Srm 2975

BALB/c mice (male, 6−8 weeks old) were obtained from Orient Bio Co., Ltd. (Gyeonggi-do, Republic of Korea). The mice were housed in animal facility with environmental conditions (temperature of 22 ± 2°C, a humidity of 60 ± 10%, and a 12-h light/dark cycle), with free access to food and water. This experimental protocol was approved by the Committee for Animal Welfare at Daejeon University (DJUARB2019-021) and was performed in accordance with the committee guidelines. The mice received intranasal administration of a mixture of PM₁₀ (ERM CZ-120, Sigma-Aldrich, USA) and diesel exhaust particles (DEP, SRM 2975, Sigma-Aldrich, St. Louis, MO, USA) on days 4, 7, and 10 and were orally treated with L. paracasei ATG-E1 every other day for 12 days. PM₁₀ (3 mg/mL) and DEP (0.6 mg/mL) were dissolved in 1% aluminum hydroxide gel adjuvant. The mice were divided into following groups (n = 8 per group): (1) 1% aluminum hydroxide gel adjuvant-treated mice (NC), (2) PM₁₀ + DEP-sensitized mice (PM₁₀D-CTL), (3) 4 × 10¹⁰ CFU of ATG-E1-treated PM₁₀ + DEP-sensitized mice (PM₁₀D-ATG-E1), and (4) 3 mg/kg dexamethasone-treated PM₁₀ + DEP-sensitized mice (PM₁₀D-Dexa, positive control). All mice were sacrificed and blood, bronchoalveolar lavage fluid (BALF), and lung and intestinal tissues were harvested on day 12.

Commercial CuO (CAS 1317-38-0) and ZnO (CAS 1314-13-2) NPs, with size <50 nm, were purchased from Sigma-Aldrich (Sigma-Aldrich, Milan, Italy). Standard Diesel Exhaust Particles (DEP) were from the National Institute of Standard and Technology (NIST) (SRM^®2975) (Sigma-Aldrich). Following preparation protocols already set up in our lab [19 (link),29 (link)], NPs and DEP were weighed in a micro-balance (Sartorius, Goettingen, Germany) in sterile condition, under a laminar flow hood, suspended in sterile ultrapure water, and sonicated in an ultrasonic bath (SONICA Soltec, Milano, Italy) for 10 min.
DEP (2 mg/mL) was sonicated with a probe-type sonicator until it reached energy 3 kJ/s (Bandelin Sonopuls, Berlin, Germany), in order to obtain a well-dispersed suspension of particles. Suspensions were stored at room temperature, while DEP was stored at −20 °C for a period no longer than 15 days. The mixtures were freshly prepared before the experiments using a subcytotoxic concentration of DEP (100 µg/mL) and increasing concentrations of CuO and ZnO NPs (10, 15, 20, 25 µg/mL). Mixtures were sonicated in the ultrasonic bath (SONICA Soltec) for 10 min. The mixtures are indicated as DEP + CuO and DEP + ZnO and were produced by mixing DEP at 100 µg/mL with metal oxide NPs to achieve the final concentrations of CuO and ZnO used for the single NPs.

The National Institute of Standard and Technology standard reference material (SRM 2975) was purchased from Sigma-Aldrich. SRM 2975 is DEPM collected from a diesel-powered industrial forklift. A detailed description of the particles can be viewed at the National Institute of Standard and Technology. The DEPM stock solution (1 mg/mL) was suspended in sterile phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA, USA) and sonicated for 2 min in an ice bath using a sonicator (Sibata; OGAWA SEIKI, Tokyo, Japan) to minimize particulate aggregation.