**4. Discussion**

#### *4.1. Determining Aluminum Concentration in Breast Tissue*

Oogoshi et al. (1994) reported that aluminum concentration was higher in the mammary glands of Sprague Dawley rats compared with other tissues by quantifying the concentration of various metals provided in their diet [59]. Furthermore, these results indicate that there was no synergistic effect of the treatments containing aluminum (AlCl3) and NMU (+2000Al/+NMU); therefore, it can be assumed that, among the effects of NMU administration, tumorigenesis does not require aluminum as an essential microelement, or NMU leads to changes in the aluminum transportation mechanism. This effect has been observed when determining that NMU administration in rats modifies the ZnT-1 zinc transporter that causes failure in the zinc transportation mechanism [60,61].

#### *4.2. Evaluation of Genomic Instability*

### 4.2.1. Histopathological Evaluation

NMU induces breast cancer in laboratory rats [62–67]. However, according to the present study, this compound caused only minimal cell proliferation in the ductal epithelium (−AL/+NMU). Reportedly, adenocarcinomas appeared 140 days after NMU application [68]. Cell proliferation of the ductal epithelium was not observed in treatment A (−Al/−NMU), which was expected because this treatment represents the negative control. The most significant histopathological change caused by treatment C (+2000Al/−NMU) was ductal epithelial cell proliferation compared with the other treatments. A previous histopathological study showed that aluminum in the form of AlCl3 can cause liver damage in Sprague Dawley rats when orally administered for 30 days [57].

### 4.2.2. Micronucleus Analysis

The present study demonstrated that NMU generates MN after 10 days of exposure under the <sup>+</sup>2000Al/−MNU treatment. A previous study assessed the genotoxicity of aluminum using the MN test and reported that genotoxic agents such as cyclophosphamide generate MN in the peripheral blood of rats within 24 h [69]. On the other hand, Balasubramanyam et al. [28,70] determined that the intraperitoneally administered nanoparticles of Al2O3 caused an increase in liver MN counts in female Wistar rats. Moreover, they determined that increasing the dosages (500, 1000, or 2000 mg of Al/kg of body weight) increased the number of MN when compared with the control group rats to which cyclophosphamide was administered as a genotoxic agent. This genotoxicity was corroborated by Klien and Godni´c-Cvar [71]. Moreover, Türkez et al. [57,72,73] assessed liver genotoxicity caused by AlCl3 solutions in Sprague Dawley rats. They observed that a sub-chronic dose of AlCl3, intraperitoneally administered for 10 weeks increased the number of MN; however, this e ffect can be reversed using boric acid or borax. Geyikoglu et al. [58] found that AlCl3 at a dose of 3 mg of aluminum/kg of body weight induced genotoxicity by forming MN in the hepatocytes of Sprague Dawley rats when aluminum was intraperitoneally administered for 10 weeks. In addition, Al-Obaidy et al. [74] demonstrated that 10, 15, and 25 mg of AlCl3 solutions per kg of body weight intraperitoneally administered to male albino rats (*Rattus norvegicus*) increased the number of MN in the bone marrow, and the number of MN increased with the increase in dose. As the dose increased, the number of cells with MN decreased, and this decrease correlates with an increase in the number of cells that die due to apoptosis [75]. In the present study, we intragastrically (gavage) administered AlCl3 solutions and the genotoxic e ffect observed was similar to that of the intraperitoneally administered aluminum solutions, as previously reported [73,74].

#### 4.2.3. Alkaline Electrophoresis Test in Individual Cells (Comet Assay)

The results obtained in treatment C (+2000Al/−NMU) implies a greater genotoxic e ffect after 15 days of exposure that is irreversible and considerably greater than the e ffect of H2O2 exposure [76,77]. A previous study reported that aluminum in the form of aluminum lactate intragastrically administered for 12 weeks could cause DNA fragmentation in the brain tissue of female Wistar rats when the genotoxic e ffect was assessed using agarose gel electrophoresis [78]. Using comet and MN tests, correlations were determined between the genotoxic damage caused by aluminum to the DNA of human lymphocytes and an increase in the dosage and exposure time [79]. The advantage of using the alkaline comet assay over the other in vitro methods is that it can detect lesions or damage to single- or double-stranded DNA as well as breaks in labile points in the DNA of single cells [80].

#### 4.2.4. Genetic Expression Assay Using RT-qPCR

*BRCA1* is a genetic marker for breast cancer, and its role in the DNA damage repair mechanism has been demonstrated. Considering that it has undergone mutations, its expression is regulated by epigenetic modification and it is overexpressed in breast cancer [81–83]. Furthermore, the product of *SCL11a2* has been identified as a divalent metal transporter gene [84–87]. The macrophage protein associated with natural resistance, Nramp, which is a product of *SCL11a2*, has recently been linked to aluminum transportation in di fferent organisms [88,89]. Therefore, other molecular biological approaches must be applied in an attempt to establish this genetic relationship. Indeed, other researchers have obtained similar results. For example, cadmium reportedly induces DNA fragmentation and tumorigenesis, but not the expression of genes such as *bcl-x* or *MT-1* in Wistar rat testis and prostate [90]. Rodrigues-Peres et al. [91] found no *ERBB2*, *C-MYC*, and *CCND1* gene instability when assessing the correlation between aluminum and genomic instability in cancerous human breast tissues and healthy tissues. Additionally, as previously reported, the changes were observed at extremely low or nanomolar concentrations of aluminum, with the ability to induce the expression of pro-inflammatory and pro-apoptotic genes. This may be related to some degree of genotoxicity [92,93], as observed by our research group in the present study.
