Male and female adult Kunming (KM) mice [⁹] (n = 24/sex), weighing 18–22 g, were obtained from the Animal Breeding Center of the Third Military Medical University (Scsk2007-0001, Chongqing, China), and maintained in the SPF-grade (equivalent to AAALAC accreditation) animal facilities at Zunyi Medical College. All animal care and experimental protocols complied with the Animal Management Guidelines of the Chinese Ministry of Health. Mice were acclimated in a temperature-humidity controlled facility with a standard 12-h light/dark schedule (lights on at 8:00 and off at 20:00). All animals had free access to rodent chow and drinking water. After acclimatizing all animals for 2 weeks, four animals per sex were sacrificed at six time points (2:00, 6:00, 10:00, 14:00, 18:00, and 22:00). Blood, liver, and kidneys were collected. Approximately 0.5 ml blood was mixed with 0.5 ml TRIzol. Livers and kidneys were frozen in liquid nitrogen and stored at −80°C prior to analysis.

Approximately 50–100 mg of liver and kidney tissues were homogenized in 1 ml TRIzol (TakaRa Biotechnology, Dalian, China). Total RNA was extracted according to the manufacturer’s instructions, followed by purification with a Total RNA(Mini) Kit (Watson Biotechonologins, Shanghai, China). The quality and quantity of RNA were determined by the 260/280 ratio and gel-electrophoresis. Purified RNA was reverse transcribed with the High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA). The primers were designed with Primer3 software and listed in Table 1. The Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used for real-time RT-PCR analysis. The expression of genes was first normalized with β-actin of the same sample, and the relative transcript levels were calculated as percentage of β-actin.

MT protein concentrations in livers were determined by the cadmium–hemoglobin assay. Liver tissues were homogenized in physiological saline (1:10, wt: vol), followed by centrifugation at 10,000 × g for 10 min. Aliquots of the supernatant (0.1 ml) were mixed with CdCl₂ solution (2 μg Cd/ml;100 μl),followed by the addition of 50 μl of 2% hemoglobin. The mixture was heated in boiling water for 90 seconds and centrifuged (12,000 g, 5 min). Then another 50 μl of 2% hemoglobin was added, boiled and centrifuged again. The supernatant (100 μl) was taken for determination of Cd by Atomic Absorption Spectrometry (Varian, Montreal, Canada).

The frozen tissue samples of the same group were pooled and put into 1 ml lysing buffer, which contain RIPA, PMSF(Beyotime, Shanghai, China), and protease inhibitor cocktail (Calbiochem, San Diego, USA). The homogenates were centrifuged (10000 g, 10 min) and the supernatants collected. Protein concentrations were determined using the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). The samples were boiled with Sample Loading Buffer for 10 min (Beyotime, Shanghai, China). Samples (100 μg) were separated on 15% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked and incubated with anti-metallothionein antibody (Abcam, Cambridge, USA) at dilution1:1000 overnight at 4°C, followed by incubation with an appropriate horseradish peroxidase conjugated secondary antibody. The membranes were detected using a chemiluminescence kit (Thermo, USA). β-Actin was used to normalize protein loading.

Special analysis of circadian rhythms has been documented and used for rhythm analysis worldwide [¹⁰]. In the present study, diurnal variation of MT was analyzed by the intergroup average cosine algorithm method with the help of Dr. Ding-Jun Cai (Chengdu Traditional Chinese Medical University, Statistics Department). Data were presented as mean and SEM. Sex-difference was analyzed by the one-way ANOVA, followed by Student^’s t-test. P < 0.05 was considered statistically significant.

To verify the circadian clock gene expression pattern in KM mice, we analyzed the clock gene Bmal1 in livers and kidneys of KM mice using real-time quantitative RT-PCR. The expression of Bmal1 had a nadir at 18:00 and a peak at 6:00. The differences of the nadirs and peaks were 14-fold for females and 54-fold for males in liver; while in kidneys were 26-fold for females and 37.5-fold for males. Statistically significant 24 h rhythms were confirmed by cosine algorithm method (p < 0.05) (Figure 1).

The expression of another clock gene Dbp was antiphase to Bmal1. Statistically significant 24 h rhythms were confirmed by the cosine algorithm method (p < 0.05) (Figure 2). The mRNA of Dbp was markedly increased at 14:00 and by 18:00 reached a peak. The peak was 264-fold higher than the nadir in the livers of female mice, and 536-fold in males. Dbp also expressed in kidney with the same pattern as in liver, with 223-fold in females and 114-fold in males.

The diurnal variation pattern of MT-1 mRNA resembles the clock gene Dbp. Statistically significant rhythms were confirmed by cosine algorithm method (p < 0.05) (Figure 3). The mRNA of MT-1 reached a peak at 18:00 and a nadir at 6:00. Females showed higher expression levels at 18:00 but males showed more dramatic circadian variations. The diurnal variation of MT-1 mRNA in the liver was 4-fold in females and 43-fold in males, while in kidney it was 7-fold in females and 2.7-fold in males.

In mammals, MT-1 and MT-2 are coordinately regulated, and have similar functions [¹¹]. The diurnal variation pattern of MT-2 mRNA expression resembles that of MT-1.The diurnal variations of MT-2 in the liver were 17-fold for females and 17-fold for males, while in the kidney were 6-fold for females and 3-fold for males. Statistically significant 24 h rhythms were confirmed by cosine algorithm method (p < 0.05) (Figure 4).

The circadian pattern of MT-2 mRNA in blood is slightly different from that of Dbp (data not shown), but resembled the clock gene cryptochrome 1(Cry1). Cry1 expression in females had a 24 h rhythms (4-fold) by cosine algorithm method (p < 0.05), but not for males (2.3-fold) (Figure 5). The nadir and peak differences for MT-2 mRNA in blood were 7.8-fold for females with 6.7-fold for males. Although no statistically significant rhythm for MT-2 mRNA in blood was detected by the cosine algorithm method due to individual variations, similar rhythm trend is clear.

MT protein was determined by western blot and the Cd/hemoglobin assay and the results were similar. Hepatic MT protein, with a peak at 22:00, showed about a 3-fold peak/trough ratio for males. MT protein reached a peak 4 h later after the peak of MT mRNA expression but to a lesser extent. The rhythms were validated by cosine algorithm method (p < 0.05) (Figure 6).

The peak expressions of the clock and MT mRNA in liver, kidney and blood of male and female mice were compared and summarized in Figure 7. In general, females showed higher expression than males (Student’s t test, p < 0.05) (Figure 7).