257

REV. HOSP. CLÍN. FAC. MED. S. PAULO 59(5):257-261, 2004

From the Discipline of Gynecology, Hospitaldas Clínicas, Faculty of Medicine, Universityof São Paulo – São Paulo/SP, Brazil.E-mail: clinab@terra.com.br

Received for publication onFebruary 27, 2004.

ORIGINAL RESEARCH

INDUCTION OF EXPERIMENTAL MAMMARYCARCINOGENESIS IN RATS WITH 7,12-DIMETHYLBENZ(A)ANTHRACENE

Alfredo Carlos S. D. Barros, Elisa Naomi K. Muranaka, Lincon Jo Mori, ChristinaHelena T. Pelizon, Kyoshi Iriya, Giovana Giocondo and José Aristodemo Pinotti

BARROS ACSD et al. Induction of experimental mammary carcinogenesis in rats with 7,12-dimethylbenz(a)anthracene.Rev. Hosp. Clín. Fac. Med. S. Paulo 59(5):257-261, 2004.

PURPOSE: To test an experimental model of chemical mammary carcinogenesis induction in rats.METHODS: Twenty young virgin Sprague-Dawley female rats, aged 47 days, received 20 mg of 7,12-

dimethylbenz(a)anthracene (DMBA) intragastrically by gavage. Afterwards, at 8 and 13 weeks, their mammary glands wereexamined. At the end of the experiment, the animals were sacrificed, and the mammary tumors were measured and weighed.Tumor fragments were analyzed using light microscopy.

RESULTS: Eight weeks after DMBA injection, 16 rats presented at least 1 breast tumor (80%). After 13 weeks, all ofthem (100%) developed breast carcinomas that were confirmed by histopathological analysis.

CONCLUSION: This experimental animal model of chemical mammary induced carcinogenesis is feasible and can beused in further experiments on the role of tumorigenic biomodulator substances.

KEY WORDS: Breast cancer. Animal models. Carcinogens. DMBA-induced tumors. Rat mammary carcinogenesis.

Breast cancer represents the mostcommon neoplastic disease in females,accounting for up to one third of newdiagnoses of women’s cancer in certainregions of the world. In developingcountries traditionally known for lowincidence of breast cancer, increases inboth incidence and mortality havebeen recently detected.1

The fundamental issue in breastcancer control is prevention (primaryand secondary), which depends onidentification of the determinants ofthe disease, in terms of initiation andpromotion.

The possibility of using biomo-dulators of breast carcinogenesis, suchas selective estrogen receptor modula-tors (SERMS), aromatase andcyclooxygenase inhibitors, and dietary

factors is very promising.2,3 Neverthe-less, before clinical investigations areconducted, all of these strategiesshould be previously validated in ani-mal models.

Mammary tumors arise spontane-ously in a few animal species, for ex-ample dogs, rats, and mice.4 For prac-tical reasons most of the studies aboutexperimental breast carcinogenesis areconducted with rodents.5

Mammary glands of several rat

strains, mainly Sprague-Dawley andWistar-Furth, are susceptible to trans-formation induced by chemical car-cinogens, and the 2 most widely usedactive chemical inductors of mammarycarcinogenesis are 7,12-dimethylbenz(a)anthracene (DMBA)and N-methylnitrosurea.6,7

Based on Russo and Russo experi-ence,8,9 we decided to test an animalmodel for chemical mammary carcino-genesis employing DMBA, given bygavage, in young female Sprague-Dawley rats.

The objectives of our study were toinvestigate the feasibility of the model,the percentage of rats that developbreast cancer, the number of glands af-fected per animal, and the histologicalaspects of the induced neoplasms.

3209.p65 15/10/2004, 15:36257

258

REV. HOSP. CLÍN. FAC. MED. S. PAULO 59(5):257-261, 2004Induction of experimental mammary carcinogenesis in ratsBarros ACSD et al.

METHODS

The group of test animals wasformed by 20 young virgin Sprague-Dawley female rats. This type of rat livesan average of 3 years, starting its repro-ductive function, which lasts for about1 year, at 50 to 60 days of age. They have7 to 10 pregnancies during this period,delivering 7 to10 pups each time.

Mammary gland tumors were in-duced by a single dose of 20 mg ofDMBA diluted in soy oil (1 mL) givenintragastrically by gavage (Figure 1).All of the rats, with an average weightof 182.9 g (161-213 g), received thechemical carcinogen at the age of 47days.

The animals were bred in our labo-ratory under ideal conditions of tem-perature, humidity, and light, and theywere fed with appropriate ration in pel-lets and filtered water.

We performed physical examina-tions weekly. Each rat had 6 pairs ofmammary glands that were checked byinspection, touching, and palpation.

For evaluation of the induction patternwe performed 2 specific analyses at 8and 13 weeks after drug injection.

At the age of 138 days (13 weeksafter DMBA), the animals were sacri-ficed in a CO

2 chamber. Complete au-

topsies were performed, mammarytumors were measured and weighed,and the findings recorded. Representa-tive fragments of the tumors were fixedin 10% formaldehyde and paraffin em-bedded. The blocks were sectionedevery 5 mm, and slides were preparedwith hematoxylin-eosin stain and ex-amined using light microscopy.

RESULTS

The first assessment 8 weeks afterDMBA administration revealed that 16animals developed at least 1 mammarytumor (80%), with an average of 3.0tumors per animal (0-6).

Thirteen weeks after DMBA ad-ministration, all of the rats (100%) de-veloped breast tumors, with an average

of 4.9 tumors per animal (1-15). Themean size of the tumors was 1.8 cm(0.5-5). The weight of the tumorsranged from 0.06 g to 29.3 g with amean weight of 2.4 g (Table 1).

Figure 2 shows an animal withchemically induced breast tumors.

In 1 rat (animal 18), tumors wereformed apart from the breasts in theneck area of the animal.

Histological examination showedthat the neoplasms were adenocarcino-mas with several morphological types.The most common type encounteredwas adenoid cystic carcinoma, whichis characterized by sheets of tumorcells separated by small cystic spaces(Figure 3). Papillary carcinoma wasalso very common, which consists ofproliferating epithelial cells with deli-cate cores of connective tissue sepa-rated by narrow spaces (Figure 4). Asmaller proportion of tumors was of themyoepithelial type, characterized by amyxoid appearance and resemblingmixed mammary tumors of dogs.

It is interesting to point out that,despite the tumor malignancy, nometastases were detected in the autop-sies.

DISCUSSION

The mammary gland is one of thefew organs that is not totally developedat birth. It undergoes intense evolutiveand functional modifications duringpuberty, pregnancy, and lactation.Russo and Russo10 have described thedevelopmental progression of humanbreast tissue and listed 4 different typesof breast lobules. Type 1 (or virginal)is the most undifferentiated lobule andoccurs in the immature female breast

Table 1 - Number of tumors per rat at 8 and 13 weeks after 7,12-dimethylbenz(a)anthracene (DMBA) administration.

ANIMAL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

8 weeks 1 1 3 1 _ 2 2 _ _ 1 4 4 7 6 2 6 1 7 _ 113 weeks 7 6 3 1 1 3 3 1 3 5 5 5 7 8 4 8 4 15 6 4

Figure 1 - Administration of DMBA by gavage.

3209.p65 15/10/2004, 15:36258

259

REV. HOSP. CLÍN. FAC. MED. S. PAULO 59(5):257-261, 2004 Induction of experimental mammary carcinogenesis in ratsBarros ACSD et al.

before menarche. Type 2 lobule has amore complex morphology, being com-posed by a higher number of ductularstructures per lobule. Type 3 has an av-erage of 80 alveoli (ductules) and isformed under gestational hormonalstimulation. Type 4 is a secreting lob-ule during lactation.

The mammary tumors in rats arisein the epithelium of the terminal endbuds, which are comparable structuresto the terminal ductal lobular units inthe human breast.12

The degree of lobular differentia-tion is of importance in the suscepti-bility to carcinogenesis. Based onstudies of the pathogenesis of humanmammary cancer, it is possible to saythat the type 1 lobule is the site of ori-gin of preneoplastic lesions. Parouswomen undergo lobular differentia-tion, whereas nulliparous women sel-dom reach the type 3 lobule stage. Thebreasts of parous women free of can-cer have the lowest percentage of type1 lobules.

Lobules type 1 and 2 are charac-terized by having a shorter doublingtime than type 3, growing faster andhaving a higher DNA labeling index.

The susceptibility of the mammarygland to DMBA carcinogenesis isstrongly age-dependent, being maxi-mal when the drug is administered torats between the ages of 45 and 60days, which is the age of the begin-ning of sexual maturity.12 Active breastorganogenesis and high rate of prolif-eration of type 1 and 2 lobules arecharacteristics of that period. Thechance of chemically inducing breastcancer in rats is greater if DMBA is ad-ministered in this phase of the life ofthe animals. This was the reason whywe injected the drug at the age of 47days. According to the literature, theinduced tumors are generally ductalcarcinomas or papillary carcinomas,but it is possible that typicalfibroadenomas, adenomas, and papil-lomas are also formed.5,11,13

Figure 2 - Breast carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA).

Figure 4 - Papillary carcinoma (HE-40x).

Figure 3 - Adenoid cystic carcinoma (HE-200x).

3209.p65 15/10/2004, 15:36259

260

REV. HOSP. CLÍN. FAC. MED. S. PAULO 59(5):257-261, 2004Induction of experimental mammary carcinogenesis in ratsBarros ACSD et al.

Epithelial and myoepithelial cellproliferation were observed in most ofthe induced tumors in our experiment.Russo et al. found the same histologi-cal aspects. They also carefully pre-sented the correlation betweenneoplastic and non-neoplastic altera-tions in the mammary gland of rats andin women, which was proposed in aconsensus meeting on this subject heldin Hannover, Germany, in 1987.11 Mostof the lesions found in rats have cor-responding lesions in humans, allow-ing the translation of basic research inrats into the clinic.14

The induced DMBA tumors in thisstudy were multifocal and locally ag-gressive, but no single case ofmetastases was identified. This fact isin agreement with studies done byother authors, and metastasis fromeven the most anaplastic inducedtumors are low in frequency.11

The absence of metastases inchemically produced mammary neo-plasms opens the door for speculation.

According to Murad and von Haam,13

the proliferation of epithelial andmyoepithelial cells in relatively equalproportions may be a protective factoragainst metastasis. In human breastcarcinomas, proliferation occurs al-most exclusively in epithelial cells.

DMBA is highly lipophilic and re-quires metabolic activation for itscarcinogenicity. Several tissues are ca-pable of activating DMBA, and theseinclude the mammary gland. In thebreast, DMBA is converted toepoxides, active metabolites with acapacity for damaging the DNA mol-ecule, the main event in carcinogen-esis initiation. With the higher cellu-lar proliferative index of types 1 and2 lobules, there is higher metabolic ac-tivity and more epoxide forma-tion.15,16,17

The first DMBA-induced tumorwas observed about 5 weeks after in-jection of the carcinogen, but indi-vidual tumoral latency was not as-sessed in our study. It is possible,

though, to grossly estimate tumor for-mation patterns, since after 8 weeks,there were tumors in 16 animals, andafter 13 weeks, tumors were detectedin all of them.

This experimental animal modelclosely mimics human breast cancerand can be used as a comparativegroup in further studies with the pur-pose of elucidating the role ofbiomodulation in mammary carcino-genesis. The effects of pretreatmentand posttreatment of rats with hor-mones, antihormones, isoflavones,celecoxib, and many other substancesthat have action on mammary carcino-genesis will be the endpoints of futureresearch in our laboratory using thisanimal model.

ACKNOWLEDGEMENT

This work was supported byFAPESP (grant 01/04169-2).

RESUMO

BARROS ACSD e col. Indução dacarcinogênese mamária experimen-tal em ratas com 7,12 – dimetil-benz(a)antraceno. Rev. Hosp. Clín.Fac. Med. S. Paulo 59(5):257-261,2004.

OBJETIVO: Testar um modelo ex-perimental de indução química decarcinogênese mamária em ratas.

MATERIAL E MÉTODOS: Com47 dias de vida, 20 ratas Sprague-Dawley, jovens e virgens, receberam

por gavagem intragástrica 20 mg de7 , 1 2 - d i m e t i l b e n z ( a ) a n t r a c e n o(DMBA). Oito e 13 semanas depois dainjeção de droga as mamas das ratasforam examinadas. Ao final os animaisforam sacrificados e fragmentos dostumores foram estudados ao microscó-pio.

RESULTADO: Oito semanas de-pois da injeção de DMBA 16 ratasapresentavam tumor nas mamas (80%).Com 13 semanas todas desenvolveramcarcinomas de mama (100%), que fo-

ram confirmados por análisehistopatológica.

CONCLUSÃO: Este modelo expe-rimental de indução química decarcinogênese mamária é factível epode ser empregado em futuras pesqui-sas para avaliar o papel de substânci-as biomoduladoras da tumorigênese.

UNITERMOS: Câncer de mama.Modelos animais. Carcinógenos. Tu-mores induzidos por DMBA.Carcinogênese mamária em ratas.

3209.p65 15/10/2004, 15:36260

261

REV. HOSP. CLÍN. FAC. MED. S. PAULO 59(5):257-261, 2004 Induction of experimental mammary carcinogenesis in ratsBarros ACSD et al.

REFERENCES

1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics 2002.CA Cancer J Clin 2002;52:23-47.

2. Powles TJ. Anti-oestrogenic chemoprevention of breast cancer-the need to progress. Eur J Cancer 2003;39:572-9.

3. Home LR,Dannenberg AJ. A role of cyclooxigenase-2 inhibitorsin the prevention and treatment of cancer. Semin Oncol2002;29(suppl11):111-19.

4. Nerurkar VR, Chitale AR, Jainapurkar BV, et al. Comparativepathology of canine mammary tumours. J Comp Pathol1989;101:389-94.

5. Russo J, Russo IH. Experimentally induced mammary tumors inrats. Breast Cancer Res Treat 1996;39:7-20.

6. Dias MF, Sousa E, Cabrita S, Patricio J, Oliveira CF.Chemoprevention of DMBA induced mammary tumors inrats by a combined regimen of alpha tocopherol, selenium,and ascorbic acid. Breast J 2000;16:14-19.

7. Giullino PM, Pettingrew HM, Grantham FIT. N-nitrosomethylureaas mammary gland carcinogen in rats. J Natl Cancer Inst1975;54:401-14.

8. Russo J. Russo IH. Biological and molecular bases of mammarycarcinogens. Lab Invest 1987;57:112-37.

9. Russo IH, Russo J. Developmental stage of the rat mammarygland as determinant of its susceptibility to 7,12-dimethylbenzan(a)thracene. J Natl Cancer Inst 1978;61:1439-42.

10. Russo J, Russo IH. Toward a physiological approach to breastcancer prevention. Cancer Epidemiol Biomark Prev1994;3:353-64.

11. Russo J, Gusterson BA, Rogers AE, et al. Biology of disease:comparative study of human and rat mammary tumorigenesis.Lab Invest 1990;62:244-77.

12. Grubbs CJ, Juliana MM, Hill DL, Whitaker LM. Suppression bypregnancy of chemically induced preneoplastic cells of the ratmammary gland. Anticancer Res 1986;6:2395-401.

13. Murad TM, von Haam E. Studies on mammary carcinoma inducedby 7,12-dimethylbenzanthracene administration. Cancer Res1972;32:1404-15.

14. Clarke R. Animal models of breast cancer: experimental designand their use in nutrition and psychosocial research. BreastCancer Res Treat 1997;46:117-33.

15. Clarke R. Issues in experimental design and endpoint analysis inthe study of experimental cytotoxic agents in vivo in breastcancer and other models. Breast Cancer Res Treat 1997;46:255-78.

16. Russo J, Larref MH, Balogh G, et al. Estrogen and its metabolitesare carcinogenic agents in human breast epithelial cells. JSteroid Biochem Mol Biol 2003;37:1-25.

17. Balogh GA, Russo IH, Russo J. Mutations in mismatch repairsgenes are involved in te neoplastic transformation of humanbreast epithelial cells. Int J Oncol 2003;23:411-9.

3209.p65 15/10/2004, 15:36261