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Free Radical Biology & Medicine 44 (2008) 1592 – 1598 www.elsevier.com/locate/freeradbiomed
Original Contribution
Chronic oxidative stress induces a tissue-specific reduction in telomere length in CAST/Ei mice Valerie Cattan a,b,1 , Nathalie Mercier a,b,1 , Jeffrey P. Gardner c , Veronique Regnault b,d , Carlos Labat a,b , Jenni Mäki-Jouppila a,b , Rosine Nzietchueng a,b , Athanase Benetos a,b , Masayuki Kimura c , Abraham Aviv c , Patrick Lacolley a,b,⁎ a
Inserm, U684, 54500 Vandoeuvre-lès-Nancy, France Henri Poincaré University, 54000 Nancy, France University of Medicine and Dentistry of New Jersey, The Center of Human Development and Aging, New Jersey Medical School, NJ, USA d Inserm, U734, 54500 Vandoeuvre-lès-Nancy, France b
c
Received 10 July 2007; revised 17 December 2007; accepted 11 January 2008 Available online 26 January 2008
Abstract We examine whether increased oxidative stress in vivo promotes telomere shortening in CAST/Ei mice. We explored the effects of L-buthionine sulfoximine treatment (BSO) on telomere length. BSO shortened telomere length in white fat, brown fat, skin, tail, and testis in concert with diminished tissue glutathione content, increased tissue carbonyl content, and increased plasma advanced oxidized protein products. Telomerase activity was mainly detected in testis but no reduction of telomerase activity was observed in response to BSO. In conclusion, BSO-mediated increase in systemic oxidative stress shortens telomeres in several tissues of the mouse. The variable effect of BSO treatment on telomere length in different tissue may result from their different adaptive antioxidative capacity. © 2008 Elsevier Inc. All rights reserved. Keywords: Telomere; Mouse; Tissue; Oxidative stress; Glutathione
Introduction The telomere hypothesis of cellular aging proposed that telomere attrition chronicles the replication of cultured somatic cells, a process that is accelerated by oxidative stress [1–3]. Enhanced telomere attrition was observed in cultured cells in response to chronic hyperoxia [4], treatment with homocysteine [5], hydrogen peroxide [6], or inhibitors of the glutathione system [7]. Telomere dynamics (length and attrition rate) is regulated by a host of factors, including the cellular anti-
Abbreviations: AOPP, advanced oxidized protein products; BSO, L-buthionine sulfoximine; LTL, leukocyte telomere length; PCR, polymerase chain reaction; TRF, terminal restriction fragments. ⁎ Corresponding author. Inserm U684, UHP Nancy, Faculté de Médecine, 54500 Vandoeuvre-lès-Nancy, France. Fax: +33 3 83683639. E-mail address: [email protected] (P. Lacolley). 1 Valerie Cattan and Nathalie Mercier contributed equally to this work. 0891-5849/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.01.007
oxidative capacity [8,9]. In vitro mechanisms by which telomeres shorten in response to oxidative stress are still poorly understood [10]. Moreover, telomere-mediated impediment of proliferation and a direct activation of the cell checkpoints to cease replication in response to oxidative stress are not mutually exclusive. In humans, leukocyte telomere length (LTL) is relatively short in a host of aging-related diseases marked by increased oxidative stress [11,12]. In addition, human LTL was found to be inversely correlated to urinary isoprostane, an index of oxidative stress [13,14]—a finding consistent with the premise that oxidative stress accelerates telomere erosion not only in vitro [8,15–17] but also in vivo. However, there has been no experimental evidence showing that a rise in systemic oxidative stress enhances telomere attrition in vivo. The objective of this study was to explore the potential effect of oxidative stress on telomere length through depleting tissue glutathione in vivo. The glutathione redox cycle is a key element
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in the antioxidative arsenal of cells [18]. Therefore, chronic oxidative stress can be produced in vivo by feeding animals with L-buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl cysteine synthase [7]. We tested the effects of BSO treatment on telomere length, tissue glutathione content, and indices of oxidative stress in wild-derived Mus musculus castaneus (CAST/ Ei) mice because telomeres are relatively short in this mouse strain and their mean length can be accurately measured using standard Southern blots. Materials and methods Animal model We used 14-week-old male CAST/Ei mice (n = 26). BSO (30 mM, Sigma Chemical Inc.) was provided in the drinking water at 8 weeks of age for 6 weeks [19]. Nontreated animals served as controls. At the end of the treatment, the heart, kidney, liver, lungs, brain, tibialis anterior muscle, skin, tail, testis, white and brown fat, spleen, and large intestine were immediately removed and frozen in liquid nitrogen. All procedures were in accordance with institutional guidelines for animal experimentation. Blood pressure measurement Carotid artery blood pressure was recorded in pentobarbitalanaesthetized mice as previously described [20]. The pressure measurement was made by using a catheter (0.5 cm of PE-10 fused to 3 cm of PE-50; Clay Adams, Parsippany, NJ) connected to a Statham pressure transducer (P23 Db) and a Gould pressure processor.
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Table 1 Effects of BSO on hemodynamic parameters, GSH contents, and indices of protein oxidation in CAST/Ei mice Control
BSO 30 mM
Hemodynamics Body weight (g) PAS (mm Hg) PAD (mm Hg) PAM (mm Hg) PP (mm Hg) FC (bpm)
15.1 ± 0.6 140 ± 10 112 ± 4 121 ± 5 28 ± 9 568 ± 32
13.4 ± 0.7∗ 140 ± 7 115 ± 7 123 ± 7 25 ± 1 572 ± 21
Total glutathione content (μM/mg of protein) White fat Skin Brain Muscle Kidney Large intestine Heart Liver
0.32 ± 0.11 0.67 ± 0.11 1.27 ± 0.20 0.78 ± 0.08 0.43 ± 0.17 1.27 ± 0.28 2.72 ± 0.27 2.99 ± 0.08
b0.1 0.25 ± 0.04∗ 1.03 ± 0.28 0.15 ± 0.02∗ 0.14 ± 0.02 0.32 ± 0.13∗ 0.18 ± 0.03∗ 0.33 ± 0.06∗
2.5 ± 1.9 2.9 ± 0.7 1.7 ± 0.2 2.0 ± 0.8 2.2 ± 0.5 3.1 ± 0.9 3.0 ± 0.7 5.9 ± 1.0 4.9 ± 1.0 4.1 ± 0.6 68 ± 4
4.8 ± 0.8 5.3 ± 0.3 5.9 ± 0.6∗ 5.7 ± 0.8∗ 7.4 ± 1.3∗ 4.8 ± 1.1 5.7 ± 0.9∗ 13.4 7.0 ± 0.5 9.8 ± 0.2∗ 98 ± 9
Carbonyl content (μmol/mg of protein) Spleen Lung Brain Muscle Testis Kidney Large intestine Tail Heart Liver Advanced oxidized protein products (mM)
Mean ± SEM, n = 7 in each group except for carbonyls in tail of BSO-treated mice (n = 2). * P b 0.001.
Tissue glutathione assay The total glutathione content was measured using a GSH assay kit (Cayman Chemical, Ann Arbor, MI). Tissues were pestled in liquid nitrogen, homogenized in 50 mM MES buffer containing 1 mM EDTA, and deproteinized with metaphosphoric acid (0.1 g/mL). Advanced oxidized protein products (AOPP) and carbonyls AOPP levels were measured in the plasma as previously described [21] and results were expressed as millimolar of chloramine T equivalents. Protein carbonyls in tissues were measured using a commercial kit (Cayman Chemical) and expressed in micromoles per milligram total protein. Measurement of the mean terminal restriction fragment length
Fig. 1. TRF length analysis in 0.3 and 0.5% agarose gels. Left panel (A) shows the high resolution of standards at intermediate molecular weights resolved on 0.3% vs 0.5% agarose gels (B).
Briefly, DNA was extracted from tissues using a standard protocol and checked for integrity on a 0.8% agarose gel. Telomere length values were determined in duplicate by measuring the mean length of the terminal restriction fragments (TRF) by a modification of a method described previously [22]. DNA samples were digested with HinfI and RsaI and electrophoresed on a
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0.3% agarose gel, blotted onto a positively charged nylon membrane, and then hybridized overnight at 65 °C with the telomeric probe [digoxigenin 3′-end-labeled 5′-(CCTAAA)3]. As illustrated in Fig. 1, resolving the TRFs on 0.3% gels (as compared with 0.5% gels) has an excellent resolution for the spectrum of TRF lengths displayed in tissues of the CAST/Ei mouse. The digoxigeninlabeled probe was detected using the digoxigenin luminescent detection procedure (Roche) and exposed on X-ray film.
calculated by subtracting individual muscle TRF lengths from those of other tissues (TRFt-m) in control and BSO-treated mice. Comparisons of ΔTRF values were adjusted for multiplicity using the Bonferroni's method. The level of statistical significance was set at P b 0.05. Results General characteristics
Telomerase activity Telomerase activity was measured using a quantitative telomerase detection kit (US Biomax, Inc.). Briefly, frozen tissues were ground in ice-cold lysis buffer, incubated on ice for 30 min, and centrifuge at 12,000 g for 30 min at 4 °C. The supernatant was used for activity measurement and determination of protein content. The real-time PCR was performed with 0.43 μg of protein. As a negative control, every sample was also tested after heat inactivation. A control template standard curve allows the calculation of the amount of template with telomeric repeat generated by telomerase. Statistical analysis Data are expressed as mean ± SEM. The analysis of the differences in TRF lengths between BSO-treated and control mice (ΔTRF) was performed using ANOVA with treatment, tissue, and their interaction as fixed effects, and mouse nested within treatment as a random effect. The ΔTRFs were tested after adjustment for skeletal muscle [22]. Adjustment was
Compared with control mice, BSO-treated mice exhibited an initial lag in weight gain but no change in hemodynamic parameters, including arterial pressure and heart rate (Table 1). After 20 days of BSO treatment the slope of increase weight with age was similar in experimental and control mice (Fig. 2). When normalized to body weight, food intake did not differ between BSO-treated and control mice. BSO-treated mice also displayed diminished water intake in the first 3 weeks of treatment. BSO treatment caused a significant decrease in total GSH content in liver, muscle, white fat, skin, large intestine, and heart but had no apparent effect in brain (Table 1). BSO treatment significantly increased carbonyl content in brain, muscle, testis, large intestine, and liver and AOPP plasma level (Table 1). TRF lengths Table 2 displays TRF lengths in different tissues of BSOtreated vs control mice. Mean TRFs varied from tissue to tissue, ranging from 11.6 kb (liver) to 17.2 kb (white fat) (P b 0.0001)
Fig. 2. Effects of BSO on weight and food and water intake in CAST/Ei mice. Mean ± SEM, n = 5 in controls and n = 7 in BSO-treated mice.
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Table 2 Effects of BSO on TRF length in CAST/Ei mice TRF length (kb)
Control
BSO 30 mM
White fat Spleen Lung Skin Brain Brown fat Muscle Testis Kidney Large intestine Tail Heart Liver All tissues
17.2 ± 0.4 17.2 ± 0.5 17.1 ± 0.4 16.6 ± 0.5 16.0 ± 0.5 15.9 ± 0.3 15.8 ± 0.6 15.8 ± 0.6 15.7 ± 0.5 15.2 ± 0.7 14.4 ± 0.4 13.2 ± 0.6 11.6 ± 0.3 15.5 ± 0.2
12.7 ± 0.9∗ 17.0 ± 0.2 17.6 ± 0.2 11.9 ± 0.5∗ 15.9 ± 0.3 14.3 ± 0.4∗ 16.3 ± 0.3 14.5 ± 0.3∗ 16.2 ± 0.3 16.0 ± 0.3 10.3 ± 0.3∗ 13.4 ± 0.2 11.8 ± 0.3 14.5 ± 0.3∗
Mean ± SEM, n = 7 in each group. * P b 0.05.
in control mice. BSO treatment significantly reduced the TRF length in testis, brown fat, tail, white fat, and skin (− 1.3 to − 4.6 kb) but not in liver, muscle, kidney, brain, spleen, lung, large intestine, and heart. For all tissues combined, mean TRF length was shortened significantly in BSO-treated compared to untreated CAST/Ei mice. To test for BSO-treated decreases in TRF lengths in proliferating tissues, we examined the difference in TRF lengths between various tissues and a reference skeletal muscle (tibialis anterior) for each mouse (TRFt-m). In this way we were able to circumvent the confounding effect of variations in TRF lengths in tissues within each mouse and among mice [23]. Fig. 3 summarizes the differences in mean adjusted ΔTRF between BSO-treated and control mice for various tissues and all tissues combined. This approach easily differentiated the ΔTRF of BSO-treated vs untreated animals, showing BSO-dependent (P b 0.0001) and tissue (P b 0.0001) effects with highly significant interaction between the two factors (P b 0.0001). Significant telomere shortening in
Fig. 3. Effects of BSO on TRF lengths adjusted to that of muscle in CAST/Ei mice. Adjustment was calculated by subtracting individual muscle TRF lengths from those of other tissues (TRFt-m) in control and BSO-treated mice. Symbols and bars represent the differences between mean-adjusted ΔTRFs and their 95% confidence intervals.
Fig. 4. Relation between oxidative stress and changes in telomere length. (A) Relation between TRF lengths and carbonyl levels in control, (B) in BSOtreated mice, and (C) differences in TRF lengths and differences in carbonyls levels for various tissues between BSO-treated and control mice. Mean ± SEM, n = 3–4 per tissue.
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Fig. 5. Telomerase activity in CAST/Ei mouse tissues. (A) Typical illustrations in muscle and testis. Line 1 shows negative control (heat-inactivated sample); line 2, positive control (telomerase standard reaction); line 3, muscle; and line 4, testis. (B) Effects of BSO on telomerase activity. Lines represent mean values (n = 5 in controls and n = 7 in BSO-treated mice).
BSO-treated mice was demonstrated for testis (P b 0.05), brown fat (P b 0.01), tail (P b 0.001), white fat (P b 0.001), and skin (P b 0.001), as well as for all tissues combined (P b 0.001). We have investigated whether increase in carbonyls was correlated with changes in telomere length. Figs. 4A and B show that there is a negative correlation between carbonyl levels and telomere length both in control (r = − 0.646; P = 0.0438) and in BSO-treated mice (r = − 0.944; P b 0.0001). The slopes were similar between the two groups. These results show that even in control mice there is a relation between carbonyl levels and TRF. Differences in TRF lengths between BSO-treated and control mice (ΔTRF) were negatively correlated with differences in carbonyl levels for various tissues (r = − 0.792; P = 0.0063) (Fig. 4C). Telomerase activity Distribution of telomerase activity is shown in Fig. 5. Telomerase activity was detectable in testis and at a low level in spleen, white fat, and brain. No activity was detected in muscle and liver. There was no significant reduction in telomerase activity in response to BSO. Discussion The central finding of this work is that BSO feeding for 6 weeks caused telomere shortening in vivo in several tissues of the CAST/Ei mouse. BSO decreased telomere length in testis, brown fat, tail, white fat, and skin in CAST/Ei mice. Unlike most mouse strains, which have very long telomeres, the CAST/ Ei strain has relatively short telomeres, though somewhat longer than those of adult humans (8–12 kb) [22]. The relatively short
telomere length in this strain makes it more suitable to study telomere dynamics in vivo. Oxidative stress heightens telomere attrition in cultured somatic cells [3] and a recent study showed that BSO treatment accelerated telomere attrition in cultured endothelial cells [7]. Based on our findings in the mouse, BSO also enhances telomere attrition in vivo. Though BSO treatment did not shorten telomeres in largely postmitotic tissues, such as skeletal muscle, heart, and brain, it also did not have an appreciable effect on telomere length in proliferative tissues such as spleen, lung, large intestine, and liver. The reason for this finding is unclear. It should be noted that among proliferative tissues, there are still considerable variations in the proliferative index. For instance, the proliferative index of the intestine is high while that of the liver is relatively low. It is noteworthy that proliferative tissues often contain cellular elements that are considered poorly proliferative, while traditionally nonproliferative tissues contain proliferative cells. For instance, the mucosa of the intestine is highly proliferative while the intestinal smooth muscle is poorly proliferative. Brain tissue is primarily nonproliferative, but glial cells do divide in vivo. One explanation for our finding is that our method was insufficiently sensitive to capture small effects of BSO treatment on TRF length in several tissues. We note, however, that the intraassay coefficient of variation of our method was 2%, which would enable a detection of a TRF length difference as low as 0.3 kb. We also examined whether GSH depletion alters the distribution of telomere lengths in the tissues in which no change in mean TRF length was observed. TRF length distribution has been previously compared with mean TRF length [23,24]. We observed no evidence for significant differences in either the short or long telomeres in tissues that showed no effect of BSO
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(data not shown). When observed, the effect of BSO treatment on the TRF length was not due to the reconfiguration of the TRF distribution, meaning that all telomeres equally displayed proportional shortening. The proliferative tissues that did not display an apparent reduction in telomere length in BSO-treated mice might have increased antioxidative capacity (compared with other tissues) or resorted to other mechanisms that shielded telomeres from the damaging effect of oxidative stress. In this context, longer telomere length in renal heart and liver tissues from female than male rats has been attributed to increased antioxidant capacity in the females [25]. Glutathione was significantly decreased in 7 out of 8 tissues in which its content was measured. We attribute the nonsignificant drop in brain glutathione content to the blood brain barrier [26]. Thus, if a decline in tissue glutathione directly caused a rise in oxidative stress, such an effect was less evident in the brain. However, the impact of the decline in tissue glutathione levels might still affect the systemic burden of oxidative stress (including the brain) due to the spillover of free radicals to the circulation. Support for this contention was provided by the increased levels of plasma AOPP in BSO-treated mice. We also found increases in the levels of protein carbonyls in various tissues. Protein carbonyls and AOPP were reported to be reliable indices of the oxidative stress state in response to BSO in vitro and in vivo [7]. In an attempt to describe the relation between oxidative stress and telomere shortening, we found that differences in TRF lengths between BSO-treated and control mice were negatively correlated with differences in carbonyl levels for various tissues (Fig. 4). These data suggest that oxidative stress, provoked by glutathione depletion, accelerates telomere shortening in mice. Our results showed that the telomerase activity was limited to a subset of tissues, with considerable activity in the testis, which harbor the germ line. Telomerase activity has been reported in different mouse strains [27–29], but to our knowledge, there are no data in CAST/Ei mice. In this strain, we show no reduction in telomerase activity in response to BSO. This result is not surprising since CAST/Ei mice have short telomeres and because low telomerase activities have been reported in mouse normal tissues whatever the strains. Therefore, the present results do not support a major role for telomerase activity in telomere shortening. Increased oxidative stress in BSO-treated CAST/Ei mice was not associated with systolic hypertension as reported previously in C57BL/6NCrl mice and Sprague Dawley rats receiving 13.5 and 30 mM BSO, respectively [19,30]. This might relate to differences between species or mouse strains [31]. We note in this regard that we did observe a blood pressure raising effect of BSO treatment in C57BL/6NCrl (unpublished data). The telomerase knockout C57BL/6 mouse with short telomeres was found to be hypertensive, presumably due to an increase in plasma endothelin-1 level [32]. These findings pointed to a link between telomerase deficiency and hypertension. Activation of NADPH oxidase and increased production of reactive oxygen species have been demonstrated in telomerase-deficient cells [32]. Overall, though, it is doubtful that short telomeres per se
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are the cause of the increased basal blood pressure observed in CAST/Ei mice. Finally, we note that BSO-treated animals were lighter than controls, which may have resulted from poor caloric intake or metabolic impairments associated with glutathione depletion in various tissues. The lag in growth in the first 3 weeks of treatment was not due to a decrease in food intake because when normalized to body weight, food intake did not differ between the two groups throughout the study. Water intake followed the same pattern. However, we cannot exclude that increased energetic expenditure occurs in the later phase in response to GSH depletion. We have also observed a marked white fat atrophy (data not shown), associated with considerable telomere length reduction. This atrophy might have resulted from reduced replicative potential of fat cells, BSO-induced insulin resistance, or diminished tissue viability, as previously suggested [33,34]. In conclusion, our results indicate that the BSO-treated CAST/Ei mouse is a useful model for exploring a causal relation between telomere dynamics and oxidative stress in vivo. Acknowledgments Grant support is from Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, and Region Lorraine. We thank Jean-Pierre Max (Inserm U734, Nancy), Gael Poitevin (Inserm U684, Nancy), Michel Thiery (CNRS, UMR 7561, Nancy) and Renaud Fay (CIC 95-01, Nancy) for help in evaluation of metabolic parameters, telomerase activity measurements, animal housing and statistical analysis. We thank Dr. Gillian Butler-Browne (Inserm 787, Paris) for helpful discussions on telomerase activity. References [1] Harley, C. B.; Vaziri, H.; Counter, C. M.; Allsopp, R. C. The telomere hypothesis of cellular aging. Exp. Gerontol. 27:375–382; 1992. [2] Blackburn, E. H. Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 579:859–862; 2005. [3] von Zglinicki, T. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27:339–344; 2002. [4] von Zglinicki, T.; Saretzki, G.; Docke, W.; Lotze, C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp. Cell Res. 220:186–193; 1995. [5] Xu, D.; Neville, R.; Finkel, T. Homocysteine accelerates endothelial cell senescence. FEBS Lett. 470:20–24; 2000. [6] Duan, J.; Duan, J.; Zhang, Z.; Tong, T. Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening. Int. J. Biochem. Cell Biol. 37:1407–1420; 2005. [7] Kurz, D. J.; Decary, S.; Hong, Y.; Trivier, E.; Akhmedov, A.; Erusalimsky, J. D. Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J. Cell. Sci. 117:2417–2426; 2004. [8] Serra, V.; von Zglinicki, T.; Lorenz, M.; Saretzki, G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J. Biol. Chem. 278:6824–6830; 2003. [9] Kimura, M.; Cao, X.; Skurnick, J.; Cody, M.; Soteropoulos, P.; Aviv, A. Proliferation dynamics in cultured skin fibroblasts from Down syndrome subjects. Free Radic. Biol. Med. 39:374–380; 2005.
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Free Radical Biology & Medicine 44 (2008) 1592 – 1598 www.elsevier.com/locate/freeradbiomed
Original Contribution
Chronic oxidative stress induces a tissue-specific reduction in telomere length in CAST/Ei mice Valerie Cattan a,b,1 , Nathalie Mercier a,b,1 , Jeffrey P. Gardner c , Veronique Regnault b,d , Carlos Labat a,b , Jenni Mäki-Jouppila a,b , Rosine Nzietchueng a,b , Athanase Benetos a,b , Masayuki Kimura c , Abraham Aviv c , Patrick Lacolley a,b,⁎ a
Inserm, U684, 54500 Vandoeuvre-lès-Nancy, France Henri Poincaré University, 54000 Nancy, France University of Medicine and Dentistry of New Jersey, The Center of Human Development and Aging, New Jersey Medical School, NJ, USA d Inserm, U734, 54500 Vandoeuvre-lès-Nancy, France b
c
Received 10 July 2007; revised 17 December 2007; accepted 11 January 2008 Available online 26 January 2008
Abstract We examine whether increased oxidative stress in vivo promotes telomere shortening in CAST/Ei mice. We explored the effects of L-buthionine sulfoximine treatment (BSO) on telomere length. BSO shortened telomere length in white fat, brown fat, skin, tail, and testis in concert with diminished tissue glutathione content, increased tissue carbonyl content, and increased plasma advanced oxidized protein products. Telomerase activity was mainly detected in testis but no reduction of telomerase activity was observed in response to BSO. In conclusion, BSO-mediated increase in systemic oxidative stress shortens telomeres in several tissues of the mouse. The variable effect of BSO treatment on telomere length in different tissue may result from their different adaptive antioxidative capacity. © 2008 Elsevier Inc. All rights reserved. Keywords: Telomere; Mouse; Tissue; Oxidative stress; Glutathione
Introduction The telomere hypothesis of cellular aging proposed that telomere attrition chronicles the replication of cultured somatic cells, a process that is accelerated by oxidative stress [1–3]. Enhanced telomere attrition was observed in cultured cells in response to chronic hyperoxia [4], treatment with homocysteine [5], hydrogen peroxide [6], or inhibitors of the glutathione system [7]. Telomere dynamics (length and attrition rate) is regulated by a host of factors, including the cellular anti-
Abbreviations: AOPP, advanced oxidized protein products; BSO, L-buthionine sulfoximine; LTL, leukocyte telomere length; PCR, polymerase chain reaction; TRF, terminal restriction fragments. ⁎ Corresponding author. Inserm U684, UHP Nancy, Faculté de Médecine, 54500 Vandoeuvre-lès-Nancy, France. Fax: +33 3 83683639. E-mail address: [email protected] (P. Lacolley). 1 Valerie Cattan and Nathalie Mercier contributed equally to this work. 0891-5849/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.01.007
oxidative capacity [8,9]. In vitro mechanisms by which telomeres shorten in response to oxidative stress are still poorly understood [10]. Moreover, telomere-mediated impediment of proliferation and a direct activation of the cell checkpoints to cease replication in response to oxidative stress are not mutually exclusive. In humans, leukocyte telomere length (LTL) is relatively short in a host of aging-related diseases marked by increased oxidative stress [11,12]. In addition, human LTL was found to be inversely correlated to urinary isoprostane, an index of oxidative stress [13,14]—a finding consistent with the premise that oxidative stress accelerates telomere erosion not only in vitro [8,15–17] but also in vivo. However, there has been no experimental evidence showing that a rise in systemic oxidative stress enhances telomere attrition in vivo. The objective of this study was to explore the potential effect of oxidative stress on telomere length through depleting tissue glutathione in vivo. The glutathione redox cycle is a key element
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in the antioxidative arsenal of cells [18]. Therefore, chronic oxidative stress can be produced in vivo by feeding animals with L-buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl cysteine synthase [7]. We tested the effects of BSO treatment on telomere length, tissue glutathione content, and indices of oxidative stress in wild-derived Mus musculus castaneus (CAST/ Ei) mice because telomeres are relatively short in this mouse strain and their mean length can be accurately measured using standard Southern blots. Materials and methods Animal model We used 14-week-old male CAST/Ei mice (n = 26). BSO (30 mM, Sigma Chemical Inc.) was provided in the drinking water at 8 weeks of age for 6 weeks [19]. Nontreated animals served as controls. At the end of the treatment, the heart, kidney, liver, lungs, brain, tibialis anterior muscle, skin, tail, testis, white and brown fat, spleen, and large intestine were immediately removed and frozen in liquid nitrogen. All procedures were in accordance with institutional guidelines for animal experimentation. Blood pressure measurement Carotid artery blood pressure was recorded in pentobarbitalanaesthetized mice as previously described [20]. The pressure measurement was made by using a catheter (0.5 cm of PE-10 fused to 3 cm of PE-50; Clay Adams, Parsippany, NJ) connected to a Statham pressure transducer (P23 Db) and a Gould pressure processor.
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Table 1 Effects of BSO on hemodynamic parameters, GSH contents, and indices of protein oxidation in CAST/Ei mice Control
BSO 30 mM
Hemodynamics Body weight (g) PAS (mm Hg) PAD (mm Hg) PAM (mm Hg) PP (mm Hg) FC (bpm)
15.1 ± 0.6 140 ± 10 112 ± 4 121 ± 5 28 ± 9 568 ± 32
13.4 ± 0.7∗ 140 ± 7 115 ± 7 123 ± 7 25 ± 1 572 ± 21
Total glutathione content (μM/mg of protein) White fat Skin Brain Muscle Kidney Large intestine Heart Liver
0.32 ± 0.11 0.67 ± 0.11 1.27 ± 0.20 0.78 ± 0.08 0.43 ± 0.17 1.27 ± 0.28 2.72 ± 0.27 2.99 ± 0.08
b0.1 0.25 ± 0.04∗ 1.03 ± 0.28 0.15 ± 0.02∗ 0.14 ± 0.02 0.32 ± 0.13∗ 0.18 ± 0.03∗ 0.33 ± 0.06∗
2.5 ± 1.9 2.9 ± 0.7 1.7 ± 0.2 2.0 ± 0.8 2.2 ± 0.5 3.1 ± 0.9 3.0 ± 0.7 5.9 ± 1.0 4.9 ± 1.0 4.1 ± 0.6 68 ± 4
4.8 ± 0.8 5.3 ± 0.3 5.9 ± 0.6∗ 5.7 ± 0.8∗ 7.4 ± 1.3∗ 4.8 ± 1.1 5.7 ± 0.9∗ 13.4 7.0 ± 0.5 9.8 ± 0.2∗ 98 ± 9
Carbonyl content (μmol/mg of protein) Spleen Lung Brain Muscle Testis Kidney Large intestine Tail Heart Liver Advanced oxidized protein products (mM)
Mean ± SEM, n = 7 in each group except for carbonyls in tail of BSO-treated mice (n = 2). * P b 0.001.
Tissue glutathione assay The total glutathione content was measured using a GSH assay kit (Cayman Chemical, Ann Arbor, MI). Tissues were pestled in liquid nitrogen, homogenized in 50 mM MES buffer containing 1 mM EDTA, and deproteinized with metaphosphoric acid (0.1 g/mL). Advanced oxidized protein products (AOPP) and carbonyls AOPP levels were measured in the plasma as previously described [21] and results were expressed as millimolar of chloramine T equivalents. Protein carbonyls in tissues were measured using a commercial kit (Cayman Chemical) and expressed in micromoles per milligram total protein. Measurement of the mean terminal restriction fragment length
Fig. 1. TRF length analysis in 0.3 and 0.5% agarose gels. Left panel (A) shows the high resolution of standards at intermediate molecular weights resolved on 0.3% vs 0.5% agarose gels (B).
Briefly, DNA was extracted from tissues using a standard protocol and checked for integrity on a 0.8% agarose gel. Telomere length values were determined in duplicate by measuring the mean length of the terminal restriction fragments (TRF) by a modification of a method described previously [22]. DNA samples were digested with HinfI and RsaI and electrophoresed on a
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0.3% agarose gel, blotted onto a positively charged nylon membrane, and then hybridized overnight at 65 °C with the telomeric probe [digoxigenin 3′-end-labeled 5′-(CCTAAA)3]. As illustrated in Fig. 1, resolving the TRFs on 0.3% gels (as compared with 0.5% gels) has an excellent resolution for the spectrum of TRF lengths displayed in tissues of the CAST/Ei mouse. The digoxigeninlabeled probe was detected using the digoxigenin luminescent detection procedure (Roche) and exposed on X-ray film.
calculated by subtracting individual muscle TRF lengths from those of other tissues (TRFt-m) in control and BSO-treated mice. Comparisons of ΔTRF values were adjusted for multiplicity using the Bonferroni's method. The level of statistical significance was set at P b 0.05. Results General characteristics
Telomerase activity Telomerase activity was measured using a quantitative telomerase detection kit (US Biomax, Inc.). Briefly, frozen tissues were ground in ice-cold lysis buffer, incubated on ice for 30 min, and centrifuge at 12,000 g for 30 min at 4 °C. The supernatant was used for activity measurement and determination of protein content. The real-time PCR was performed with 0.43 μg of protein. As a negative control, every sample was also tested after heat inactivation. A control template standard curve allows the calculation of the amount of template with telomeric repeat generated by telomerase. Statistical analysis Data are expressed as mean ± SEM. The analysis of the differences in TRF lengths between BSO-treated and control mice (ΔTRF) was performed using ANOVA with treatment, tissue, and their interaction as fixed effects, and mouse nested within treatment as a random effect. The ΔTRFs were tested after adjustment for skeletal muscle [22]. Adjustment was
Compared with control mice, BSO-treated mice exhibited an initial lag in weight gain but no change in hemodynamic parameters, including arterial pressure and heart rate (Table 1). After 20 days of BSO treatment the slope of increase weight with age was similar in experimental and control mice (Fig. 2). When normalized to body weight, food intake did not differ between BSO-treated and control mice. BSO-treated mice also displayed diminished water intake in the first 3 weeks of treatment. BSO treatment caused a significant decrease in total GSH content in liver, muscle, white fat, skin, large intestine, and heart but had no apparent effect in brain (Table 1). BSO treatment significantly increased carbonyl content in brain, muscle, testis, large intestine, and liver and AOPP plasma level (Table 1). TRF lengths Table 2 displays TRF lengths in different tissues of BSOtreated vs control mice. Mean TRFs varied from tissue to tissue, ranging from 11.6 kb (liver) to 17.2 kb (white fat) (P b 0.0001)
Fig. 2. Effects of BSO on weight and food and water intake in CAST/Ei mice. Mean ± SEM, n = 5 in controls and n = 7 in BSO-treated mice.
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Table 2 Effects of BSO on TRF length in CAST/Ei mice TRF length (kb)
Control
BSO 30 mM
White fat Spleen Lung Skin Brain Brown fat Muscle Testis Kidney Large intestine Tail Heart Liver All tissues
17.2 ± 0.4 17.2 ± 0.5 17.1 ± 0.4 16.6 ± 0.5 16.0 ± 0.5 15.9 ± 0.3 15.8 ± 0.6 15.8 ± 0.6 15.7 ± 0.5 15.2 ± 0.7 14.4 ± 0.4 13.2 ± 0.6 11.6 ± 0.3 15.5 ± 0.2
12.7 ± 0.9∗ 17.0 ± 0.2 17.6 ± 0.2 11.9 ± 0.5∗ 15.9 ± 0.3 14.3 ± 0.4∗ 16.3 ± 0.3 14.5 ± 0.3∗ 16.2 ± 0.3 16.0 ± 0.3 10.3 ± 0.3∗ 13.4 ± 0.2 11.8 ± 0.3 14.5 ± 0.3∗
Mean ± SEM, n = 7 in each group. * P b 0.05.
in control mice. BSO treatment significantly reduced the TRF length in testis, brown fat, tail, white fat, and skin (− 1.3 to − 4.6 kb) but not in liver, muscle, kidney, brain, spleen, lung, large intestine, and heart. For all tissues combined, mean TRF length was shortened significantly in BSO-treated compared to untreated CAST/Ei mice. To test for BSO-treated decreases in TRF lengths in proliferating tissues, we examined the difference in TRF lengths between various tissues and a reference skeletal muscle (tibialis anterior) for each mouse (TRFt-m). In this way we were able to circumvent the confounding effect of variations in TRF lengths in tissues within each mouse and among mice [23]. Fig. 3 summarizes the differences in mean adjusted ΔTRF between BSO-treated and control mice for various tissues and all tissues combined. This approach easily differentiated the ΔTRF of BSO-treated vs untreated animals, showing BSO-dependent (P b 0.0001) and tissue (P b 0.0001) effects with highly significant interaction between the two factors (P b 0.0001). Significant telomere shortening in
Fig. 3. Effects of BSO on TRF lengths adjusted to that of muscle in CAST/Ei mice. Adjustment was calculated by subtracting individual muscle TRF lengths from those of other tissues (TRFt-m) in control and BSO-treated mice. Symbols and bars represent the differences between mean-adjusted ΔTRFs and their 95% confidence intervals.
Fig. 4. Relation between oxidative stress and changes in telomere length. (A) Relation between TRF lengths and carbonyl levels in control, (B) in BSOtreated mice, and (C) differences in TRF lengths and differences in carbonyls levels for various tissues between BSO-treated and control mice. Mean ± SEM, n = 3–4 per tissue.
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Fig. 5. Telomerase activity in CAST/Ei mouse tissues. (A) Typical illustrations in muscle and testis. Line 1 shows negative control (heat-inactivated sample); line 2, positive control (telomerase standard reaction); line 3, muscle; and line 4, testis. (B) Effects of BSO on telomerase activity. Lines represent mean values (n = 5 in controls and n = 7 in BSO-treated mice).
BSO-treated mice was demonstrated for testis (P b 0.05), brown fat (P b 0.01), tail (P b 0.001), white fat (P b 0.001), and skin (P b 0.001), as well as for all tissues combined (P b 0.001). We have investigated whether increase in carbonyls was correlated with changes in telomere length. Figs. 4A and B show that there is a negative correlation between carbonyl levels and telomere length both in control (r = − 0.646; P = 0.0438) and in BSO-treated mice (r = − 0.944; P b 0.0001). The slopes were similar between the two groups. These results show that even in control mice there is a relation between carbonyl levels and TRF. Differences in TRF lengths between BSO-treated and control mice (ΔTRF) were negatively correlated with differences in carbonyl levels for various tissues (r = − 0.792; P = 0.0063) (Fig. 4C). Telomerase activity Distribution of telomerase activity is shown in Fig. 5. Telomerase activity was detectable in testis and at a low level in spleen, white fat, and brain. No activity was detected in muscle and liver. There was no significant reduction in telomerase activity in response to BSO. Discussion The central finding of this work is that BSO feeding for 6 weeks caused telomere shortening in vivo in several tissues of the CAST/Ei mouse. BSO decreased telomere length in testis, brown fat, tail, white fat, and skin in CAST/Ei mice. Unlike most mouse strains, which have very long telomeres, the CAST/ Ei strain has relatively short telomeres, though somewhat longer than those of adult humans (8–12 kb) [22]. The relatively short
telomere length in this strain makes it more suitable to study telomere dynamics in vivo. Oxidative stress heightens telomere attrition in cultured somatic cells [3] and a recent study showed that BSO treatment accelerated telomere attrition in cultured endothelial cells [7]. Based on our findings in the mouse, BSO also enhances telomere attrition in vivo. Though BSO treatment did not shorten telomeres in largely postmitotic tissues, such as skeletal muscle, heart, and brain, it also did not have an appreciable effect on telomere length in proliferative tissues such as spleen, lung, large intestine, and liver. The reason for this finding is unclear. It should be noted that among proliferative tissues, there are still considerable variations in the proliferative index. For instance, the proliferative index of the intestine is high while that of the liver is relatively low. It is noteworthy that proliferative tissues often contain cellular elements that are considered poorly proliferative, while traditionally nonproliferative tissues contain proliferative cells. For instance, the mucosa of the intestine is highly proliferative while the intestinal smooth muscle is poorly proliferative. Brain tissue is primarily nonproliferative, but glial cells do divide in vivo. One explanation for our finding is that our method was insufficiently sensitive to capture small effects of BSO treatment on TRF length in several tissues. We note, however, that the intraassay coefficient of variation of our method was 2%, which would enable a detection of a TRF length difference as low as 0.3 kb. We also examined whether GSH depletion alters the distribution of telomere lengths in the tissues in which no change in mean TRF length was observed. TRF length distribution has been previously compared with mean TRF length [23,24]. We observed no evidence for significant differences in either the short or long telomeres in tissues that showed no effect of BSO
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(data not shown). When observed, the effect of BSO treatment on the TRF length was not due to the reconfiguration of the TRF distribution, meaning that all telomeres equally displayed proportional shortening. The proliferative tissues that did not display an apparent reduction in telomere length in BSO-treated mice might have increased antioxidative capacity (compared with other tissues) or resorted to other mechanisms that shielded telomeres from the damaging effect of oxidative stress. In this context, longer telomere length in renal heart and liver tissues from female than male rats has been attributed to increased antioxidant capacity in the females [25]. Glutathione was significantly decreased in 7 out of 8 tissues in which its content was measured. We attribute the nonsignificant drop in brain glutathione content to the blood brain barrier [26]. Thus, if a decline in tissue glutathione directly caused a rise in oxidative stress, such an effect was less evident in the brain. However, the impact of the decline in tissue glutathione levels might still affect the systemic burden of oxidative stress (including the brain) due to the spillover of free radicals to the circulation. Support for this contention was provided by the increased levels of plasma AOPP in BSO-treated mice. We also found increases in the levels of protein carbonyls in various tissues. Protein carbonyls and AOPP were reported to be reliable indices of the oxidative stress state in response to BSO in vitro and in vivo [7]. In an attempt to describe the relation between oxidative stress and telomere shortening, we found that differences in TRF lengths between BSO-treated and control mice were negatively correlated with differences in carbonyl levels for various tissues (Fig. 4). These data suggest that oxidative stress, provoked by glutathione depletion, accelerates telomere shortening in mice. Our results showed that the telomerase activity was limited to a subset of tissues, with considerable activity in the testis, which harbor the germ line. Telomerase activity has been reported in different mouse strains [27–29], but to our knowledge, there are no data in CAST/Ei mice. In this strain, we show no reduction in telomerase activity in response to BSO. This result is not surprising since CAST/Ei mice have short telomeres and because low telomerase activities have been reported in mouse normal tissues whatever the strains. Therefore, the present results do not support a major role for telomerase activity in telomere shortening. Increased oxidative stress in BSO-treated CAST/Ei mice was not associated with systolic hypertension as reported previously in C57BL/6NCrl mice and Sprague Dawley rats receiving 13.5 and 30 mM BSO, respectively [19,30]. This might relate to differences between species or mouse strains [31]. We note in this regard that we did observe a blood pressure raising effect of BSO treatment in C57BL/6NCrl (unpublished data). The telomerase knockout C57BL/6 mouse with short telomeres was found to be hypertensive, presumably due to an increase in plasma endothelin-1 level [32]. These findings pointed to a link between telomerase deficiency and hypertension. Activation of NADPH oxidase and increased production of reactive oxygen species have been demonstrated in telomerase-deficient cells [32]. Overall, though, it is doubtful that short telomeres per se
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are the cause of the increased basal blood pressure observed in CAST/Ei mice. Finally, we note that BSO-treated animals were lighter than controls, which may have resulted from poor caloric intake or metabolic impairments associated with glutathione depletion in various tissues. The lag in growth in the first 3 weeks of treatment was not due to a decrease in food intake because when normalized to body weight, food intake did not differ between the two groups throughout the study. Water intake followed the same pattern. However, we cannot exclude that increased energetic expenditure occurs in the later phase in response to GSH depletion. We have also observed a marked white fat atrophy (data not shown), associated with considerable telomere length reduction. This atrophy might have resulted from reduced replicative potential of fat cells, BSO-induced insulin resistance, or diminished tissue viability, as previously suggested [33,34]. In conclusion, our results indicate that the BSO-treated CAST/Ei mouse is a useful model for exploring a causal relation between telomere dynamics and oxidative stress in vivo. Acknowledgments Grant support is from Agence Nationale de la Recherche, Fondation pour la Recherche Médicale, and Region Lorraine. We thank Jean-Pierre Max (Inserm U734, Nancy), Gael Poitevin (Inserm U684, Nancy), Michel Thiery (CNRS, UMR 7561, Nancy) and Renaud Fay (CIC 95-01, Nancy) for help in evaluation of metabolic parameters, telomerase activity measurements, animal housing and statistical analysis. We thank Dr. Gillian Butler-Browne (Inserm 787, Paris) for helpful discussions on telomerase activity. References [1] Harley, C. B.; Vaziri, H.; Counter, C. M.; Allsopp, R. C. The telomere hypothesis of cellular aging. Exp. Gerontol. 27:375–382; 1992. [2] Blackburn, E. H. Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 579:859–862; 2005. [3] von Zglinicki, T. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27:339–344; 2002. [4] von Zglinicki, T.; Saretzki, G.; Docke, W.; Lotze, C. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp. Cell Res. 220:186–193; 1995. [5] Xu, D.; Neville, R.; Finkel, T. Homocysteine accelerates endothelial cell senescence. FEBS Lett. 470:20–24; 2000. [6] Duan, J.; Duan, J.; Zhang, Z.; Tong, T. Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening. Int. J. Biochem. Cell Biol. 37:1407–1420; 2005. [7] Kurz, D. J.; Decary, S.; Hong, Y.; Trivier, E.; Akhmedov, A.; Erusalimsky, J. D. Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J. Cell. Sci. 117:2417–2426; 2004. [8] Serra, V.; von Zglinicki, T.; Lorenz, M.; Saretzki, G. Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening. J. Biol. Chem. 278:6824–6830; 2003. [9] Kimura, M.; Cao, X.; Skurnick, J.; Cody, M.; Soteropoulos, P.; Aviv, A. Proliferation dynamics in cultured skin fibroblasts from Down syndrome subjects. Free Radic. Biol. Med. 39:374–380; 2005.
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