2.9.2. Micronucleus Analysis

To examine MN, three female Sprague Dawley rats weighing 200 ± 20 g were used per treatment group. Peripheral blood sampling was performed according to the present regulations [34,37] at 5, 10, and 15 days of experimental treatment. Briefly, 0.5 mL of peripheral blood was collected in 1.5-mL Eppendorf tubes and three blood smears (extensions) were prepared on clean slides with blood from each rat. The smears were stained with Wright's stain [46] and MN were subsequently counted in 2000 erythrocytes (1 MN, 2 MN, or >2 MN) [28] under a LEICA DME compound microscope (Bu ffalo, NY, 14240, USA).

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

Cell viability was assessed using trypan blue dye, and cells were counted in a Neubauer chamber/hematocytometer [47,48]. The comet test was performed according to Singh's method [49] at an exploratory level. Briefly, we used three female Sprague Dawley rats from treatments A (−Al/−NMU) and C (+2000Al/−NMU), which were anesthetized in a halothane chamber, to extract 3–4 mL of blood by intracardiac puncture, and these rats were subsequently euthanized by cervical dislocation. A total of 30 μL of whole blood was added to 300 μL of 1% low melting point agarose at 37 ◦C. A total of 75 μL of this mixture was extracted and placed on a slide pre-coated with a 150-μL layer of 1% regular agarose, which was immediately covered with a coverslip and maintained at 4 ◦C for 10 min. The coverslip was removed and 75 μL of 1% low melting point agarose was added at 37 ◦C to create another layer and form a sandwich, which was protected with a coverslip and maintained at 4 ◦C for 10 min. The cells were subsequently lysed in a lysis solution (2.5 mM NaCl, 1% KOH, 100 mM EDTA, 10 mM Trizma base, 1% Triton X-100, and 10% DMSO) for 1 h at 4 ◦C. The samples were then placed in a dark electrophoresis chamber containing cold, alkaline running bu ffer at pH > 13 (300 mM NaOH, 1 nM EDTA, pH adjusted to >13). Samples were stored in the refrigerator for 20 min. The conditions of the electrophoretic run were 25 mV and 300 mA for 20 min.

Following electrophoresis, the slides were removed and washed three times with a neutralization bu ffer (0.4 mM Tris bu ffer adjusted to pH 7.5) for 5 min/wash. Finally, the slides were washed twice with anhydrous absolute ethanol for 5 min/wash. Excess alcohol was removed, and the slides were allowed to dry, following which they were then stained with 25 μL ethidium bromide (20 μg/mL in deionized water) and covered with coverslips. All stages of the comet assay were performed under indirect yellow light or in the dark. Comet observations were made under a LEICA DM2500 fluorescence microscope equipped with an excitation filter (515–560 nm) and barrier filter (590 nm). Photographs were captured with a 5-megapixel LEICA model DFC450C digital camera cooled with monochromatic light, with a C-mount adapter. The comet evaluations were performed using the TriTek CometScore software. DNA damage was reported as % Olive Tail Moment (% OTM) [50,51].

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

Chomczynski and Sacchi's method (1987) [52] was used for total RNA extraction. The mammary gland tissues (0.050 ± 0.008 g) collected from the rats was weighed and used for total RNA extraction using TRIzol. In addition, PolyTron equipment was used to homogenize the samples. Purity and concentration of the total RNA obtained was estimated by absorbance at 260/280 nm using Nanodrop equipment [53], and the integrity was determined by electrophoresis in 1.5% agarose gel under denaturing conditions and then stained with 1.5% ethidium bromide according to Jacobs Protocol

(2017) [54]. A reverse transcription reaction was performed with the RNA obtained from each sample using a QuantiTect Reverse Transcription kit to obtain the cDNA, under the operating conditions recommended by the provider (Qiagen, 2016; cat. no. 205311). PCR reactions were conducted in a T100 Thermocycler (BioRad, 2015, Hercules, CA, USA).

On obtaining the cDNA, genetic expressions of *BRCA1* and *SCL11a2* (FAM fluorophore) were evaluated using 30 ng of cDNA and TaqMan genetic expression assay (Applied Biosystems). RT-qPCR reactions were conducted in the StepOneTM v2.3 device using *GAPDH* (VIC fluorophore) as the reference gene (housekeeping gene). Duplex reactions (*BRCA1*/*GAPDH* and *SCL11a2*/*GAPDH*) were performed under the conditions recommended by the provider (Applied Biosystems 2016).
