*4.3. Micronuclei Assay*

For the MN evaluations, sequential peripheral blood samples (5 μL) were obtained from the same individuals (0 to 72 h), and 0–h samples were designated as a negative control. The samples were placed directly onto slides previously treated with AO, as described by Hayashi et al. [74]. Two slides were prepared for each mouse and were stored in the dark at 4 ◦C for 24 h. The assessments were performed by identifying PCE, NCE, and MN in PCE using a fluorescence microscope (NikonTM OPTIPHOT-2; Tokyo, Japan) with blue excitation (480 nm) and a barrier filter emission (515–530 nm) at 100× magnification. MN analysis was based upon 4000 PCE per mouse, and the presence of MN was considered to indicate genotoxic damage. The relative proportion of PCE to NCE was also analyzed for 2000 erythrocytes.

In this study, underlying metabolic processes such as 8-OHdG adduct repair, endogenous antioxidant component system, apoptosis, and cell viability analyses were also evaluated because these processes may be involved in preventing Cr(VI)-induced DNA damage. These parameters were measured using the same peripheral blood samples obtained at 48 h after treatments, in which MN were measured since this is the time when the greatest genotoxic damage induced by Cr(VI) has been observed [26].

#### *4.4. Plasma 8-Hydroxydeoxyguanosine Levels*

Plasma 8-OHdG levels were determined using an enzyme-linked immunosorbent assay. Peripheral blood samples (50 μL) were centrifuged (15 min at 2500× *g*) at room temperature. The plasma was collected and immediately analyzed according to the manufacturer's instructions using Trevigen's HT 8-oxo-dG ELISA Kit II (No. 4380-192-K; Gaithersburg, MD, USA). The absorbance at 450 nm of each well was determined using a Multiskan™ FC microplate reader (Thermo Scientific™, Vantaa, Finland). Product formation is inversely proportional to the amount of 8-OHdG present in the sample. The 8-OHdG levels were determined in duplicate (per sample) and according to the 8-OHdG standard curve.

#### *4.5. Antioxidant System*

The antioxidant system was evaluated by determining the activities of SOD, GPx, and CAT, as well as the GSH levels.

#### 4.5.1. Superoxide Dismutase Activity

SOD activity was evaluated in peripheral blood erythrocytes. Fifty-μL samples were diluted in phosphate-buffered saline. The erythrocytes were separated using FicollPaque™ (Sigma Chemical Co., St. Louis, MO, USA). (800× *g* for 25 min at 12 ◦C). The precipitate was separated, and cold distilled water (4 ◦C) was added (10:1). Then, it was incubated (0 ◦C for 15 min) to lyse. Hemoglobin was then precipitated by adding ethanol and chloroform (10,000× *g* for 10 min at 4 ◦C). The SOD activity was determined according to

the manufacturer's instructions using Trevigen's HT Superoxide Dismutase Assay Kit (No. 7501-500-K; Gaithersburg, MD, USA). The absorbance was read at 450 nm at 1-min intervals for 10 min in a Multiskan™ FC microplate reader (Thermo Scientific™, Vantaa, Finland). One unit of SOD activity was defined as the amount of protein that inhibited tetrazolium salt (WST-1)-formazan, up to a maximum of 50%. The protein concentration was determined according to the instructions for the Cayman Chemicals Protein Determination Kit (No. 704002; Ann Arbor, MI, USA). SOD activity was measured in duplicate (per sample) and according to the SOD standard curve.
