Cadmium-Induced Disruption in 24-h Expression of Clock and Redox Enzyme Genes in Rat Medial Basal Hypothalamus: Prevention by Melatonin

Abstract

In a previous study we reported that a low daily p.o. dose of cadmium (Cd) disrupted the circadian expression of clock and redox enzyme genes in rat medial basal hypothalamus (MBH). To assess whether melatonin could counteract Cd activity, male Wistar rats (45 days of age) received CdCl(2) (5 ppm) and melatonin (3 μg/mL) or vehicle (0.015% ethanol) in drinking water. Groups of animals receiving melatonin or vehicle alone were also included. After 1 month, MBH mRNA levels were measured by real-time PCR analysis at six time intervals in a 24-h cycle. In control MBH Bmal1 expression peaked at early scotophase, Per1 expression at late afternoon, and Per2 and Cry2 expression at mid-scotophase, whereas neither Clock nor Cry1 expression showed significant 24-h variations. This pattern was significantly disrupted (Clock, Bmal1) or changed in phase (Per1, Per2, Cry2) by CdCl(2) while melatonin counteracted the changes brought about by Cd on Per1 expression only. In animals receiving melatonin alone the 24-h pattern of MBH Per2 and Cry2 expression was disrupted. CdCl(2) disrupted the 24-h rhythmicity of Cu/Zn- and Mn-superoxide dismutase (SOD), nitric oxide synthase (NOS)-1, NOS-2, heme oxygenase (HO)-1, and HO-2 gene expression, most of the effects being counteracted by melatonin. In particular, the co-administration of melatonin and CdCl(2) increased Cu/Zn-SOD gene expression and decreased that of glutathione peroxidase (GPx), glutathione reductase (GSR), and HO-2. In animals receiving melatonin alone, significant increases in mean Cu/Zn and Mn-SOD gene expression, and decreases in that of GPx, GSR, NOS-1, NOS-2, HO-1, and HO-2, were found. The results indicate that the interfering effect of melatonin on the activity of a low dose of CdCl(2) on MBH clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression.

Keywords: cadmium; circadian rhythms; clock genes; medial basal hypothalamus; melatonin; redox enzyme genes.

Figure 1

Effect of melatonin on Cd-induced changes in 24-h pattern in expression of Clock, Bmal1, Per1, Per2, Cry1, and Cry2 in rat MBH. The rats received CdCl₂ (5 ppm) and melatonin (3 μg/mL) or vehicle in drinking water for 1 month. Two groups given melatonin or vehicle in tap water were also included. Groups of six to eight rats were killed by decapitation at six different time intervals throughout a 24-h cycle. mRNA levels encoding the clock genes were measured as described in the text. Shown are the mean ± SEM of mRNA determination as measured by triplicate real-time PCR analyses of RNA samples. Letters denote significant differences in a one-way ANOVA followed by a Bonferroni’s multiple comparison test performed at every time interval, as follows: ^ap < 0.05 vs. Cd and melatonin groups; ^bp < 0.02 vs. Cd and Cd + melatonin groups; ^cp < 0.05 vs. Cd; ^dp < 0.01 vs. the remaining groups; ^ep < 0.01 vs. Cd and control groups; ^fp < 0.05 vs. control. One-way ANOVAs within each experimental group indicated significant time-related changes in clock gene expression as follows: Controls: Bmal1, Per1, Per2, and Cry2 (F = 11.3, p < 0.0001, F = 2.54, p < 0.04, F = 7.94, p < 0.001, and F = 3.41, p < 0.03, respectively). Cd: Clock, Per1, Per2, and Cry2 (F = 4.71, p < 0.008, F = 4.12, p < 0.01, F = 5.84, p < 0.03, and F = 19.1, p < 0.0001, respectively). Cd + melatonin: Bmal1, Per1, Per2, Cry1, and Cry2 (F = 10.1, p < 0.0001, F = 3.11, p < 0.04, F = 4.48, p < 0.009, F = 7.82, p < 0.001, and F = 19.6, p < 0.0001, respectively. Melatonin: Per2 and Cry2 (F = 14.2 and 50.8, p < 0.0001, respectively). For further statistical analysis, see text.

Figure 2

Effect of melatonin on Cd-induced changes in 24-h pattern in expression of mRNA for Cu/Zn-SOD, Mn-SOD, catalase, Gpx, and GSR in rat MBH. For experimental details see legend to Figure 1. mRNA levels encoding the enzymes were measured as described in the text. Shown are the mean ± SEM of mRNA determination as measured by triplicate real-time PCR analyses of RNA samples. Letters denote significant differences in a one-way ANOVA followed by a Bonferroni’s multiple comparison test performed at every time interval, as follows: ^ap < 0.02 vs. Cd and control groups; ^bp < 0.05 vs. melatonin and control groups; ^cp < 0.01 vs. the remaining groups; ^dp < 0.05 vs. melatonin and Cd groups; ^ep < 0.05 vs. melatonin and control groups; ^fp < 0.05 vs. the remaining groups; ^gp < 0.01 vs. Cd; ^hp < 0.02 vs. control; One-way ANOVAs within each experimental group indicated significant time-related changes in enzyme gene expression as follows: Controls: Cu/Zn-SOD, Mn-SOD, catalase, and GPx (F = 6.21, p < 0.002, F = 3.33, p < 0.03, F = 3.17, p < 0.04, and F = 2.98, p < 0.04, respectively). Cd: Cu/Zn-SOD and Mn-SOD (F = 6.25, p < 0.002, and F = 7.01, p < 0.001, respectively). Cd + melatonin: Cu/Zn-SOD, Mn-SOD, catalase, and GSR (F = 14.1, p < 0.0001, F = 15.5, p < 0.0001, F = 10.8, p < 0.0001, and F = 8.24, p < 0.0001, respectively). Melatonin: Mn-SOD, catalase, GPx, and GSR (F = 4.49, p < 0.002, F = 2.46, p < 0.05, F = 15.9, p < 0.0001, and F = 8.35, p < 0.0001, respectively). For further statistical analysis, see text.

Figure 3

Effect of melatonin on Cd-induced changes in 24-h pattern of expression of mRNA for NOS-1, HO-1, NOS-2, and NOS-2 in rat MBH. For experimental details see legend to Figure 1. mRNA levels encoding the enzymes were measured as described in the text. Shown are the mean ± SEM of mRNA determination as measured by triplicate real-time PCR analyses of RNA samples. Letters denote significant differences in a one-way ANOVA followed by a Bonferroni’s multiple comparison test performed at every time interval, as follows: ^ap < 0.05 vs. Cd + melatonin group; ^bp < 0.05 vs. the remaining groups; ^cp < 0.02 vs. Cd; ^dp < 0.02 vs. control and Cd groups; ^ep < 0.01 vs. the remaining groups; ^fp < 0.02 vs. control and Cd + melatonin groups; ^gp < 0.01 vs. control; ^hp < 0.01 vs. melatonin. One-way ANOVAs within each experimental group indicated significant time-related changes in enzyme gene expression as follows: Controls: HO-1, NOS-2, and HO-2 (F = 7.54, p < 0.001, F = 10.1, p < 0.0001, and F = 5.64, p < 0.002, respectively). Cd: NOS-1, HO-1, NOS-2, and HO-2 (F = 5.88, p < 0.002, F = 8.41, p < 0.0001, F = 18.3, p < 0.0001, and F = 7.42, p < 0.001, respectively). Cd + melatonin: HO-1, NOS-2, and HO-2 (F = 8.45, p < 0.0001, F = 6.12, p < 0.002, and F = 4.04, p < 0.03, respectively). Melatonin: NOS-1, NOS-2, and HO-1 (F = 10.7, p < 0.0001, 12.9, p < 0.0001, and 5.92, p < 0.004, respectively). For further statistical analysis, see text.

Figure 4

Effect of melatonin on Cd-induced changes in mRNA of redox enzymes in rat MBH. Shown are the mean 24 h values ± SEM. For experimental details see legend to Figure 1. Letters denote significant differences in a one-way ANOVA followed by a Bonferroni’s multiple comparison as follows: ^ap < 0.01 vs. control and Cd groups; ^bp < 0.05 vs. control; ^cp < 0.02 vs. control; ^dp < 0.02 vs. the remaining groups; ^ep < 0.02 vs. control and Cd groups; ^fp < 0.03 vs. the remaining groups.