Diurnal variation of hepatic antioxidant gene expression in mice

Abstract

Background: This study was aimed to examine circadian variations of hepatic antioxidant components, including the Nrf2- pathway, the glutathione (GSH) system, antioxidant enzymes and metallothionein in mouse liver.

Methods and results: Adult mice were housed in light- and temperature-controlled facilities for 2 weeks, and livers were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Hepatic mRNA levels of Nrf2, Keap1, Nqo1 and Gclc were higher in the light-phase than the dark-phase, and were female-predominant. Hepatic GSH presented marked circadian fluctuations, along with glutathione S-transferases (GST-α1, GST-µ, GST-π) and glutathione peroxidase (GPx1). The expressions of GPx1, GST-µ and GST-π mRNA were also higher in females. Antioxidant enzymes Cu/Zn superoxide dismutase (Sod1), catalase (CAT), cyclooxygenase-2 (Cox-2) and heme oxygenase-1 (Ho-1) showed circadian rhythms, with higher expressions of Cox-2 and CAT in females. Metallothionein, a small non-enzymatic antioxidant protein, showed dramatic circadian variation in males, but higher expression in females. The circadian variations of the clock gene Brain and Muscle Arnt-like Protein-1(Bmal1), albumin site D-binding protein (Dbp), nuclear receptor Rev-Erbα (Nr1d1), period protein (Per1 and Per2) and cryptochrome 1(Cry1) were in agreement with the literature. Furthermore, acetaminophen hepatotoxicity is more severe when administered in the afternoon when hepatic GSH was lowest.

Conclusions: Circadian variations and gender differences in transcript levels of antioxidant genes exist in mouse liver, which could affect body responses to oxidative stress at different times of the day.

Figure 1. Circadian variations of mRNA levels of the clock gene Brain and Muscle Arnt-like Protein-1(Bmal1), albumin site D-binding protein (Dbp), nuclear receptor Rev-Erbα (Nr1d1), cryptochrome 1 (photolyase-like) (Cry1), period protein 1 (Per1) and period protein 2 (Per2) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 2. Circadian variations of mRNA levels of the Nrf2 pathway-related genes, including the nuclear factor erythroid-2-related factor-2 (Nrf2), Kelch-like ECH associating protein 1 (Keap1), NADPH quinone oxidase 1 (Nqo1), and glutamate-cysteine ligase catalyze subunit (Gclc) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 3. Circadian variations of hepatic glutathione (GSH) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 4. Circadian variations of hepatic GSH-related genes (Gpx1, GST-α1, GST-µ, GST-π) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 5. Circadian variations of cycloooxygenae-2 (Cox-2), antioxidant enzymes Cu/Zn superoxide dismutase (Sod1), catalase (CAT), and heme oxygenase-1 (Ho-1) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 6. Circadian variations of the small antioxidant protein metallothionein gene (Mt-1 and Mt-2) in adult female and male mouse livers (n = 4 for each time point).

*Significant circadian rhythm p<0.05; #Significant sex difference p<0.05.

Figure 7. Circadian variation in acetaminophen hepatotoxicity.

Mice (n = 10) were administered acetaminophen (500 mg/kg, ip, in 20 ml/saline) at 6∶00 and 18∶00, respectively. Hepatotoxicity was evaluated by serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 12 h after acetaminophen administration. Data are mean ± SEM, *Significant difference between 6∶00 and 18∶00, p<0.05.