3.2.2. Micronucleus Analysis

MN analysis is a valuable indicator for the partial assessment of genotoxicity via ruptures in the chromosomes [57,58]. The complete MN count is shown in Table 2, which indicates that the presence of MNs was not detected in the negative control (−Al/−MNU) at 5, 10, or 15 days. This is normal to some extent because these rats were not administered aluminum solutions or NMU, a cancer inducing agent. As denoted in Figure 2, an effect from the treatment and exposure time (*p* < 0.05) was observed for counts with 1 MN. Our result suggests that there was no synergistic effect between aluminum (AlCl3) and NMU, as previously noted in the results obtained for aluminum concentration in the mammary glands as well as from the histological evaluation of the breast. In the <sup>+</sup>2000Al/−NMU and −Al/+NMU treatments, a higher number of MN (*p* < 0.05) were observed. Although the average number of MN was higher for the <sup>+</sup>2000Al/−NMU treatment than for the –Al/+NMU treatment, there was no significant difference (*p* > 0.05) because of the substantial variance among the <sup>+</sup>2000Al/−NMU treatment replications. Nevertheless, the study proved that the treatment containing only aluminum could independently cause genotoxicity in rats.


**Table 2.** Genotoxicity caused by experimental treatments by micronucleus count (MN) in peripheral blood erythrocytes of female Sprague Dawley rats at 5, 10, and 15 days of exposure.

**Figure 2.** Genotoxicity (1 MN) caused by experimental treatments in the peripheral blood of Sprague Dawley rats at 5, 10, and 15 days of exposure.

In addition, the genotoxic effects started to significantly manifest (*p* < 0.05) on day 10 and showed greater values at day 15 (effect from exposure time), indicating a subacute effect due to aluminum bioconcentration. Additionally, the results revealed that the apparent effect was intermediate in treatments with aluminum and NMU (+2000Al/+NMU).

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

The 96% ± 2% cell viability identified by trypan blue was similar to the results obtained by other researchers [47,48]. This study was conducted at an exploratory level to determine whether the genotoxicity caused by aluminum in the form of AlCl3 caused DNA damage or fragmentation in rat leukocytes. Only three rats were used as a negative control (−Al/−NMU) and three rats were subjected to the <sup>+</sup>2000Al/−NMU experimental treatment, thereby proving that this experimental treatment could independently induce intraductal cell proliferation in mammary glands and lead to a higher number of comet and clouds as exposure time increased. Figure 3 denotes these results, indicating that there was no genotoxic damage at 5, 10, and 15 days of exposure after the −Al/−NMU treatment (negative control). Conversely, no comets were observed in the <sup>+</sup>2000Al/−NMU experimental treatment, and only nucleoids or unrolled DNA were noted after five days of exposure. The test was consistent with the lack of genotoxicity observed in the formation of MN after five days of exposure. However, genotoxicity was distinctly observed after 10 and 15 days, when comets were observed. Although no significant differences (α = 0.05) were observed in terms of the number of comets and % OTM after 10 and 15 days of aluminum exposure, there were significant differences in terms of cloud formation because there were three and 73 clouds per 100 comets detected at 10 and 15 days of aluminum exposure, respectively.

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

The results of genetic expression obtained for *BRCA1* and *SCL11a2* using RT-qPCR indicate that no experimental evidence demonstrating the expression of both genes was present; therefore, it can be inferred that the product of *BRCA1* does not participate in the DNA damage repair mechanism as observed in the comet test. In addition, it can be determined that the product of *SCL11a2* does not play a role in the aluminum transport process in Sprague Dawley rats under the proposed experimental conditions.

**Figure 3.** Genotoxic effect caused by the <sup>+</sup>2000Al/−NMU treatment in the peripheral blood of Sprague Dawley rats at 5, 10, and 15 days of exposure, evaluated by the comet test.
