Diurnal-and sex-related difference of metallothionein expression in mice
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 9 June 2012
Accepted: 16 July 2012
Published: 24 July 2012
Metallothionein (MT) is a small, cysteine-rich, metal-binding protein that plays an important role in protecting against toxicity of heavy metal and chemicals. This study was aimed to define diurnal and sex variation of MT in mice.
Adult mice were maintained in light- and temperature-controlled facilities for 2 weeks with light on at 8:00 and light off at 20:00. The blood, liver, and kidneys were collected every 4 h during the 24 h period. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis and MT protein was determined by western blot and the Cd/hemoglobin assay.
The diurnal variations in mRNA levels of MT-1 and MT-2in liver were dramatic, up to a 40-foldpeak/trough ratio. MT mRNA levels in kidneys and blood also showed diurnal variation, up to 5-fold peak/trough ratio. The diurnal variation of MT mRNAs resembled the clock gene albumin site D-binding protein (Dbp), and was anti-phase to the clock gene Brain and Muscle ARNT-like Protein 1 (Bmal1) in liver and kidneys. The peaks of MT mRNA levels were higher in females than in males. Hepatic MT protein followed a similar pattern, with about a 3-fold difference.
MT mRNA levels and protein showed diurnal- and sex-variation in liver, kidney, and blood of mice, which could impact the body defense against toxic stimuli.
Metallothionein (MT) was discovered in 1957 from equine kidney, and was characterized by its metal-binding capacity and high sulfhydryl content . MT has four isoforms termed MT-1, MT-2, MT-3 and MT-4 and is ubiquitous in mammals . MT has been shown to have multiple functions such as in the homeostasis of tissue copper and zinc, in the detoxification of heavy metals, in the modulation of immune function, and in the scavenge of free radicals .
When cadmium was administered during the dark phase of the daily cycle, the kidneys and liver contained more cadmium and higher levels of MT . It has been shown that circadian clock-controlled genes in Neurosporacrassa are late-night to early morning specific. One of them, ccg-12, encodes copper metallothionein . Two reports indicate that cadmium-induced mortality showed a circadian variation in mice [6, 7]. The circadian variation to cadmium toxicity could be associated with diurnal variation of metallothionein. However, no information on diurnal variation of MT in mammals is available. This study was initiated to fill that gap.
We have recently demonstrated the circadian variations in hepatic antioxidant components including the Nrf2 pathways, glutathione systems, and antioxidant enzymes in male and female mice , thus, it would be of interest to examine whether the diurnal variations exist for the basal levels of MT mRNA. We firstly verified the circadian rhythms of 3 clock genes, followed by examination of diurnal variations of the major MT isoforms (MT-1 and MT-2) in major organs (liver, kidney, and blood) of Kunming mice. The results revealed for the first-time a dramatic diurnal variation of the MT mRNA, which could have important pharmacological and toxicological significance.
Materials and Methods
Male and female adult Kunming (KM) mice  (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.
RNA Isolation and Real-time RT-PCR analysis
Primer sequence for real-time RT-PCR analysis
Hepatic MT protein determination
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).
Western blot analysis
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 . 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.
Diurnal variations of clock gene Bmal1 and Dbp mRNA
Diurnal variations of hepatic MT-1 mRNA in the liver and kidney
Diurnal variations of MT-2 mRNA in liver and kidney
Diurnal variations of MT-2 and Cry1 mRNA in blood
Diurnal variations of hepatic MT protein
Sex differences of clock and MT mRNA expression in liver, kidney and blood
The present study depicts the diurnal variations of MT during a 24 h period in liver (40-fold), kidney (7-fold), and blood (7-fold) of KM mice. The diurnal variations of MT genes resemble the clock genes Dbp and Cry1, but were antiphase to Bmal1. MT protein also showed diurnal variation, but to a lesser extent (3-fold) than mRNA. In general, female mice had higher MT mRNA levels than males. Similar findings were also observed in the inbred male C57BL/6 mouse liver (data not shown), fortifying this phenomenon.
Circadian rhythms in mammals are under control of a rhythm generator located in the hypothalamic suprachiasmatic nuclei (SCN) [12, 13]. At the cellular level, circadian rhythms are driven by the transcription/translation-based negative feedback loops regulating the cyclic expression of clock genes (such as Cry1, Bmal1, Dbp, etc.) . Our study verified he rhythms of the above 3 classic clock genes (Bmal1, Dbp and Cy1) in KM mice (Figures 1, 3, and 6), and the patterns of their expression were in agreement with the literature . The clock gene expression pattern verifications validate the further examination of diurnal variations of metallothionein.
The liver is the major organ of drug metabolism and detoxification. MT acts as a part of the antioxidant defense mechanism against oxidative damage caused by toxic stimuli [3, 16]. Mice deficient in MT were more susceptible than wild-type mice to hepatotoxicity of cadmium, CCl4, acetaminophen , arsenic , and rifampicin . These data demonstrate that MT plays an important role in the defense against toxicity stimuli. In contrast, down-regulation of MT has been reported in hepatocellular carcinoma [19, 20]. Thus, to define the circadian variations of MT is of toxicological and clinical significance. To our knowledge, this is the first report on circadian variation of MT in livers of mice.
It has long been known that kidney is a target organ of cadmium and long-term Cd exposure causes renal tubular dysfunction in occupational exposed and the general population . Renal cadmium is detoxified by MT in the kidney [3, 16], and the present study also showed circadian variation of MT in the kidney. This could impact not only Cd nephrotoxicity , but also other nephrotoxicants as well. MT has been implicated to play important roles in protecting against oxidative stress induced not only by cadmium , but also by organic chemicals such as adriamycin .
Cd-induced sensitivity to Paramecium tetraurelia also showed diurnal-variations . Whether the circadian variations of organisms to cadmium are due to MT requires further investigation. The circadian variations of MT gene expression could influence the sensitivity of mammals to environmental chemicals, but also to oxidative stress.
The blood MT-mRNA could be used as a sensitive biomarker for exposure to cadmium , arsenic intoxication  and several other heavy metals . The MT levels in blood can be considered as a promising biomarker for diagnosis, prognosis and therapeutic efficiency evaluation of childhood tumors . The potential role of MT as a prostate cancer marker has been proposed [29, 30]. This study depicts that mRNA levels of MT in blood also showed a diurnal variation, with a peak at 14:00,and peak/trough differences up to 7.8-fold. Thus it is important to realize for blood sample collection at designated times of the day to avoid circadian variations in MT gene expression when using MT mRNA as a biomarker.
In summary, these results demonstrate dramatic diurnal variations of MT-1 and MT-2gene expression in liver, kidney, and blood of KM mice. The diurnal variations in MT mRNA could be an important factor affecting the magnitude and progression in responses to toxic stimuli.
This study was supported by the Science and Technology Foundation of Guizhou (No. 2008–002, 2009–70019 and 2010–5) and Foundation of Zunyi Medical College.
- Margoshoes M, Vallee BL: Cadmium protein from equine kidney cortex. J Am Chem Soc 1957, 79:4813–4814.View ArticleGoogle Scholar
- Thirumoorthy N, Shyam Sunder A, Manisenthil Kumar K, Senthil Kumar M, Ganesh G, Chatterjee M: A review of metallothionein isoforms and their role in pathophysiology. World J Surg Oncol 2011, 9:54–61.PubMedView ArticleGoogle Scholar
- Klaassen CD, Liu J, Diwan BA: Metallothionein protection of cadmium toxicity. Toxicol Appl Pharmacol 2009, 238:215–220.PubMedView ArticleGoogle Scholar
- Cahill AL, Nyberg D, Ehret CF: Tissue distribution of cadmium and metallothionein as a function of time of day and dosag. Environ Res 1983, 31:54–65.PubMedView ArticleGoogle Scholar
- Bell-Pedersen D, Shinohara ML, Loros JJ, Dunlap JC: Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific. Proc Natl Acad Sci USA 1986, 93:13096–13101.View ArticleGoogle Scholar
- Cambar J, Cal JC, Desmouliere A, Guillemain J: Circadian variations of the mortality of mice due to cadmium sulfate. CR Seances Acad Sci III 1983, 296:949–952.Google Scholar
- Miura N, Yanagiba Y, Ohtani K, Mita M, Togawa M, Hasegawa T: Diurnal variation of cadmium-induced mortality in mice. J Toxicol Sci 2012, 37:191–196.PubMedView ArticleGoogle Scholar
- Xu YQ, Zhang D, Jin T, Cai DJ, Wu Q, Lu YF, Liu J, Klaassen CD: Diurnal variation of hepatic antioxidant components in mice. PLoS One (under revision).Google Scholar
- Lu YF, Wu Q, Liang SX, Miao JW, Shi JS, Liu J: Evaluation of hepatotoxicity potential of cinnabar-containing An-Gong-Niu-Huang Wan, a patent traditional Chinese medicine. Regul Toxicol Pharmacol 2011, 60:206–211.PubMedView ArticleGoogle Scholar
- Refinetti R, Cornélissen G, Halberg F: Procedures for numerical analysis of circadian rhythms. Biol Rhythm Res 2007, 38:235–325.View ArticleGoogle Scholar
- Kägi JH: Overview of metallothionein. Methods Enzymol 1991, 205:613–626.PubMedView ArticleGoogle Scholar
- Lincoln GA, Clarke IJ, Hut RA, Hazlerigg DG: Characterizing a mammalian circannual pacemaker. Science 2006, 314:1941–1944.PubMedView ArticleGoogle Scholar
- Dibner C, Schibler U, Albrecht U: The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol 2010, 72:517–549.PubMedView ArticleGoogle Scholar
- Levi F, Okyar A, Dulong S, Innominato PF, Clairambault J: Circadian timing in cancer treatments. Annu Rev Pharmacol Toxicol 2010, 50:377–421.PubMedView ArticleGoogle Scholar
- Zhang YK, Yeager RL, Klaassen CD: Circadian expression profiles of drug-processing genes and transcription factors in mouse liver. Drug Metab Dispos 2009, 37:106–115.PubMedView ArticleGoogle Scholar
- Klaassen CD, Liu J, Choudhuri S: Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 1999, 39:267–294.PubMedView ArticleGoogle Scholar
- Liu J, Liu Y, Goyer RA, Achanzar W, Waalkes MP: Metallothionein-I/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of chronic oral or injected inorganic arsenicals. Toxicol Sci 2000, 55:460–467.PubMedView ArticleGoogle Scholar
- Lian Y, Xu PY, Ouyang ZH, Zhao J, Wang YM, Peng SQ: Effects of metallothionein on rifampicin (RFP)-induced hepatotoxicity in mice. Sichuan Da Xue Xue Bao Yi Xue Ban 2010, 41:609–612–647.PubMedGoogle Scholar
- Huang GW, Yang LY: Metallothionein expression in hepatocellular carcinoma. World J Gastroenterol 2002, 8:650–653.PubMedGoogle Scholar
- Tao X, Zheng JM, Xu AM, Chen XF, Zhang SH: Downregulated expression of metallothionein and its clinicopathological significance in hepatocellular carcinoma. Hepatol Res 2007, 37:820–827.PubMedView ArticleGoogle Scholar
- Jarup L, Berglund M, Elinder CG, Nordberg G, Vahter M: Health effects of cadmium exposure–a review of the literature and a risk estimate. Scand J Work Environ Health 1998, 24:1–51.PubMedView ArticleGoogle Scholar
- Huang M, Choi SJ, Kim DW, Kim NY, Bae HS, Yu SD, Kim DS, Kim H, Choi BS, Yu IJ, Park JD: Evaluation of factors associated with cadmium exposure and kidney function in the general population. Environ Toxicol 2011.Google Scholar
- Kang YJ: Antioxidant defense against anthracycline cardiotoxicity by metallothionein. Cardiovasc Toxicol 2007, 7:95–100.PubMedView ArticleGoogle Scholar
- Hinrichsen RD, Tran JR: A circadian clock regulates sensitivity to cadmium in Paramecium tetraurelia. Cell BiolToxicol. 2010, 26:379–89.Google Scholar
- Chang X, Jin T, Chen L, Nordberg M, Lei L: Metallothionein I isoform mRNA expression in peripheral lymphocytes as a biomarker for occupational cadmium exposure. Exp Biol Med (Maywood) 2009, 234:666–672.View ArticleGoogle Scholar
- Liu J, Cheng ML, Yang Q, Shan KR, Shen J, Zhou Y, Zhang X, Dill AL, Waalkes MP: Blood metallothionein transcript as a biomarker for metal sensitivity: low blood metallothionein transcripts in arsenicosis patients from Guizhou, China. Environ Health Perspect 2007, 115:1101–1106.PubMedView ArticleGoogle Scholar
- Yamada H, Koizumi S: Lymphocyte metallothionein-mRNA as a sensitive biomarker of cadmium exposure. Ind Health 2001, 39:29–32.PubMedView ArticleGoogle Scholar
- Krizkova S, Masarik M, Majzlik P, Kukacka J, Kruseova J, Adam V, Prusa R, Eckschlager T, Stiborova M, Kizek R: Serum metallothionein in newly diagnosed patients with childhood solid tumours. Acta Biochim Pol 2010, 57:561–566.PubMedGoogle Scholar
- Krizkova S, Ryvolova M, Gumulec J, Masarik M, Adam V, Majzlik P, Hubalek J, Provaznik I, Kizek R: Electrophoretic fingerprint metallothionein analysis as a potential prostate cancer biomarker. Electrophoresis 2011, 32:1952–1961.PubMedView ArticleGoogle Scholar
- Gumulec J, Masarik M, Krizkova S, Hlavna M, Babula P, Hrabec R, Rovny A, Masarikova M, Sochor J, Adam V, Eckschlager T, Kizek R: Evaluation of alpha-methylacyl-CoA racemase, metallothionein and prostate specific antigen as prostate cancer prognostic markers. Neoplasma 2012, 5:191–201.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.