2.1. Chemicals and Animals
Chamomile essential oil was obtained from Gritman Co. (Frendswood, Tex., U.S.). It was extracted from flowers of Chamomilla recutita
by steam distillation, and then it was analyzed by gas chromatography to identify the chemical species. Thirteen compounds were determined with this assay, including bisabolol and its oxides, β-farnecene, chamazulene, germacrene, and sesquiterpenes (Table 1
For the SCE assay, the following chemicals were used: activated charcoal, purchased from Sigma Chemicals (St. Louis, Mo., U.S.), sifted in a sieve (number 200), and neutralized to pH 7.4 with continuous washes of chloridric acid 1M (J.T. Baker, Mexico City), sodium hydroxide 1M (J.T. Baker, Mexico City), and deionized water (Hycell, Mexico City). Corn oil, colchicine, bromodeoxyuridine (BrdU), Hoescht 33258, PBS, EDTA, trypsin, DPPH, α–tocopherol, phosphate buffer, α-cariophylene, and dextrose were purchased from Sigma Chemicals (St. Louis, Mo., U.S.). Linoleic acid was obtained from Fluka Chemicals (St. Louis, Mo., U.S.). Ferrous chloride, iron free ethanol and methanol, amonium thiocyanate, chlorhidric acid, potassium chloride, dibasic sodium phosphate, monobasic potassium phosphate, sodium citrate, citric acid, sodium bicarbonate, sodium chloride, and acetic acid were acquired from J.T. Baker (Mexico City). The Giemsa stain was obtained from Merck (Mexico City), and DAU, 97% pure, from Lemery Laboratories (Mexico City).
The animals used for the SCE study were male mice strain NIH with a mean weight of 25 g. They were obtained from the National Laboratories of Public Health (Mexico City), and maintained in our laboratory in metallic cages at a temperature of 23 °C and 60% humidity, with food (Nutrimix, Mexico City) and water ad libitum, in a 12 h light-dark period, adjustment time period for the animals was of one week before the administration of the treatments. The protocol was approved by the Committee of Ethics and Biosecurity of the National School of Biological Sciences.
2.2. SCE Assay
Six groups with five animals each were organized for this assay: a negative control group that was administered corn oil orally (0.1 mL), a group positive treated with corn oil (0.1 mL) and DAU administered by intramuscular injection (10 mg/kg), a group administered with 500 mg/kg of CEO, and three groups treated with DAU and CEO (5, 50, and 500 mg/kg) respectively.
Immediately after administration of corn oil and CEO, all animals were treated by intraperitoneal route with an aqueous suspension of BrdU adsorbed to activated charcoal (1.2 mg/kg) so as to obtain the chromatid differential labeling [13
]. Twelve hours afterward, 10 mg/kg of DAU was injected to mice of the aforementioned groups. This schedule and route were tested earlier and found to be appropriate to detect the SCE induction by DAU. Colchicine (7.5 mg/kg) was subcutaneously injected 51 h after the beginning of the CEO administration to stop cell division. To obtain spermatogonial cells in metaphase, we followed the technique described by Madrigal-Bujaidar et al.
]. The animals were killed by cervical dislocation and their testes were dissected; the tunica albuginea of each organ was removed, and the seminiferous tubules were fragmented and washed several times with PBS; then, the tissue was put in a 0.1% trypsin solution made in trypsin medium free of calcium and magnesium, plus EDTA; the tissue was agitated for 5 min at 37 °C, dissagregated, centrifuged for 8 min at 2,000 rpm, and the sediment was washed several times with PBS to be finally resuspended in a solution of KCL 0.075 M for 5 min at 37 °C. The supernatant was recovered and centrifuged 5 min at 500 rpm; finally, the cell suspension was fixed three times in a mixture of methanol-acetic acid (3:1) and dropped onto iced slides that were briefly flamed. Differentiation of the sister chromatids was made according to the Hoescht-Giemsa method [13
]. Six drops of Hoescht 33250 (2.5 μg/mL) were placed on the slide and expanded with a coverslide and protected from light for 20 min. Buffer solution (phosphate-citrate) was added prior to their exposure to black light for 120 min at a distance of 1 cm. Coverslides were removed by immersion in distilled water and the slides were placed in a Coplin jar with saline citrate solution at 60 °C for 15 min, and washed in hot and cold distilled water prior to staining with a 4% Giemsa solution for 15 min. The microscopic scoring was made in 30 second division metaphases per animal to determine the rate of SCE.
2.4. Ferric Thiocyanate Assay
This assay was made according to the one described by Ono et al. (1999). Ethanolic solutions of CEO and α–tocopherol were prepared (14, 140, and 280 mg/mL), then, 200 μL of each solution were added to the reaction mixture: 800 μL of ethanolic solution of linoleic acid 2.25%, 1600 μL of phosphate buffer pH = 7, 600 μL of absolute ethanol (iron free), and 800 μL of deionized water. The mixture was placed in a vial protected from light, and stirred in a vortex. Then, five vials were prepared for each tested concentration of CEO, α-tocopherol, in the linoleic acid control group. The mixtures were incubated at 40 °C for 24, 48, 72, 96, 120, 168, 192, and 226 h, and at each time, 50 μL of each vial was diluted in 4.85 mL of 75% ethanol. Then, we added 50 μL of a 30% ammonium thiocyanate solution, as well as 50 μL of a 20 mM solution of ferrous chloride made in 3.5% chlorhidric acid. The absorbance was measured at 500 nm, 3 min after the addition of ferrous chloride.