Permethrin (40 60 cis trans isomer ratio, 95% purity) was purchased from Ronch Chemical Co. (Nanjing, China). Hematoxylin, eosin, and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Animals and treatment
Ten male Wistar rats (200 ± 20 g) were obtained from Weitong Lihua Laboratory Animal Technology Company (Beijing, China) and were housed individually in cages. All animals were acclimatized for at least 1 week prior to the study . During the experiment, the animal rooms were maintained at 22 ± 2 °C temperature and 50–60% humidity and a light/dark cycle of 12 h. Animals had free access to water and the diet. The rats were randomly divided into 2 groups (control and permethrin treatment groups) with 5 animals in each group. Previous studies showed that acute oral half-lethal dose (LD50) of permethrin was 1500 mg/kg for male rats . In this study we chose the value of 1/20 LD50 as the dose (75 mg/kg body weight/day) for the pesticide treatment rats.
The pesticide was dissolved in corn oil (it is difficult to be dissolved in water) and then orally administered daily via gavage to rats (1 ml/kg body weight). Rats were given permethrin daily (once a day) for 90 days. The rats in control group received daily an equivalent dose of corn oil. The body weight of each rat was recorded daily, and symptoms and conditions of the rats were monitored daily throughout the experiment.
All animal procedures were performed in accordance with current China legislation and approved by the Animal and Medical Ethics Committee from Institute of Zoology, Chinese Academy of Sciences.
Samples collection and preparation
The sample collection and preparation were carried out in the similar way as our earlier report . Briefly, after the last administration, 24-hour urine samples of each rat were collected into an ice-cold vessel containing 0.1 ml of 1% sodium azide to prevent bacterial contamination and the samples were stored at − 80 °C prior to biochemical analysis.
All rats were anesthetized with pentobarbital sodium and decapitated twenty-four hours after the last administration. Blood samples were collected and then centrifuged to obtain the serum for biochemical assays. Weights of organs were immediately measured after they were isolated from the body.
For histopathological analysis, the kidney and liver tissues of the rats were dissected immediately at the end of the 90-day exposure to the pesticide, and then were fixed in a solution of 10% formalin. After fixation, the specimens were dehydrated with 80, 95, 100% gradient alcohol, and then soaked in melted wax, and finally embedded in paraffin . After that, the paraffin blocks were cut into 4-μm-thickness sections using a microtome (Microm HM 340E, Thermo Fisher Scientific, USA), then were stained with hematoxylin and eosin. The histological changes in the kidney and liver tissue sections were examined in a microscope (Olympus, Tokyo, Japan) and evaluated by a pathologist blinded to the different treatment groups.
Serum and urine biochemistry
All biochemical parameters of serum and urine samples were analyzed on an Autolab-PM4000 automatic analyzer (AMS Co.), according to the standard spectrophotometric methods. Values of the biochemical parameters were expressed as the mean ± SD.
Student’s t-test was used to assess the statistical significance of differences in measured parameters between the two groups. P < 0.05 was considered statistically significant. SPSS 18.0 software (SPSS, Inc., Chicago, MI, USA) was employed for the statistical evaluation.