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Am J Pathol. Jan 2009; 174(1): 63–70.
PMCID: PMC2631319

The Protective Role of Per2 Against Carbon Tetrachloride-Induced Hepatotoxicity

Abstract

Period 2 (Per2) is a key component of the core clock oscillator and is involved in regulating a number of different biological processes and pathways. Here we report that Per2 plays a protective role in carbon tetrachloride (CCl4)-induced hepatotoxicity via the modulation of uncoupling protein-2 (Ucp2) gene expression in mice. Hepatic injury after acute CCl4 injection was monitored in both wild-type and Per2-null mice. At the 12-hour time point after CCl4 treatment, many more vacuolations were observed in the liver tissues of Per2-null mice whereas fatty tissue degeneration primarily occurred in the liver tissues of wide-type mice. Serum alanine and aspartate aminotransferase activities were elevated in Per2-null mice compared with wide-type mice at 24 hours after CCl4 treatment, which was in agreement with the observation of significantly larger areas of centrilobular necrosis in the livers of Per2-null mice. A deficit of the Per2 gene enhanced Ucp2 gene expression levels in the liver. As a consequence, intracellular levels of ATP markedly decreased in the liver, allowing increased production of toxic CCl4 derivatives. The absence of Per2 expression caused a dramatic elevation of Clock expression and influenced Ucp2 through a mechanism that involved a Clock-controlled PPAR-α signal transduction pathway. Our studies suggest that the Per2 gene functions in hepatocyte protection from chemical toxicants via the regulation of hepatic Ucp2 gene expression levels.

Circadian rhythms in mammals are endogenous oscillators that drive the daily oscillations of multiple biological processes.1,2,3 Disruption in circadian timing, either by mutation in mice or by shiftwork in people, is associated with metabolic disease.4,5 The master circadian clock is located in the suprachiasmatic nuclei of the anterior hypothalamus.6 In recent years, the existence of peripheral oscillators has been discovered and found to be critically important for organizing the metabolism of the whole body.7 Tissue-based clocks control the local circadian transcriptome, and are in turn synchronized to each other and to solar time by behavioral and neuroendocrine cues emanating from the suprachiasmatic nuclei.2 The molecular circadian clockwork consists of interwoven positive and negative feedback loops, in which two transcription factors, CLOCK and BMAL1, form heterodimers and directly activate the transcription of Per1, Per2, Cry1, and Cry2, and so forth. These genes products are thought to enter the nucleus, inhibit the transcription activity of CLOCK-BMAL1 heterodimers, and generate a negative feedback loop.8 The circadian clock controls downstream events by regulating the expression of clock-controlled genes that function in the rate-limiting steps of various biological pathways.9

Several lines of evidence indicate that circadian rhythms are involved in both incidence and severity of drug-induced hepatic injury,10,11 implying that the clock genes might play a role in modulating the action of hepatotoxic effects such as fatty degeneration and hepatocellular death. Carbon tetrachloride (CCl4) is the classic hepatotoxicant that causes acute, reversible liver injury in a range of laboratory animals.12 It is now accepted that liver injury induced by CCl4 involves initially the metabolism of CCl4 to trichloromethyl(CCl3*)-free radical by cytochrome P450 isozymes such as CYP2E1. Once the free radical is formed, it reacts with various biologically important substances such as amino acids, nucleic acids, and lipids, and then causes lipid peroxidation, membrane damage, and loss of hepatocellular calcium homeostasis.13 CCl4 treatment causes morphological changes of liver mitochondria at early stages and decreases liver ATP content because of uncoupling of oxidative phosphorylation of liver mitochondria.14

Hepatotoxicity induced by CCl4 increases with age.15 Circadian functions are also impaired in old animals,16 in parallel to increase in drug-induced hepatic injury. That provoked us to investigate whether there is molecular relationship between circadian gene and CCl4-induced hepatotoxicity. In this study, we found that mice deficient in the Per2 gene, a key component of core clock oscillators, displayed increased sensitivity to CCl4 by enhanced Ucp2 gene expression and lowered ATP level in livers. Our findings suggest that the clock gene Per2 plays a protective role in hepatic injury induced by hepatotoxicants.

Materials and Methods

Animals and CCl4 Treatment

Male, 6- to -8-week-old Per2-null and wild-type C57BL/6 mice were used in this study. The Per2-null mice used in this study have been characterized previously Fu and colleagues.17 The wild-type (C57BL/6) and Per2-null mice (on a co-isogenic C57BL/6 background) were maintained in 12-hour light/dark cycles with lights on at 7:00 AM and off at 7:00 PM, and with free access to regular chow food and water. Mice were administrated a single intraperitoneal injection of CCl4 in a dose of 0.3 ml/kg in olive oil. Control animals were injected with the same volume of olive oil. The time for injection and mice sacrifice is shown in Table 1. All animals had free access to food and water, only fasting 12 hours before they were sacrificed. All animal care and use procedures were in accordance with the guidelines of the Institutional Animal Care and Use Committee at the Nanjing University of Science and Technology.

Table 1
Time for Injection and Mouse Sacrifice

Serum Aminotransferase Activity

Serum was collected at 0, 12, and 24 hours after CCl4 treatment and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were measured by an Olympus (Tokyo, Japan) AU2700 automatic biochemical analyzer.

Histological Analysis

At 0, 12, and 24 hours after CCl4 treatment, a small piece of liver tissue was collected and fixed in 10% formalin. The sample was then embedded in paraffin, sliced into 5-μm-thick sections, and stained with hematoxylin and eosin (H&E) for histological analysis under a light microscope. For liver fat analysis, samples were frozen in Tissue-Tek Cry03 DM230 (Sakura, Tokyo, Japan); 5-μm-thick sections were loaded on poly-l-lysine-coated slides and stained with Sudan IV and counterstained with hematoxylin to show lipids in red. Necrosis areas were quantitated and percentage of necrosis was calculated using Scion Image β 4.0.3 (Scion Corp., Frederick, MD).

Quantitative Real-Time Polymerase Chain Reaction (PCR)

Total RNA from liver samples was extracted using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Reverse transcript reaction using KeyGene (Wageningen, The Netherlands) reverse transcript enzyme according to the manufacturer’s protocol. The real-time PCR reaction was performed on an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA) with a cDNA sample and amplified in a 20-μl reaction volume containing 1× SYBR Green PCR master mix (Applied Biosystems). Primers of each gene are as follows: Cyp2e1: 5′-CAAGTCTTTAACCAAGTTGGCAAA-3′ (forward) and 5′-CCACGATGCGCCTCTGA-3′ (reverse) and Ucp-2: 5′-GCTGGTGGTGGTCGGAGATA-3′ (forward) and 5′-ACAGTTGACAATGGCATTACGG-3′ (reverse) and Clock: 5′-CGGCGAGAACTTGGCATT-3′ (forward) and 5′-AGGAGTTGGGCTGTGATCA-3′ (reverse) and PPAR-α: 5′-GGGTAC CACTACGGAGTTCACG-3′(forward) and 5′-CAGACAGGCACTTGTGAAAACG-3′(reverse) and Gapdh: 5′-CATCCACTGGTGCTGCCAAGGCTGT-3′ (forward) and 5′-ACAACCTGGTCCTCAGTGTAGCCCA-3′ (reverse). Relative expression in comparison with Gapdh was calculated by the comparative CT method.

Measurement of Liver Nucleotides

Liver tissue (100 mg) was homogenized and extracted from liquid nitrogen frozen samples using 0.4 N of perchloric acid. Extracts were separated and quantified by using reversed-phase high performance liquid chromatography (Waters 1525 system; Millipore Corp., Bedford, MA) analysis on a Partisphere bonded phase C18 (reverse phase) cartridge column at a flow rate of 1.0 ml/minute. As previously described by Smolenski and colleagues,18 the mobile phase was 150 mmol/L KH2PO4 and 150 mmol/L KCl, pH 6.0, with a superimposed 15% acetonitrile gradient: 0% for 0 to 20 seconds, 0 to 9% for 20 seconds to 7 minutes, 9 to 100% for 7 to 10 minutes, 100% for 10 to 14 minutes, 100 to 0% for 14 to 15 minutes and 0% for 15 to 20 minutes. ATP, ADP, and 5′-AMP standards were purchased from Sigma. (St. Louis, MO).

Cell Culture and Plasmid Transfections

Murine H22 cells (murine carcinoma of hepatoma 22) were maintained in Dulbecco’s modified Eagle’s medium supplemented with antibiotics and 15% fetal calf serum in an atmosphere of humidified 95% air and 5% CO2 at 37°C. The cells were transfected with the mPer2 cDNA plasmids, indicated using transfection reagent. Cells were prepared 24 hours later for total RNA extraction and Ucp2, PPAR-α, and Clock expressions were examined by quantitative real-time RT-PCR as described above.

Statistics

Results are expressed as mean ± SEM. Statistical evaluation was performed using a parametric Student’s t-test. A P value less than 0.05 was considered a statistically significant difference.

Results

Increased CCl4-Induced Liver Injury in Per2-Null Mice

Per2-null mice display impaired clock resetting and differ from wild-type mice in their behavioral and physiological response to exterior signaling or factors.17,19 Therefore, we searched for a difference in liver injury induced by CCl4 in Per2-null and wild-type mice. At 9:00 AM (at 2 hours after lights on), mice were injected with a dose of CCl4 or oil, and serum aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured as markers of liver injury (Figure 1, A and B). Compared with control animals injected with oil, serum ALT and AST activities were elevated in mice injected with CCl4. The increased levels of ALT and AST activities were similar between wild-type and Per2-null mice at 12 hours after CCl4 injection, indicating no significant difference in liver injury between the two mouse genotypes at this stage. Interestingly, Per2-null mice had approximately two and three times higher levels of serum ALT and AST activities than wild-type mice at 24 hours after treatment with CCl4. Moreover, in mice injected with CCl4 at 9:00 PM (2 hours after lights off), ALT and AST activities were more strongly increased in Per2-null mice compared with wild-type mice at 24 hours after injection (Figure 1, C and D). Meanwhile, control animals showed no difference in ALT and AST activities after oil injection (insets in Figure 1, C and D). These data suggest that mice deficient in Per2 gene are more sensitive to CCl4 hepatotoxicity.

Figure 1
Serum ALT (A) and AST (B) activities were monitored after acute CCl4 treatment. Injection times: 9:00 AM (A and B); 9:00 PM (C and D). Significant difference between Per2-null and WT mice was observed at 24 hours (inset, oil control). All values were ...

To further assess the CCl4-induced hepatocyte injury in Per2-null and wild-type mice, liver samples at 0, 12, and 24 hours after CCl4 treatment were obtained and histological analysis was taken at each point (Figure 2). Livers from both groups of animals had similar weights (data not shown). We compared lipid degeneration and extent of necrosis in H&E-stained sections from Per2-null and wild-type mice, respectively. As shown in Figure 1, no obvious difference was observed between Per2-null (Figure 2, A, E, and I) and wild-type animals (Figure 2, B, F, and J) at various time points after injection with oil as a control or at the time of injection with CCl4 (Figure 2, C and D). Unexpectedly, Per2-null mice displayed many more vacuolations at 12 hours after CCl4 treatment (Figure 2G), whereas fatty degeneration mainly occurred in wide-type mice (Figure 2H). At 24 hours after injection with CCl4, centrilobular necrosis was observed in both groups of mice and the hepatocyte necrosis of Per2-null mice was much more predominant compared with that of wild-type mice (Figure 2, K and L). Adequate quantifications for areas of necrosis from multiple sections showed the areas of necrosis were ~2.5-fold larger in Per2-null mice than in wild-type mice (Figure 3A). Moreover, fat-specific Sudan staining analysis confirmed that fatty degeneration was obviously present in wild-type mice at 12 hours after CCl4 treatment, whereas Per2-null mice displayed a negative reaction to Sudan staining (Figure 3B).

Figure 2
Histological analysis of WT and Per2-null livers after CCl4 treatment. Paraffin-embedded sections from livers taken 0, 12, and 24 hours after CCl4 treatments or olive oil control were stained with H&E. No differences were observed in both liver ...
Figure 3
Triglyceride distribution and necrosis quantitation associated with Per2-null and wild-type mice after CCl4 treatment. A: Liver sections from mice at 24 hours after CCl4 treatment were analyzed by morphometric image analysis, digitized, and quantified ...

Cyp2e1 Down-Regulation Was Similar in Wild-Type and Per2-Null Mice

The enzyme Cyp2e1 is the major enzyme responsible for CCl4 metabolism, and it plays an important role in the modulation of CCl4-induced liver injury in the early stage.20 Down-regulation of Cyp2e1 after CCl4 injection is considered as an adaptive mechanism for decreasing toxicity.13 Increased or stabilized Cyp2e1expression could result in more severe liver injury induced by CCl4,21 and mice deficient in Cyp2e1 are resistant to CCl4-induced hepatotoxicity.20,22 So we first examined whether Per2 influences Cyp2e1 expression in CCl4-induced liver injury. However, at various stages (0, 12, and 24 hours) of CCl4 treatment, Cyp2e1 expression had no significant difference between wild-type and Per2 mice (Figure 4). The Cyp2e1mRNA down-regulation after CCl4 treatment also occurred in Per2-null mice and had a similar tendency to that in wild-type mice. These observations suggest that Per2 is not involved in regulating Cyp2e1 expression in the early stage of liver injury.

Figure 4
Cyp2e1 gene expression. Liver Cyp2e1 expression level was detected at 0, 12, and 24 hours after acute CCl4 treatment. No statistical difference was observed between WT and Per2-null mice. (n = 4 to 5). Data are presented as mean ± SEM. ...

Increased Hepatic Ucp2 Gene Expression and Lowered Liver ATP Content in Per2-Null Mice

Ucp2 is an inner mitochondrial membrane protein that uncouples respiration from oxidative phosphorylation.23 Overexpression of Ucp2 can reduce the efficiency of cellular ATP synthesis, resulting in a precipitous drop of cellular ATP level.24,25 The resulting ATP depletion has been proposed as causative factor in CCl4-induced cell death.13,25,26 CCl4 induces the expression of Ucp2 mRNA and Ucp2 protein in hepatocytes.27 Analysis of expression of Ucp2 gene at the mRNA level indicated that Per2-null mice displayed a marked elevation both at 0 and 24 hours after CCl4 injection compared with wild-type mice (Figure 5A), suggesting Per2 functions as suppressor of Ucp2 gene expression. Therefore overexpression of Per2 should lower Ucp2 gene expression. Transfecting Per2 cDNA into H22 culture cells, a mouse hepatic tumor cell line, reduced Ucp2 gene expression at 24-hour time points (Figure 5B). Then, we next measured liver ATP in both Per2-null and wild-type mice at 24 hours after CCl4 injection. High performance liquid chromatography analysis demonstrated the ATP peak was lower in Per2-null mice than in wild-type mice (Figure 6, A and B). Quantification showed that levels of liver ATP were ~50% decreased in Per2-null mice compared with those in wild-type mice (Figure 6C). Furthermore, the ratio of hepatic ADP and ATP was significantly decreased in CCl4-treated Per2-null mice compared with wild-type mice (Figure 6D), indicating the precipitous drop of cellular ATP was associated with enhanced Ucp2 gene expression because of the absence of Per2. These results provided evidence of a protective role for Per2 by suppressing Ucp2 gene expression in CCl4-induced hepatotoxicity.

Figure 5
Ucp2 gene expression in vivo and in vitro. Liver Ucp2 gene expression was detected at the 0- and 24-hour point after CCl4 treatment. A: High levels of Ucp2 expression were observed at both time points (n = 4 to 5) in Per2-null mice. B: Ucp2 expression ...
Figure 6
Liver ATP content at 24 hours after CCl4 injection. High performance liquid chromatography profile of ATP (peak 1), ADP (peak 2), and AMP (peak 3) in livers at 24 hours after CCl4 injection in wild-type (A) and Per2-null mice (B). Quantitation of nucleotides ...

Per2 Regulated Ucp2 Gene Expression Through a Mechanism that Involves a Clock-Controlled PPAR-α Signal Transduction Pathway

In in vitro and in vivo experiments, Ucp2 gene expression is induced by stimulating peroxisome proliferator-activated receptor-α (PPAR-α) signal transduction pathways.28,29 The circadian gene Clock is involved in the transactivation of PPAR-α mRNA in peripheral tissues.30 We postulated that clock genes functioned in Ucp2 gene regulation with PPAR-α, and that they would be up-regulated in Per2-null mice. To test this hypothesis, total RNA was extracted from the livers of wild-type and Per2-null mice. The levels of mRNAs of PPAR-α and other clock genes were examined by quantitative real-time PCR analysis. The results of three independent experiments were summarized and are presented in Figure 7. In Per2-null livers, the PPAR-α mRNA showed a higher expression compared with that in wild-type mice (Figure 7A). Thus, PPAR-α was a potential modulator and its expression was up-regulated in mPer2-null mice. We next examined the expression of the circadian gene Clock by mRNA level at day and night times. In Per2-null livers, Clock mRNA expression still had day and night variation but reached a 1.5-fold higher level in wild-type livers (Figure 7B). To confirm that the role of mPer2 in regulating Ucp2 expression was really through a mechanism that involves a Clock-controlled PPAR-α transduction pathway, we analyzed gene expression in hepatic tumor H22-cultured cell lines that overexpress Per2 cDNA. As expected, the cells showed decreased expression of PPAR-α after 24 hours of transfection of Per2 cDNA (Figure 7C). Also, as shown in Figure 7D, transfection of Per2 cDNA impaired Clock mRNA expression. These results indicated that loss of Per2 function resulted in abnormal transcription by Clock-controlled PPAR-α pathway, which modulates Ucp2 gene expression.

Figure 7
Analysis of the Clock-controlled PPAR-α signal transduction. A: Increased PPAR-α mRNA expression in livers of Per2-null mice (n = 5). Clock gene expression was detected at 11:00 AM and 11:00 PM, respectively. B: Per2-null mice ...

Discussion

Recent studies have demonstrated that multiple genes are controlled by the circadian clock in a tissue-specific manner and that many of these clock-controlled genes function in the rate-limiting steps of major physiological process.9,31 It is commonly accepted that circadian disorder could influence the stress response. In our studies, we focused on investigating the circadian clock-controlled PPAR-α and Ucp2 gene expression, and highlighted the fundamental role that the circadian clock played in protecting liver injury from oxidization stress. A nonfunctional Per2 gene led to a feedback increase of Clock expression and as a consequence, live ATP content was decreased by activated Clock-controlled PPAR-α and Ucp2 gene expression. In many cases, decreases in ATP level would inactivate Na+-K+ ATPase activity, resulting in an unbalance of extra- and intracellular sodium-potassium homeostasis.32,33 At the physiological level, this led to increased vacuolations in liver tissue in the early stage of CCl4 treatment, as we observed. In addition, PPAR-α activation stimulated β-oxidation of fatty acids and decreased fat accumulations by amplifying the expression of lipoprotein lipase.34 Thus, after CCl4 treatment, the Per2-null mice had no obvious fatty degeneration because of a higher level of PPAR-α expression compared with wild-type mice. This implied, in Per2-null mice, that fatty degeneration was not a dominating factor in hepatotoxicity induced by CCl4.

Studies in Cyp2e1-knockout mice, in which no liver necrosis and no increase in serum ALT and AST activities after CCl4 treatment were seen, strongly support the contention that Cyp2e1 was required for the CCl4-induced hepatotoxicity.20,22 CCl4 metabolism by Cyp2e1 in hepatocytes produces high levels of oxidative stress.13 In this stage, loss of Per2 function had no obvious effects in elevation of ALT and AST activities compared with wild-type mice. The oxidation stress induced by CCl4 metabolites was correlated to induction of Ucp2 via the stimulation of PPAR-α expression.27,29 Ucp2 is a member of a family of proton transporters located in the mitochondrial inner membrane and involved in the regulation of superoxide and ATP synthesis.23 The up-regulation of Ucp2 enhanced the cyanide-induced mitochondrial dysfunction, leading to execution of necrosis.35 Inhibiting Ucp2 gene expression attenuated some drug-induced neurotoxicity.36 In our observation, loss of Per2 function resulted in resetting circadian clock functions, especially, feedback stimulated Clock gene expression. PPAR-α, one of the clock-controlled genes, was up-regulated in hepatocytes, leading to activation of Ucp2 expression. Increase in Ucp2 mRNA in the liver of CCl4-treated animals was associated with a rise in Ucp2 protein.27 Thus, as we observed, the elevation Ucp2 expression caused a more severe drop in ATP content of Per2-null livers, which was obligated for death of hepatocytes after CCl4 treatment.

The circadian rhythm has been implicated in susceptibility of rats to chemical-induced liver damage.10 An increase in CCl4 hepatotoxicity occurs in old animals,15 indicating genetic compounds are potential causes of age-dependent changes in chemically induced liver damage. Age-dependent changes in the circadian gene expression in laboratory rodents and humans have been described extensively.37,38 An age-dependent difference was found in the case of Per2 mRNA expression39; also Ucp2 expression increased with age.40 Thus, the phenotype of increased CCl4 hepatotoxicity regulated by increased Ucp2 levels because of Per2 gene deficiency could reflect the natural phenomena of increasing the sensitivity to toxicant with age in old animals.

Collectively, our data established Ucp2 as a link between dysfunction of circadian clock gene Per2 and increased CCl4-induced liver injury.

Acknowledgments

We thank Dr. C.C. Lee for providing Per2-deficient mice.

Footnotes

Address reprint requests to Dr. Jianfa Zhang, the Center for Molecular Metabolism, Nanjing University of Science and Technology, Nanjing 210094, China. E-mail: nc.ude.tsujn.liam@gnahzfj.

Supported by the National Science Foundation of China (grant 30730030) and the Ministry of Education of China to (grant 706031 to J.Z.).

P.C. and C.L. contributed equally to this study.

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