene-induced rat epithelial ovarian tumors

Ovarian carcinoma is the second most common malignant tumor of the female reproductive system and an notable cause of cancer death. The detection and diagnosis of early ovarian carcinomas are still clinical challenges, which calls for imaging studies using early ovarian carcinoma animal models. The present study aimed to optimize the 7,12-dimethylbenz(a)anthracene (DMBA)-induced model of rat ovarian tumors by investigating the delivery methods, induction dose and time of DMBA exposure, and explored the morphological features of tumors using MRI. Three schemes were performed. In scheme one the ovary was covered with absorbable hemostatic gauze loaded with a high concentration of liquid DMBA. For this scheme, 150 Sprague-Dawley rats were divided into three groups depending on the DMBA dose (1.0, 2.0 and 3.0 mg). In scheme two DMBA solution was injected under the ovarian capsule. For this scheme, 159 rats were divided into 0.5, 1.0 and 1.5 mg DMBA groups. In scheme three the ovary was covered with absorbable gauze loaded with a high concentration of solid DMBA. For this scheme 161 rats were divided into 1.0, 2.0 and 3.0 mg DMBA groups. Each group of the three schemes was further subdivided into 60-, 90-, 120-, 150- and 180-day groups. In scheme two, the tumor formation rate was 75.6% (99/131), which was the highest in the 1.5 mg group (86.4%, 38/44) and reached 100% (10/10) on day 120. The induced tumors were serous in 93.9% (93/99) of tumors. Borderline ovarian tumors accounted for 19.2% (19/99) of all tumors, and ovarian cancer accounted for 46.5% (46/99). The mean maximum diameter (MMD) of borderline ovarian tumors was 10.29±3.41 mm, and that of ovarian cancer was 15.19±7.10 mm. MMD of the solid components increased with increasing malignancy. Cystic, cystic-solid and solid tumors were observed. The ovarian subcapsular injection of 1.5 mg DMBA was the best scheme for the rat ovarian tumor model. The present model is ideal for investigating the occurrence, development and imaging of ovarian tumors.     

Specific N-ras mutation in bone marrow within 48 H of 7,12-dimethylbenz[a]anthracene treatment in Huggins-Sugiyama rat leukemogenesis

7,12-Dimethylbenz[a]anthracene (DMBA)-induced leukemias in Long-Evans rats consistently have an A→T transversion at the second base of codon 61 in the N-ras gene. This mutation is also detected in the preleukemic stage. To determine when this specific N-ras mutation occurs in the early stages of leukemogenesis, we designed the mutant allele-specific amplification method, which was sensitive enough to detect one mutant cell among 10⁶ normal cells. In the study reported here, N-ras mutation was found in bone-marrow cells 2 d after a single DMBA injection and thereafter throughout the preleukemic stage. These results show that DMBA induces a specific N-ras mutation soon after one DMBA injection and that this mutation is probably the first event in DMBA leukemogenesis. © 1996 Wiley-Liss, Inc.     

Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridizations detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occured in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration. © 1994 Wiley-Liss, Inc.     

Chemopreventive efficacy of Moringa oleifera pods against 7, 12-dimethylbenz[a]anthracene induced hepatic carcinogenesis in mice

Oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage in a variety of liver disorders. Hence there is a great demand for the development of agents with potent antioxidant effect. The aim of the present investigation is to evaluate the efficacy of Moringa oleifera as a hepatoprotective and an antioxidant against 7, 12-dimethylbenz[a]anthracene induced hepatocellular damage. Single oral administration of DMBA (15 mg/kg) to mice resulted in significantly (p<0.001) depleted levels of xenobiotic enzymes like, cytochrome P450 and b5. DMBA induced oxidative stress was confirmed by decreased levels of reduced glutathione (GSH) and glutathione-S-transferase (GST) in the liver tissue. The status of hepatic aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) which is indicative of hepatocellular damage were also found to be decreased in DMBA administered mice. Pretreatment with the Moringa oleifera (200 and 400 mg/kg) orally for 14 days significantly reversed the DMBA induced alterations in the liver tissue and offered almost complete protection. The results from the present study indicate that Moringa oleifera exhibits good hepatoprotective and antioxidant potential against DMBA induced hepatocellular damage in mice that might be due to decreased free radical generation.     

Activated Ki-ras genes in bladder epithelial cell lines transformed by treatment of primary mouse bladder explant cultures with 7,12-dimethylbenz[a]anthracene

DNA from five lines of transformed bladder epithelial cells derived from cultures of primary cells that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) can transform NIH 3T3 mouse fibroblasts in DNA transfection experiments. Southern analysis of DNA from NIH 3T3 primary and secondary transformants established that four of the DMBA-transformed cell lines contained activated cellular Ki-ras, while the remaining cell line contained a transforming gene that is unrelated to Ki-ras, N-ras, and Ha-ras. The point mutations responsible for Ki-ras activation were detected using oligonucleotide probes following selective amplification of Ki-ras specific sequences using the polymerase chain reaction. The results showed that activation of Ki-ras invariably involved a GC----AT transition mutation of the first position of codon 12. Surprisingly, a Ki-ras gene that was activated by a GC----AT transition mutation at the same position was also detected in a single transformed bladder urothelial cell line derived from control cultures of mouse bladder cells. Together, our results indicate that Ki-ras activation in the DMBA-transformed bladder cell lines may not be a direct consequence of interaction of activated DMBA metabolites with the Ki-ras gene.     

Modulation of 7,12-Dimethylbenz[a]anthracene-induced Transmammary Carcinogenesis by Disulfiram and Butylated Hydroxyanisole in Mice

The individual as well as combined chemopreventive actions of disulfiram (DSF) and butylated hydroxyanisole (BHA) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced transmammary car-cinogenesis in mice were examined. When nursing mothers receiving normal diet were treated with DMBA (1 mg/mouse) on days 6, 8 and 10 postpartum , the tumor incidence in their 50-week-old F₁ progeny was 44.1%. When nursing mothers receiving 0.75% BHA diet, 0.5% DSF diet and 0.75% BHA+0.5% DSF diet were similarly treated with DMBA, the tumor incidences in their 50-week-old F₁ progeny were 14.7% ( P <0.05), 12.5% ( P <0.05) and 5.8% ( P <0.01), respectively. It is concluded that diets containing BHA (0.75%) and DSF (0.5%), singly or in combination, can inhibit trans-mammary carcinogenesis in Swiss albino mice.     

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0–2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 281 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA ≥ DMBA trans-5,6-dihydrodiol ≥ 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Inhibitory Effects of Dietary Protocatechuic Acid and Costunolide on 7,12-Dimethylbenz[a]anthracene-induced Hamster Cheek Pouch Carcinogenesis

The modifying effects of dietary exposure to two natural products, protocatechuic acid (PCA) and Costunolide during the development of neoplasms in oral carcinogenesis initiated with 7,12-dimethyl-benz[ a ]anthracene (DMBA) were investigated in male Syrian golden hamsters. All hamsters except those in the test chemical alone and control groups received DMBA (0.5%) in mineral oil to the right buccal pouch 3 times per week for 4 or 6 weeks. At 13 weeks of age, the groups exposed to DMBA were fed diet containing PCA or Costunolide at a dose of 0.2 g/kg diet (200 ppm) for 17 weeks. The other groups consisted of hamsters given mineral oil alone for 6 weeks, or given 200 ppm PCA or Costunolide alone, or untreated. All animals were necropsied at the termination of the experiment (week 24). PCA or costunolide significantly decreased the tumor burden (P<0.001- P <0.05) and the extent of dysplastic areas (%) (P<0.001-P<0.05). PCA significantly decreased the mean number of AgNORs/nucleus (P<0.05). The BrdUrd-labeling index was reduced by dietary administration of test compounds, though not significantly. These results suggest that PCA and costunolide inhibited hamster buccal pouch carcinogenesis and such inhibition may be related to suppression of cell proliferation in the buccal mucosa. It was also found that telomerase activity expressed in neoplastic and preneoplastic lesions of hamster buccal pouch epithelium after DMBA treatment correlated with the histopathological degree of malignancy.