*2.1. Chemicals*

NMU N1517-1G (lot no. SLBF6813V) reagen<sup>t</sup> was purchased from Sigma Chemical Co. (St. Louis, MO, USA), TRIzolTM reagen<sup>t</sup> was acquired from Life Technologies (Thermo Fisher Scientific, cat. no. 1596026, (Waltham, MA, USA), and the QuantiTect Reverse Transcription Kit manufactured by Qiagen (cat. no. 205311), (Germantown, MD, USA) was obtained. In addition, the TaqMan gene expression assays were purchased from Applied Biosystems (cat. nos. 4331182 and 4308313), and aluminum chloride (AlCl3) was procured from Sigma Chemical Co. (St. Louis, MO, USA). All other chemical products were of analytical grade.

### *2.2. Experimental Animals*

We selected 32 female Sprague Dawley rats (weight between 180 and 220 g) from the biotery at the Food Sciences Graduate Studies and Research Department at the University of Sonora.

#### *2.3. Experimental Design (Treatments)*

Groups of eight rats were randomly assigned to one of the following experimental treatments: treatment A rats not fed with aluminum and with no breast cancer induction (negative control) (−Al/−NMU); treatment B rats fed with 2000 mg/<sup>L</sup> aluminum (AlCl3) and breast cancer induced by NMU (+2000Al/+NMU); treatment C rats fed with 2000 mg/<sup>L</sup> aluminum (AlCl3) with no breast cancer induction (+2000Al/−NMU); and treatment D rats not fed with aluminum and breast cancer induced by NMU (positive control) (−Al/+NMU). Rats not fed with aluminum (AlCl3) and with no breast cancer induction (NMU) were treated with 0.98% physiological saline to match the experimental

conditions. Rats were intragastrically (gavage) fed 1 mL AlCl3 5 days/week for 90 days, whereas NMU was intraperitoneally administered at 50 and 70 days of age [28,29]. A volume of 1 mL of AlCl3 solution with a concentration of 2000 mg/<sup>L</sup> is equivalent to administering 2 mg of aluminum to rats, considering that the mean weight of rats is 0.2 kg. This is equivalent to a dose of 10 mg Al/day/kg of body weight.

#### *2.4. Breast Cancer Induction in Rats*

The rats were treated with NMU (N1517-1G Sigma-Aldrich, St. Louis, MO, USA) at doses of 50 mg/kg of body weight administered at 50 and 70 days of age to induce breast adenocarcinoma [30,31].

#### *2.5. Biotery Handling Conditions*

Biotery handling conditions were as follows: 12 h light/dark cycles, humidity ranging from 40% to 70%, temperature between 18 ◦C and 22 ◦C, and ad libitum access to water and food [32].
