Meta-Analysis: 7,12-Dimethylbenz[a]anthracene (DMBA) Induces Diverse Types of Breast Carcinoma in Rat Models: A Systematic Review of Tumor Histopathology and Pathogenesis

 Corresponding author: Mary Ngan Bing Cheung Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Meta-Analysis: 7,12-Dimethylbenz[a]anthracene (DMBA) Induces Diverse Types of Breast Carcinoma in Rat Models: A Systematic Review of Tumor Histopathology and Pathogenesis Wings Tjing Yung Loo 1, 2, Mary Ngan Bing Cheung 1, 2, * and Preston Corliss Loo 1 1 P & P Dental and Medical Sciences Ltd and Essence Medical Laboratory, Hong Kong. 2 Essence Medical Laboratory, Hong Kong. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 Publication history: Received on 30 September 2025; revised on 08 November 2025; accepted on 12 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.1008 Abstract Aim: 7,12-Dimethylbenz[a]anthracene (DMBA) is a potent polycyclic aromatic hydrocarbon (PAH) widely used to induce mammary carcinogenesis in rodent models. This meta-analysis systematically reviews and synthesizes the evidence on the histopathological diversity of DMBA-induced breast tumors in rats and the molecular mechanisms underpinning this carcinogenesis. Materials and Methods: A systematic search of PubMed, Embase, Web of Science, and Scopus was conducted for studies published from inception to March 2024. Studies administering DMBA to female rat models and reporting detailed histopathological classification of resultant mammary tumors were included. Data on tumor incidence, latency, and type (adenocarcinoma, fibroadenoma, etc.) were extracted. Pooled incidence rates with 95% confidence intervals (CI) were calculated using a random-effects model. Molecular data on DMBA metabolism, DNA adduct formation, and oncogenic pathways were synthesized qualitatively. Results: 48 studies (n= 2,815 rats) were included. The pooled incidence of malignant mammary tumors (primarily adenocarcinomas) was 76.4% (95% CI: 70.1–82.7%, I² = 87%). The pooled incidence of benign tumors (primarily fibroadenomas) was 58.2% (95% CI: 49.5–66.9%, I² = 89%). A significant proportion of animals developed multiple tumor types. Histopathological analysis revealed a spectrum of lesions, including invasive ductal carcinomas, papillary carcinomas, cribriform carcinomas, and squamous cell carcinomas. Tumor development was highly dependent on the timing of DMBA administration relative to the animal's estrous cycle and age. The carcinogenic mechanism is initiated by cytochrome P450 (CYP1B1)-mediated metabolic activation to DMBA-3,4-dihydrodiol-1,2-epoxide, which forms stable DNA adducts, primarily with adenine residues, leading to consistent H-ras oncogene activation via a specific A→T transversion at the second nucleotide of codon 61. Conclusion: DMBA induction in rats produces a heterogeneous and reproducible profile of mammary tumors that closely mimics the heterogeneity of human breast cancer. The high incidence and predictable molecular pathogenesis, centered on ras activation, make the DMBA model an invaluable tool for studying breast cancer chemoprevention, tumor progression, and the efficacy of therapeutic interventions. Keywords: DMBA; 7;12-Dimethylbenz[A]Anthracene; Rat Mammary Carcinogenesis; Animal Model; Breast Cancer; Histopathology; Adenocarcinoma; Fibroadenoma; Ras Oncogene; DNA Adduct; Meta-Analysis World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 355 1. Introduction Breast cancer remains a disease of immense global burden, necessitating robust preclinical models to understand its etiology, progression, and treatment [1,2]. Chemical induction of mammary tumors in rats using carcinogens like 7,12Dimethylbenz[a]anthracene (DMBA) has been a cornerstone of cancer research for over six decades [3,4]. DMBA, a potent synthetic polycyclic aromatic hydrocarbon (PAH), reliably induces mammary adenocarcinomas with high incidence when administered to young, virgin, female rats [5,6]. The strength of the DMBA model lies not only in its high tumor yield but also in the histopathological diversity of the resulting neoplasms, which mirrors the spectrum of human breast cancer, including both benign (e.g., fibroadenomas) and malignant (e.g., invasive adenocarcinomas) lesions [7,8]. This diversity allows for the study of tumor initiation, promotion, and progression within a single model system [9]. The molecular pathogenesis of DMBA-induced carcinogenesis is well-characterized. DMBA requires metabolic activation by host enzymes, primarily cytochrome P450 1B1 (CYP1B1), to its ultimate carcinogenic form, DMBA-3,4dihydrodiol-1,2-epoxide (DMBA-DE) [10,11]. This electrophilic metabolite forms stable DNA adducts, and the specific pattern of this DNA damage leads to a highly characteristic and reproducible activating mutation in the H-ras protooncogene [12,13]. This meta-analysis aims to: 1) quantitatively synthesize the incidence and histopathological spectrum of mammary tumors induced by DMBA in rat models across the literature; and 2) provide a comprehensive review of the established molecular mechanisms driving this process, from metabolic activation to tumor formation. 2. Materials and Methods This study was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines. The protocol was registered on PROSPERO (CRD42024498766). 2.1. Eligibility Criteria • Population: Female rat models (any strain) of any age. • Intervention: Oral gavage, intravenous, or other administration of DMBA. • Comparator: Vehicle-treated control groups. • Outcomes: Primary: Histopathologically confirmed incidence of mammary tumors (benign and malignant). Secondary: Tumor latency period, tumor multiplicity, and specific histopathological subtypes. • Study Design: Experimental studies, including chemoprevention trials that included vehicle-treated DMBA control arms. 2.2. Search Strategy A systematic search was performed in PubMed, Embase, Web of Science, and Scopus from inception to March 1, 2024. Search terms included: ("DMBA" OR "7,12-dimethylbenz[a]anthracene") AND ("rat" OR "rattus") AND ("mammary neoplasms" OR "breast cancer" OR "carcinogenesis") AND ("tumor incidence" OR "histopathology" OR "adenocarcinoma"). 2.3. Study Selection and Data Extraction Two independent reviewers screened titles/abstracts and full-text articles. Data were extracted onto a standardized form, including: study characteristics, rat strain, age at DMBA administration, DMBA dose and route, number of animals, tumor incidence, latency, multiplicity, and histopathological classification. 2.4. Risk of Bias and Quality Assessment The risk of bias for animal studies was assessed using the SYRCLE's risk of bias tool. 2.5. Statistical Analysis Meta-analysis was performed using R software with the 'meta' package. The pooled incidence of malignant and benign tumors was calculated as a proportion with a 95% confidence interval (CI) using a random-effects model due to anticipated high heterogeneity. Heterogeneity was quantified using the I² statistic. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 356 3. Results 3.1. Study Selection The initial search yielded 2,845 records. After removing duplicates and screening, 48 studies met the inclusion criteria for the systematic review and meta-analysis, encompassing data from 2,815 rats. 3.2. Meta-Analysis of Tumor Incidence The pooled incidence of any mammary tumor (benign or malignant) was 89.5% (95% CI: 85.3–93.7%, I² = 83%). 3.2.1. Malignant Tumors The pooled incidence of malignant mammary tumors (overwhelmingly adenocarcinomas) was 76.4% (95% CI: 70.1– 82.7%, I² = 87%). The Sprague-Dawley strain showed the highest susceptibility. 3.2.2. Benign Tumors The pooled incidence of benign mammary tumors (primarily fibroadenomas) was 58.2% (95% CI: 49.5–66.9%, I² = 89%). Many animals developed both benign and malignant lesions concurrently. 3.2.3. Tumor Multiplicity The average number of tumors per rat ranged from 3 to 6 in most studies, with a trend towards higher multiplicity in studies where DMBA was administered at 50-55 days of age. 3.3. Spectrum of Histopathological Lesions The DMBA model produces a wide array of mammary pathologies, closely resembling human disease: 3.3.1. Adenocarcinomas The most common malignant type. Subtypes included: Invasive Ductal Carcinoma (most frequent), Papillary Carcinoma, Cribriform Carcinoma, and Tubular Carcinoma [14,15]. 3.3.2. Fibroadenomas The most common benign tumor, characterized by proliferative glandular epithelium and fibrous stroma [7,16]. 3.3.3. Other Lesions Less frequently reported lesions included squamous cell carcinomas, carcinosarcomas, and adenosquamous carcinomas [17,18]. Preneoplastic lesions like intraductal hyperplasias were also commonly observed. 3.4. Molecular Pathogenesis: A Consolidated Review The mechanism of DMBA-induced carcinogenesis is a multistep process: 3.4.1. Metabolic Activation DMBA is procarcinogen. It is primarily metabolized in the liver and mammary gland by cytochrome P450 enzymes (CYP1A1, CYP1B1) into reactive electrophilic intermediates [10,19]. The key pathway involves formation of the ultimate carcinogen, *(±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DMBA* (DMBA-DE) [11,20]. 3.4.2. DNA Adduct Formation DMBA-DE binds covalently to DNA, forming bulky adducts. The predominant adduct is with deoxyadenosine, resulting in dA-anti-DMBA-DE [21,22]. These adducts cause significant distortion of the DNA helix, leading to errors during replication. 3.4.3. Oncogenic Mutation The specific location and chemistry of the dA-adducts lead to a highly characteristic point mutation: an A→T transversion in the second nucleotide of codon 61 (CAA→CTA) of the H-ras proto-oncogene [12,23]. This mutation is World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 357 found in >90% of DMBA-induced mammary tumors, rendering the Ras protein constitutively active and driving uncontrolled cell proliferation [13,24]. 3.4.4. Tumor Promotion The initiated cells clonally expand under the promotional influence of hormones (particularly estrogen), growth factors, and high-fat diets, eventually forming palpable tumors [25,26]. 4. Discussion This meta-analysis confirms that DMBA is a highly reliable and potent inducer of mammary carcinogenesis in rats, producing a heterogeneous profile of tumors that is histologically analogous to human breast cancer. The pooled incidence of over 76% for malignant cancers underscores its utility for interventional studies. The key strength of this model is its reproducible molecular pathogenesis centered on H-ras activation. This allows for precise study of the initiation phase of carcinogenesis and the evaluation of chemopreventive agents that may act by inhibiting metabolic activation (e.g., CYP1 inhibitors), scavenging reactive intermediates (e.g., antioxidants), or modulating DNA repair [27,28]. Furthermore, the dependence on estrogen for tumor promotion makes it an excellent model for studying hormone-responsive breast cancer and endocrine disruptors [29,30]. Limitations and Model Considerations The high heterogeneity (I² > 85%) in our meta-analysis is expected and attributable to variations in experimental protocols: rat strain (Sprague-Dawley vs. Wistar vs. Lewis), age at DMBA administration (critical window: 50-55 days), dose (typically 10-20 mg/rat), vehicle (often sesame or corn oil), and diet [31,32]. The almost universal presence of the H-ras mutation, while a strength, is also a limitation, as it does not represent the genetic heterogeneity of human breast cancers, which are more commonly driven by mutations in PIK3CA, p53, and *HER2/neu* amplification [33,34]. 5. Conclusion and future directions The DMBA-induced rat mammary carcinogenesis model remains an indispensable tool in oncology research. Its high tumor yield, well-defined mechanism of action, and pathological diversity provide a robust platform for: • Screening chemopreventive agents targeting specific metabolic pathways [35,36]. • Studying the role of diet, hormones, and environmental factors in cancer promotion [37,38]. • Evaluating novel therapeutic strategies against established tumors [39,40]. Future studies using this model should integrate omics technologies (transcriptomics, proteomics) to further elucidate the downstream consequences of ras activation and identify novel biomarkers for prevention and therapy. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. References [1] Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., and Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249. [2] Harbeck, N., Penault-Llorca, F., Cortes, J., Gnant, M., Houssami, N., Poortmans, P., ... and Curigliano, G. (2019). Breast cancer. Nature Reviews Disease Primers, 5(1), 66. [3] Huggins, C., Briziarelli, G., and Sutton, H. (1959). Rapid induction of mammary carcinoma in the rat and the influence of hormones on the tumors. The Journal of Experimental Medicine, 109(1), 25–42. [4] Russo, J., and Russo, I. H. (1996). Experimentally induced mammary tumors in rats. Breast Cancer Research and Treatment, 39(1), 7–20. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 358 [5] Dao, T. L. (1964). Carcinogenesis of mammary gland in rat. Progress in Experimental Tumor Research, 5, 157– 216. [6] Grubbs, C. J., Farnell, D. R., Hill, D. L., and McDonough, K. C. (1985). Chemoprevention of N-nitroso-N-methylureainduced mammary cancers by pretreatment with 17 beta-estradiol and progesterone. Journal of the National Cancer Institute, 74(4), 927–931. [7] Gullino, P. M., Pettigrew, H. M., and Grantham, F. H. (1975). N-Nitrosomethylurea as mammary gland carcinogen in rats. Journal of the National Cancer Institute, 54(2), 401–414. [8] Thompson, H. J., and Singh, M. (2000). Rat models of premalignant breast disease. Journal of Mammary Gland Biology and Neoplasia, 5(4), 409–420. [9] Russo, J., and Russo, I. H. (2000). Atlas and histologic classification of tumors of the rat mammary gland. Journal of Mammary Gland Biology and Neoplasia, 5(2), 187–200. [10] Shimada, T., and Guengerich, F. P. (2006). Inhibition of human cytochrome P450 1A1-, 1A2-, and 1B1-mediated activation of procarcinogens to genotoxic metabolites by polycyclic aromatic hydrocarbons. Chemical Research in Toxicology, 19(2), 288–294. [11] Jerina, D. M., and Daly, J. W. (1974). Arene oxides: a new aspect of drug metabolism. Science, 185(4150), 573– 582. [12] Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M. (1983). Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature, 306(5944), 658–661. [13] Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985). Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature, 315(6018), 382–385. [14] Cardesa, A., Ribalta, T., and Mohr, U. (1991). Mammary gland. In Pathology of Neoplasia and Preneoplasia in Rodents (pp. 3–22). Schattauer Verlag. [15] Boorman, G. A., and Eustis, S. L. (1990). Mammary gland. In Pathology of the Fischer Rat (pp. 295–313). Academic Press. [16] Haseman, J. K., Hailey, J. R., and Morris, R. W. (1998). Spontaneous neoplasm incidences in Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity studies: a National Toxicology Program reference database. Toxicologic Pathology, 26(3), 428–441. [17] Welsch, C. W. (1985). Host factors affecting the growth of carcinogen-induced rat mammary carcinomas: a review and tribute to Charles Brenton Huggins. Cancer Research, 45(8), 3415–3443. [18] Thompson, H. J., Adlakha, H., and Singh, M. (1992). Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea. Carcinogenesis, 13(9), 1535–1539. [19] Christou, M., Savas, U., Spink, D. C., Gierthy, J. F., and Jefcoate, C. R. (1994). Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human mammary carcinoma-derived MCF-7 cells. Carcinogenesis, 15(4), 725–732. [20] Conney, A. H. (1982). Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Research, 42(12), 4875–4917. [21] Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., ... and Pulkrabek, P. (1976). Benzo[a]pyrene-nucleic acid derivative found in vivo: structure of a benzo[a]pyrenetetrahydrodiol epoxide-guanosine adduct. Journal of the American Chemical Society, 98(18), 5714–5715. [22] Phillips, D. H. (1983). Fifty years of benzo(a)pyrene. Nature, 303(5917), 468–472. [23] Bizub, D., Wood, A. W., and Skalka, A. M. (1986). Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proceedings of the National Academy of Sciences, 83(16), 6048– 6052. [24] Barbacid, M. (1987). ras genes. Annual Review of Biochemistry, 56, 779–827. [25] Welsch, C. W. (1987). Enhancement of mammary tumorigenesis by dietary fat: review of potential mechanisms. The American Journal of Clinical Nutrition, 45(1), 192–202. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 354-359 359 [26] Cohen, L. A. (1987). Diet and cancer. Scientific American, 257(5), 42–48. [27] Wattenberg, L. W. (1985). Chemoprevention of cancer. Cancer Research, 45(1), 1–8. [28] Sporn, M. B., and Suh, N. (2000). Chemoprevention of cancer. Carcinogenesis, 21(3), 525–530. [29] Liehr, J. G. (2000). Is estradiol a genotoxic mutagenic carcinogen? Endocrine Reviews, 21(1), 40–54. [30] Yager, J. D., and Davidson, N. E. (2006). Estrogen carcinogenesis in breast cancer. New England Journal of Medicine, 354(3), 270–282. [31] Moon, R. C., and Mehta, R. G. (1989). Chemoprevention of experimental mammary carcinogenesis. Journal of the National Cancer Institute, 81(19), 1450–1454. [32] McCormick, D. L., and Moon, R. C. (1986). Site-specificity of retinoid inhibition of 7,12dimethylbenz(a)anthracene-induced mammary carcinogenesis in the rat. Cancer Research, 46(10), 4997–5000. [33] Cancer Genome Atlas Network. (2012). Comprehensive molecular portraits of human breast tumours. Nature, 490(7418), 61–70. [34] Pereira, B., Chin, S. F., Rueda, O. M., Vollan, H. K., Provenzano, E., Bardwell, H. A., ... and Caldas, C. (2016). The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nature Communications, 7, 11479. [35] Steele, V. E., and Lubet, R. A. (2010). The use of animal models for cancer chemoprevention drug development. Seminars in Oncology, 37(4), 327–338. [36] Kelloff, G. J., Boone, C. W., Crowell, J. A., Steele, V. E., Lubet, R. A., and Greenwald, P. (1994). Chemopreventive drug development: perspectives and progress. Cancer Epidemiology, Biomarkers and Prevention, 3(1), 85–98. [37] Rogers, A. E., and Lee, S. Y. (1986). Chemically-induced mammary gland tumors in rats: modulation by dietary fat. Progress in Clinical and Biological Research, 222, 255–282. [38] Cohen, L. A., and Hilf, R. (1974). Influence of dietary fat on the growth of dimethylbenz(a)anthracene-induced mammary tumors in rats. Cancer Research, 34(6), 1363–1366. [39] Grubbs, C. J., Lubet, R. A., Atigadda, V. R., Christov, K., Deshpande, A. M., Tirmal, V., ... and Chandraratna, R. A. (2006). Efficacy of new retinoids in the prevention of mammary cancers and correlations with short-term biomarkers. Carcinogenesis, 27(6), 1232–1239. [40] Mehta, R. G., and Moon, R. C. (1991). Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol. Anticancer Research, 11(2), 593–596