Citronellol

For olfactory stimulation, we used a custom-built olfactometer [11 (link),30 (link)]. A constant air stream (1.5 m s⁻¹) was provided via a Teflon tube (Ø 6 mm). Syringes (5 ml) were inserted into the tube to function as odour chambers. Farnesol and citronellol (Sigma-Aldrich Chemie GmbH) were diluted in paraffin oil (Sigma-Aldrich Chemie GmbH) to a 0.01 concentration (volume/volume). Filter papers (2 cm²) were soaked with 10 µl of odour solution and placed in the syringes. During the three seconds of stimulation, air volumes of only half of the syringes were injected into the air stream. An exhaust hood was placed behind the bee to quickly remove all odour molecules.

Methyl methacrylate (MM) (≥99%), methacryloyl chloride (97%), geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol, 98%), citronellol (trans-3,7-dimethyl-2,6-octadien-1-ol, 95%), and trimethylamine (≥99.5%) were purchased in Sigma-Aldrich (St. Louis, MO, USA). Irgacure 651 (2,2,-dimethoxy-1,2-diphenylethan-1-one), methanol, chloroform, hexane, toluene, butanol, and silica gel were obtained from Merck. Sodium carbonate, magnesium sulfate, sodium hydroxide, carbon tetrachloride, hydrochloric acid, magnesium sulfate, and buffer solutions (pH 5, 7, and 9) were obtained from POCh, Gliwice, Poland.

The study used 32 male BALB/c Albino mice weighing 25-32 gr. The mice had free access to water and a standard diet. Rhabdomyolysis was induced in the mice by subjecting them to 24 hours of water deprivation, followed by a single intramuscular injection of glycerol 50% (obtained from Merk, Germany) at a dose of 10 ml/kg [2 (link), 8 (link)]. The mice were randomly divided into four groups, each consisting of 8 mice. The control group received normal saline for 4 consecutive days. The model group received a single intramuscular injection of glycerol 50% at a dose of 10 mg/kg to induce rhabdomyolysis. The treatment groups were administered citronellol (obtained from Sigma-Aldrich, Germany) orally at doses of 50 mg/kg and 100 mg/kg for 4 days, followed by a single intramuscular injection of glycerol 50% at a dose of 10 mg/kg on the fourth day [6 (link)]. All mice were euthanized 24hrs after glycerol injection by diethyl ether followed by cervical dislocation [8 (link)]. The right kidneys were extracted and homogenized [9 (link), 10 (link)] and kept frozen for later measurement of myoglobin, cleaved caspase-3, and BAX using ELISA (MY BioSource, USA). The left kidneys were fixed in formalin for histopathological analysis using hematoxylin and eosin (H&E) staining [9 (link), 10 (link)].

Benzaldehyde, ®-citronellol or Isoamyl alcohol (Sigma-Aldrich) were diluted as described in water. Odor-exposures were done by suspending a 4 μl drop of these dilutions on the lids of worm culture dishes at least during 24 h from the egg stage at 20°C, covering the critical plasticity period corresponding to the first 12 h of post-hatch development (Remy and Hobert, 2005 (link)).

The standards of volatile compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA), including isobutanol, 3-methyl-1-butanol, cis-3-hexen-1-ol, 1-hexanol, heptanol, octanol, phenethyl alcohol, 2,6-nonadien-1-ol, dodecanol, ethyl acetate, isoamyl acetate, ethyl lactate, ethyl hexanoate, ethyl octanoate, diethyl succinate, phenethyl acetate, ethyl decanoate, butanoic acid, hexanoic acid, octanoic acid, dodecanoic acid, octanal, (2e,6z)-2,6-nonadienal, benzaldehyde, furfural, citronellol, linalool, rose oxide, geraniol, nerol, β-damascenone, β-ionone, nerolidol, hexanal, guaiacol, 4-Ethylphenol, and 2-octanol which were used as the internal standard.
Folin–Ciocalteu reagent and sodium chloride were purchased from Beijing Chemical Works (Beijing, China). Potassium hydrogen tartrate were purchased at Kemiou Chemical Reagent Co. (Tianjin, China). Bovine serum albumin was obtained from Asahi Kasei Corporation (Tokyo, Japan). Deionized water (<18 MW resistance) was purified by using a Milli-Q purification system (Molecular, Chongqing, China). Bentonite, soybean protein and potassium metabisulfite were purchased from Lallemand Company (Lallemand, Toulouse, France).

The following items were purchased from Sigma Aldrich Darmstadt for the current study: citronellol, doxorubicin (DOX), dexrazoxane, phosphate buffer, hydrogen peroxide (H₂O₂), trichloroacetic acid, sodium phosphate buffer, glacial acetic acid, n-butanol, and acetic acid. All of these items were of analytical grade.

Phanta super-fidelity DNA polymerase (Vazyme, Nanjing, China) was used for DNA amplification. Alkaline phosphatase, calf intestinal (CIP) and restriction enzymes NotI and Sau3AI were purchased from New England BioLabs (Ipswich, MA, USA). Other restriction enzymes and T4 DNA ligase were supplied by MBI Fermentas (Baltimore, MD, USA). The p-nitrophenyl ester series, p-nitrophenol, citronellol, geraniol, cinnamyl alcohol, and isoamyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other commercially available chemicals and solvents were of analytical or higher grade.