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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:232-241 (2008)
© 2008 The Gerontological Society of America

Fibroblasts From Naked Mole-Rats Are Resistant to Multiple Forms of Cell Injury, But Sensitive to Peroxide, Ultraviolet Light, and Endoplasmic Reticulum Stress

Adam B. Salmon, Amir A. Sadighi Akha, Rochelle Buffenstein and Richard A. Miller

1 Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor.
2 Department of Pathology and the Geriatrics Center, University of Michigan, Ann Arbor.
3 Department of Biology, City College of New York, New York.
4 The Barshop Institute for Aging and Longevity Studies and 5 Department of Physiology, University of Texas Health Science Center, San Antonio.
6 Ann Arbor VA Medical Center, Michigan.

Address correspondence to Richard A. Miller, MD, PhD, Room 3001 BSRB Box 2200, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200. E-mail: millerr{at}umich.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Fibroblasts from long-lived mutant mice are resistant to many forms of lethal injury as well as to the metabolic effects of rotenone and low-glucose medium. Here we evaluated fibroblasts from young adult naked mole-rats (NMR; Heterocephalus glaber), a rodent species in which maximal longevity exceeds 28 years. Compared to mouse cells, NMR cells were resistant to cadmium, methyl methanesulfonate, paraquat, heat, and low-glucose medium, consistent with the idea that cellular resistance to stress may contribute to disease resistance and longevity. Surprisingly, NMR cells were more sensitive than mouse cells to H2O2, ultraviolet (UV) light, and rotenone. NMR cells, like cells from Snell dwarf mice, were more sensitive to tunicamycin and thapsigargin, which interfere with the function of the endoplasmic reticulum (ER stress). The sensitivity of both Snell dwarf and NMR cells to ER stress suggests that alterations in the unfolded protein response might modulate cell survival and aging rate.

Key Words: Longevity • Comparative biology • Naked mole-rat • Stress resistance • Oxidation • Endoplasmic reticulum (ER) stress

THE natural variation of life span among the species of mammals may prove to be a valuable resource for understanding the molecular mechanisms involved in regulating the aging process. Among mammals, maximum life span can differ by more than 100 years, and even within a single order the longest-lived rodents can in some cases live more than 10-fold longer than the shortest-lived (1–3). These differences in mammalian longevity are much greater than those that can be created through single-gene mutations or dietary restriction (4). Species and mutant strains within species with exceptionally long life span tend to exhibit delayed rates of age-rated physical decline in multiple organ systems (2,5). Therefore, studies examining differences in the physiology and cellular biology of species that vary in their maximum life span may provide insights into the mechanisms that modulate the rate of age-related physiological decline.

It has long been proposed that the mechanisms that regulate the aging process may be similar to those that regulate resistance to stress (6,7). Further support for this idea has come from experimental evidence that single-gene mutations that extend life span in the nematode Caenorhabditis elegans typically render these animals resistant to numerous forms of lethal cellular insults, including oxidative stress, ultraviolet (UV) light, heavy metals, and heat (8–11). To a very limited extent, similar results have been seen in mammals; mutations in the insulin-like growth factor-I (IGF-I) signaling pathway that extend mouse longevity also tend to enhance survival of mice injected with paraquat, an agent that induces oxidative stress (12–14).

In our own laboratory, we have found that fibroblasts derived from the long-lived Snell dwarf (Pit1dw/dw), Ames dwarf (Prop1dw/dw), and growth hormone receptor knockout (GHR-KO) mice are resistant to cell death induced by multiple agents including heat, external and internal oxidative stressors (specifically H2O2 and paraquat), heavy metal, UV light, and the DNA alkylating agent methyl methanesulfonate (MMS) (15,16). All three of these mutant mouse lines share a phenotype of very low circulating levels of IGF-I as adults and a life-span extension of 40%–60% (17–19). Cells derived from newborn Snell dwarf mice show little or no difference in stress resistance in comparison to cells from newborn controls, suggesting that the level of fibroblast stress resistance is regulated by postnatal factors, presumably hormonal, in these endocrine-deficient mice (16). Snell dwarf mouse-derived fibroblasts also exhibit metabolic abnormalities independently elicited by exposure to nonlethal forms of stress. For example, Snell dwarf mouse-derived fibroblasts are relatively resistant to the metabolic effects of depleting glucose from the growth medium and to incubation with the mitochondrial inhibitor rotenone (20). Although changes in stress resistance of skin-derived fibroblasts are unlikely to play an important causal role in the control of mouse longevity, these in vitro data suggest a model in which abnormalities of the hormonal milieu during development may lead to augmented stress resistance in a variety of cell types from long-lived mutant mice and thus contribute to the unusual delayed pathology of multiple forms of late-life illnesses in these animals (18,21,22).

Tests of stress resistance of fibroblasts derived from the skin of adult donors have also shown that maximum species longevity is correlated with cellular resistance to some forms of stress and some forms of metabolic inhibition when evaluated in multiple long-lived and short-lived species of rodents (23). In that study, we found that fibroblasts from long-lived species tended to be more resistant to the lethal effects of cadmium, H2O2, heat, and MMS and also more resistant to the metabolic effects of glucose withdrawal and treatment with rotenone. These data suggest that in vitro studies of cellular stress resistance might help to evaluate the hypothesis that shared mechanisms contribute to the regulation of longevity across the mammalian evolutionary tree.

Prompted by the results of the rodent comparison, we have now tested to see if cells from the longest-living rodent known, the naked mole-rat (NMR) (24), are resistant to various forms of in vitro stress. The NMR (Heterocephalus glaber) has been observed to live to an age of at least 28 years in captivity, about seven times longer than the maximum captive life span of laboratory mice (5,24). These animals are roughly the same size as mice, yet live about 10 times longer than predicted by linear regression of life span against size for nonvolant eutherians (i.e., those that neither fly nor glide) (25,26). NMRs show little physiological or reproductive decline even when approaching 30 years of age, have never been observed to develop any spontaneous neoplasm, and do not show the typical age-associated acceleration in mortality risk that characterizes nearly every other species for which detailed survival data are available (5,27,28). Thus, physiological and biochemical mechanisms have evolved in this species that can dramatically extend life span compared to rodents of comparable size. As such, we thought it of interest to learn if cells from the NMR, like those of long-lived mutant mice, are resistant to the effects of cellular stress. Fibroblast cell lines were derived from the skin of young adult NMRs to test the hypothesis that the long life span of these animals is accompanied by cellular resistance to stress. Our results show that cells from these animals are, as predicted, resistant to some agents, but are, unexpectedly, quite sensitive to others.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals
NMR skin samples were obtained from a captive colony maintained in the Department of Biology at City College of New York. The husbandry conditions of these animals were as presented previously (5,28–32). Mouse skin samples were prepared from the genetically heterogeneous DC stock. DC mice are the intercrossed progeny of UM-HET3 mice, themselves generated by a cross between females of the (BALB/c x C57BL/6)F1 stock and males of the (DBA/2 x C3H/He)F1 stock. Biopsies for this study were taken from the G4 generation of DC mice. Skin samples were taken from male NMRs of approximately 2 years of age and from male DC mice of 3–4 months of age. These ages were chosen as representatives of a physiologically equivalent age representing postpubertal young adults of each species (33); 2-year-old NMRs have reached an age of about 7% of the maximal life span of the species, and 3-month-old mice have reached an age of about 6% of the maximal life span of the species.

Snell dwarf (Pitdw/dw) mice and heterozygote (Pitdw/+) controls were bred as the progeny of (DW/J x C3H/HeJ)-dw/+ females and (DW/J x C3H/HeJ)F1-dw/dw males. Their sires had been previously treated with growth hormone and thyroxine to increase body size and fertility. Tail skin biopsies were taken from male mice at approximately 3 months of age.

Cell Culture and Establishment of Cryopreserved Cell Lines
Tails from NMRs were sterilized with 70% ethanol wipes, and biopsies of 3–5 mm in length were obtained from the distal half of the tail and placed in complete medium (CM) made of Dulbecco's modified Eagle medium (DMEM, high-glucose variant; Gibco-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics (penicillin at 100 U/mL and streptomycin at 100 µg/mL; Sigma, St. Louis, MO), and fungizone at 0.25 µg/mL (Biowhittaker-Cambrex Life Sciences, Walkersville, MD) on ice and shipped overnight from New York to Michigan. For laboratory-raised DC mice, Snell dwarf mice, and controls, tails were washed with 70% ethanol, and tail-skin biopsies of 3–5 mm in length obtained from the distal half of the tail were placed in CM prior to fibroblast isolation.

Fibroblast cultures from NMR and DC biopsies were developed using methods similar to those previously reported (15,16) except that the present study used 10% CO2 in air and an incubator temperature of 33°C. CM described above was used to subculture cells from both NMRs and mice. We, like others (34), have found that fibroblasts from NMRs grow poorly at the standard 37°C incubation temperature, perhaps because the animals have a body temperature several degrees below this in their natural environment (35). For this reason, both NMR and DC fibroblast cultures were incubated at 33°C. Initial cultures of cells derived from NMRs ("P0") grew to confluence at a slower rate than those of DC-derived cells (data not shown) so all cells were trypsinized for subculturing when NMR cells reached approximately 90% confluence. Cell cultures were fed at Day 3 (replacing 2/3 of the medium), again at Day 6, and subcultured at Day 10 at a density of 7.5 x 105 in tissue culture flasks of 75 cm2 surface area to produce P1 cultures. This protocol was retained for all subsequent subculturing. Following an additional passage using the same protocol, P2 cells were harvested and cryopreserved as in our previous studies (23). These cryopreserved cells were stored in liquid N2 for up to 24 months before assessment of stress resistance. Cell aliquots were thawed and grown two further passages as previously shown, with all incubations at 33°C and 10% CO2 in air (23). For studies of thapsigargin and tunicamycin effects, fibroblasts from Snell dwarf mice and littermate controls were established and cultured at 37°C as previously described (15,16).

Assessment of Fibroblast Resistance to Lethal Stress
NMR and DC cells lines were evaluated in two sets of experiments, each of which evaluated equal numbers of NMR and DC donors. P4 cultures were used to assess resistance to all agents. Tests for resistance to the lethal stresses cadmium, H2O2, paraquat, UV light, MMS, and heat were performed as previously described (15,16,23,36). After treatment, cells were washed at 33°C with 1X phosphate-buffered saline (PBS), and incubated at 33°C in serum-free DMEM supplemented with 2% bovine serum albumin (BSA), antibiotics, and fungizone; survival was measured 18 hours later using conversion of the extracellular tetrazolium dye WST-1 to its colored formazan product (15,16,23,36). All incubations (except for heat) were at 33°C in a humidified incubator with 10% CO2 in air.

Assessment of Cell Metabolism in Low-Glucose Medium and Rotenone Effects
For each set of glucose or rotenone assay experiments, representatives of P4 cell lines from both NMRs and DC mice were assayed in parallel to minimize the effects of day-to-day variation. Tests for the effect of glucose withdrawal and rotenone were performed as previously shown (20,23). All incubations were at 33°C in a humidified incubator with 10% CO2 in air. It is important to note that, although WST-1 is used in tests of both lethal stresses and in the evaluation of responses to low glucose, the decline in WST-1 reduction under low glucose conditions or at the rotenone concentrations used is not accompanied by cell death (20).

Assessment of Fibroblast Resistance to Endoplasmic Reticulum Stress
For Snell dwarf mouse and littermate control experiments, P3 cells were prepared in 96-well plates as previously described. These cells had not been previously cryopreserved, and all assays with them were performed in a humidified incubator with 5% CO2 in air and at a temperature of 37°C. Thapsigargin and tunicamycin were suspended in a solution of dimethyl sulfoxide (DMSO; Sigma), and doses of these agents were diluted in DMEM with a final DMSO concentration <0.5%. Cells were treated for 24 hours after which they were washed with 1X PBS and incubated with serum-free DMEM supplemented with 2% BSA, antibiotics, and fungizone; survival was measured 18 hours later using WST-1. DC and NMR assays for resistance to these agents were performed similarly, except all incubations and treatments were at 33°C in a humidified incubator with 10% CO2 in air.

Calculation of Lethal Dose and Effective Dose Values and Statistical Analysis
For calculation of the resistance of each cell line to chemical stressors, at each dose of chemical stressor, mean survival was calculated for duplicate wells for each cell line. The lethal dose (LD50, i.e., dose of stress agent that led to survival of 50% of the cells) was then calculated using probit analysis as implemented in NCSS software (NCSS, Kaysville, UT). For this analysis, extremely low doses of stress agents that caused no cell death in fibroblasts, as measured by the WST1 assay, were censored from all data sets. Effective dose values (ED50, i.e., dosage of glucose or rotenone associated with a 50% reduction in cellular metabolic activity) were calculated in a similar manner. Because some groups of data were not normally distributed, differences between groups in mean LD50 levels were evaluated by Mann–Whitney U nonparametric tests, with each day's work containing equal numbers of cultures from each species or each genotype. For some agents, the day of assay seemed to affect the calculated LD50 or ED50; therefore, data were also analyzed by two-way analysis of variance (ANOVA) with species and day of assay as the factors.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
To see if fibroblasts from NMRs resembled cells from other long-lived rodent species or from long-lived mutant mice, we evaluated their resistance to a series of lethal stresses and nonlethal inhibitors of cellular metabolism. Skin fibroblasts were derived in each case from young adult animals, in which approximately 5%–10% of the maximal life span of each species had been completed, and cell cultures for DC and NMR cultures were maintained at 33°C (a temperature at which NMR cells proliferate and survive).

The data in Figure 1 and Table 1 represent the results from cell lines tested for their resistance to the lethal agents cadmium, paraquat, heat, MMS, H2O2, and UV light. Our working hypothesis, based on the longer life span of NMRs, was that fibroblasts from NMRs would be relatively resistant to all these agents, similar to cells from long-lived Snell dwarf mice (15,16). Our data supported this notion for experiments assaying resistance to cadmium (Figure 1A), to paraquat (Figure 1B), to heat (Figure 1C), and to MMS (Figure 1D) in cell lines from 11 individual animals of each species. The difference in resistance to lethal stressors ranged from 2.7-fold for paraquat to 1.2-fold for MMS. These differences reached statistical significance by nonparametric Mann–Whitney test, with p <.04 in each case.


Figure 01
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  		Figure 1. Skin-derived fibroblasts from naked mole-rats (NMR) are resistant to many forms of lethal stress, but sensitive to H2O2 and ultraviolet (UV) light. Each symbol represents fibroblasts isolated from an individual NMR or laboratory mouse (DC); horizontal line indicates the mean value for each group. All data values were collected from assays performed on two separate days; the day of assay is represented in these figures by open or closed triangles. The p values for the Mann–Whitney U statistic are shown in Table 1; N = 11 for each group. LD50 = lethal dose (dose of stress agent that led to survival of 50% of the cells); MMS = methyl methanesulfonate

 

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  		Table 1. Resistance to Lethal and Nonlethal Agents of Skin-Derived Fibroblasts From the Naked Mole-Rat (NMR) and Laboratory Mice (DC).

 
In contrast, however, we found that NMR fibroblasts were markedly more sensitive than DC mouse fibroblasts to H2O2 (approximately 4-fold more sensitive, Figure 1E) and to irradiation with UVC light (approximately 3-fold more sensitive, Figure 1F). In both cases, these differences reached statistical significance by Mann–Whitney U test, with p <.001. ANOVA calculations that adjust for day-to-day variations, as compiled in Table 1, confirmed the conclusion that NMR fibroblasts were more resistant to cadmium, paraquat, heat, and MMS and more sensitive than mouse cells to H2O2 and UV light, with p <.01 in each case.

The data in Figure 2 (and also included in Table 1) represent the results from cell lines tested for their resistance to the metabolic inhibition caused by withdrawal of glucose from the growth medium or by the mitochondrial inhibitor rotenone. Fibroblasts from the NMR, like Snell dwarf cells and cells from the long-lived rodents previously tested, were relatively resistant to the effect of glucose withdrawal from the growth medium (Figure 2A, Mann–Whitney U test, p =.01). Surprisingly, however, NMR-derived fibroblasts were approximately 2-fold more sensitive than DC fibroblasts to rotenone, at the nonlethal doses used for these tests (Figure 2B, Mann–Whitney U test, p =.003). An ANOVA approach confirmed both inferences (Table 1).


Figure 02
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  		Figure 2. Skin-derived fibroblasts from naked mole-rats (NMR) are resistant to glucose withdrawal from growth medium (A), but sensitive to rotenone (B). Each symbol represents fibroblasts isolated from an individual NMR or laboratory mouse (DC); horizontal line indicates the mean value for each group. All data values were collected from assays performed on two separate days; the day of assay is represented in these figures by open or closed triangles. The p values for the Mann–Whitney U statistic are shown in Table 1. N = 10 for glucose; N = 11 for rotenone. ED50, dosage of glucose or rotenone associated with a 50% reduction in cellular metabolic activity

 
The LD50 and ED50 values calculated for the resistance of mouse cells to most agents were similar to those noted in previous studies that had used cultures grown at 37°C, except for somewhat increased resistance to cadmium and rotenone (15,16,20,23,36). In a set of experiments not shown, cell lines from DC mice were assayed in parallel under growth conditions of 33°C or 37°C, with 10% CO2 in air. The 4°C temperature decrease alone resulted in a mean LD50 and ED50 that were approximately 10-fold higher for cadmium and rotenone resistance and 2-fold higher for peroxide resistance; there was no difference in resistance to UV light.

It is speculated that some of the agents used in this and our other studies might partly act by inducing endoplasmic reticulum (ER) stress (37–39), a term coined to signify an imbalance between cellular demand for ER function and ER capacity (40). As a prelude to assessing NMR cells for resistance to ER stress agents, we compared the lethal effects of thapsigargin and tunicamycin on fibroblasts from Snell dwarf mice and their littermate controls. Thapsigargin disrupts ER Ca2+ homeostasis through irreversible inhibition of the ER Ca2+-ATPase (41), which in turn interferes with the function of Ca2+-sensitive chaperones such as calreticulin and ERp57 and hampers the post-translational processing, folding, and export of proteins from the ER (42). Tunicamycin abolishes N-linked glycosylation of proteins in eukaryotic cells by blocking the function of N-acetylglucosamine-1-transferase, and thus interferes with the assembly and transport of glycoproteins from the ER to the Golgi complex (43,44). Figure 3 shows the results of six experiments involving cell lines derived from eight or nine pairs of Snell dwarf and control mice, showing that, in contrast to their relative resistance to most sources of lethal injury, Snell fibroblasts are significantly more sensitive than cells from littermate controls to cell death induced by thapsigargin (Figure 3A, Mann–Whitney U test, p =.002) or tunicamycin (Figure 3B, Mann–Whitney U test, p =.03). To see if NMR cells were, like Snell dwarf fibroblasts, unusually sensitive to agents that induced death via ER stress, we evaluated six pairs of NMR and mouse (DC) cultures. Figure 4 shows that NMR cells were indeed significantly more sensitive to both thapsigargin (Figure 4A, Mann–Whitney U test, p =.01) and tunicamycin (Figure 4B, Mann–Whitney U test, p =.004) compared to cells from DC mice tested in parallel. These data are also included in Table 1. Table 2 presents a summary of these stress resistance data, together with published data, for comparison, on cells from long-lived mutant mice and from species of long-lived rodents.


Figure 03
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  		Figure 3. Skin-derived fibroblasts from long-lived Snell dwarf mice are sensitive to the lethal effects of agents that induce endoplasmic reticulum (ER) stress. Each symbol in the figure represents a cell line isolated from a separate Snell dwarf (Dwarf) or littermate control (Control) assayed for resistance to thapsigargin (N = 8; A) or tunicamycin (N = 9; B). Horizontal lines represent mean values for each group. LD50 = lethal dose (dose of stress agent that led to survival of 50% of the cells)

 

Figure 04
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  		Figure 4. Skin-derived fibroblasts from naked mole-rats (NMR) are sensitive to the lethal effects of agents that induce endoplasmic reticulum (ER) stress. Each symbol in the figure represents a cell line isolated from a separate NMR or laboratory mouse (DC) that was assayed for resistance to thapsigargin (A) or tunicamycin (B). Horizontal lines represent the mean value for each group. The p values for the Mann–Whitney U statistic are shown in Table 1; N = 6 for each group. LD50 = lethal dose (dose of stress agent that led to survival of 50% of the cells)

 

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  		Table 2. Relationship Between Donor Longevity and Cellular Resistance to Multiple Agents.

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In this study, we tested the hypothesis that fibroblasts derived from the skin of the longest-lived rodent, the NMR, exhibit enhanced resistance to lethal and nonlethal cellular stresses as has been reported for cell lines from long-lived mutant mice (15,16,20,36) and for cell lines from long-lived rodent species (23). We found that NMR-derived cells were indeed significantly more resistant than mouse cells to some forms of stress, e.g., cadmium, paraquat, heat, and MMS, and relatively resistant to the effects of low glucose concentrations. Surprisingly, however, we found that cells from NMRs were significantly more susceptible than mouse cells to the effects of H2O2, UV light, and rotenone. This unexpected pattern of differential resistance raises a number of questions about the extent to which regulated responses to various forms of stress are coordinated, and provides some insights into the potential role of stress resistance in the evolution of NMR longevity.

Interspecies comparisons can sometimes be confounded by differences in body size and the plethora of parameters that scale with size, such as metabolic rate. Larger species of mammals tend to live longer, so that almost any trait that is linked to body size is likely to show a correlation with life span (26,45). Interpretation of NMR results, however, is not confounded in this manner, because adult NMR (~35 g) are similar to mice in size (~40 g for adult DC mice). Our design also focused on comparisons between cells taken from mice and NMRs that were at equivalent stages of development and at physiological equivalent ages, i.e., from young adult animals that had reached full maturity and had lived ~6% of their life span. At this physiological age (3–4 months for mice and ~2 years for NMRs), neither mice nor NMRs have yet begun to show any of the signs of aging, including increased mortality risk (5). Furthermore, many of the confounding comparative metabolic data are attenuated in cell studies because metabolic rate of fibroblasts from different species rapidly converge (46).

Our previous work has shown that fibroblast resistance to the lethal effects of cadmium, heat, and MMS toxicity and to the metabolic effects of glucose withdrawal from growth medium show significant or suggestive correlations with maximum longevity of the donor species (23). Fibroblasts from NMRs are relatively resistant to each of these agents and are also resistant to paraquat. These data suggest that there may be pathways that regulate the resistance to these agents and that the level of activity could vary with maximum life span among rodent species. These same hypothetical pathways may be those that produce resistance to these forms of stress in cells from Snell and Ames dwarf mice and from GHR-KO mice. Studies of cell lines from normal, genetically heterogeneous mice have shown that those individual mice whose skin-derived fibroblasts are resistant to cadmium are also resistant to low glucose, again consistent with the idea that resistance to these two forms of stress is coregulated, perhaps involving effects of polymorphic loci (20).

Many experts have proposed that cellular damage due to oxidative stress contributes to many of the diseases of aging, and that resistance to oxidative damage is a key element in extended longevity within or across species (47,48). Studies similar to those presented here have shown that primary fibroblasts from long-lived animals tend to be resistant to peroxide damage in comparisons of mammals across many taxonomic orders (49), within the rodents alone (23), and in long-lived mutant mouse strains (15,16). Some tissues from the NMR also seem to be resistant to oxidative injury, including arterial endothelial tissue and mitochondrial membranes from the liver and muscle (28,32). Our results showing that fibroblasts from NMRs are resistant to cell death induced by paraquat are consistent with this model (Figure 1B). It is thought that paraquat causes acute cellular toxicity through the production of superoxide radicals in the mitochondria, which in turn result in disruption of NADPH-requiring biochemical processes (50,51). In contrast, however, NMR cells were dramatically more sensitive than mouse cells to the lethal effects of H2O2, and are indeed more sensitive to this agent than is any other rodent we have previously studied (23). This result is provocative because it does not support most models of oxidative stress and longevity, and its basis may lie in the particulars of both species-specific oxidative damage and the conditions under which our assays were performed. The toxicity of H2O2 differs from that of paraquat in that H2O2 causes oxidative stress through the production of hydroxyl radicals that damage the integrity of membranes, lipids, and nucleic acids (52), conditions that may be exacerbated by the serum-free conditions of our assay (53,54). Recent studies have shown higher levels of oxidative damage to proteins and nucleic acids and particularly to lipids in tissues from NMRs compared to mice of the same chronological or physiological age (29–31). If NMR-derived fibroblasts exhibit a similar phenotype, these cells may be sensitive to the effects of further oxidative damage, such as that caused by H2O2. In theory, this sensitivity could be distinct from the actions of paraquat and other oxidative stressors upon NADPH-dependent processes. These results may also represent our use of standard culture conditions of atmospheric oxygen levels (approximately 20% in air), which are higher than typical tissue oxygen levels (55) and could potentially induce either oxidative damage or compensatory responses to damage that would not have been seen in cells grown under lower oxygen tension (56,57). Alterations of the oxygen radicals produced by cellular metabolism due to cell culture at a lower temperature might affect the stress sensitivity of fibroblasts.

NMRs exhibit many characteristics that are contrary to those predicted by the oxidative stress theory of aging in addition to the relatively high levels of oxidative molecular damage just mentioned. For example, mice and NMRs show similar levels of reactive oxygen species production in heart mitochondria despite their disparities in maximum longevity (58). Similarly, activity levels of the antioxidants superoxide dismutase, catalase, and glutathione in the NMR liver are similar to those levels seen in mice (29–31). In addition, glutathione peroxidase activity is much lower in NMR livers compared to that observed in livers from mice (29). Peroxide treatment in vitro induces lipid peroxidation and DNA oxidation that cause cell death (59); these effects can be modulated in part by the activity of glutathione peroxidase (60). It is not known if NMR fibroblasts have lower levels of superoxide dismutase, catalase, and glutathione peroxidase than do mouse cells, but such differences might in principle contribute to the sensitivity of these cells to peroxide exposure in vitro. It seems clear from the NMR fibroblast data that resistance to H2O2 and resistance to paraquat are likely to be regulated by different defense mechanisms. Our data support the general conclusion that the exceptional life span of NMRs is not likely to reflect merely a dramatic resistance to all forms of oxidative damage (29–31).

Loss of cellular resistance to H2O2 and UV light may reflect evolutionary adaptations to the subterranean niche of the NMR in its natural environment, where the gaseous atmosphere within the burrows is both hypoxic and hypercapnic (61). Fossil records reveal that Bathyergidae ancestors have inhabited a subterranean milieu since the early Miocene (ca 24 million years ago) (62). These animals have evolved a set of morphological and physiological phenotypes well suited to life underground, such as a markedly atrophied visual cortex and expanded somatosensory cortex, in keeping with sensory needs in the dark, and their skin is poorly pigmented (63). Physiological adaptation has included low rates of gas exchange and heat production leading to low body temperatures, as well as tolerance of hypoxia and hypercapnia (64,65). Adaptation to a hypoxic environment also may have driven the loss of cellular defenses to peroxide discussed above. Similarly, sensitivity to UV light may result from evolving in an underground environment in which sunlight-induced damage rarely occurs.

The sensitivity of NMR-derived fibroblasts to UV light presents a contrast to the UV resistance of fibroblasts from long-lived Snell dwarf mice. We have found (66) that dwarf mouse fibroblasts have an elevated rate of nucleotide excision repair (NER) after UV damage to DNA, and speculate that this mechanism may contribute to their resistance to UV-induced cell death. The repair of UV genomic lesions is closely correlated with survival of cells following irradiation and occurs primarily through NER, the rate of which is thought to be limited by the recognition of genomic lesions (67,68). It will be of interest to learn more about the biochemistry of NER, in both transcribed and nontranscribed regions, in various cells of the NMR. NER also recognizes lesions caused by agents other than UV light, and it has been shown that cells that have lost the ability to repair UV-induced damage nevertheless can still repair these other lesions with varying levels of efficiency (69). We show here that NMR cells are resistant to cell death caused by a different form of DNA damage and DNA methylation caused by MMS, and NMR cells are also resistant to several other DNA-damaging agents (Mele J, Buffenstein R, personal communication, 2008). It would be of interest to test if skin-derived NMR cells, which we show here are sensitive to UV light, are able to resist the toxic effects of other DNA lesions repaired by NER such as the interstrand cross-links induced by cisplatin. It would also be of interest to test if cells derived from NMR internal organs display sensitivity to UV light compared to cells derived from the internal organs of other rodents. NMR cells also seem to show slower progression into cell cycle relative to mouse cells following a DNA-damaging insult (Mele J, Buffenstein R, personal communication, 2008), suggesting that NMR cells might have tightly regulated cellular processes to minimize the accumulation of cells with DNA damage or mutation. Thus, it might be possible that the UV sensitivity of NMR-derived cells contributes to the remarkably low rate of spontaneous cancer in these animals (5); enhanced apoptosis associated with cells with damaged genomes might inhibit the development and progression of neoplasia (70). It will be of interest to further dissect how DNA repair mechanisms may differ between short-lived, cancer-prone species (such as the mouse) and long-lived, cancer-resistant species (such as the NMR).

NMR-specific cellular responses to some of these agents may result from altered cellular metabolism. Cells from poikilothermic reptiles tend to exhibit small inner mitochondrial surface area and greater membrane permeability of the inner mitochondria compared to mammals, and both traits are associated with decreased mitochondrial activity (71,72). Because NMRs are thermally labile mammals outside the warm confines of their thermally buffered equatorial burrows, they may exhibit mitochondrial phenotypes similar to those seen in other poikilotherms in other classes of vertebrates which may result in decreased metabolic activity and metabolite levels. The nonlethal effects measured by the rotenone assay may be regulated by cues from the mitochondria, including the levels of metabolic products such as NADH and NADPH (20), and thus relate to metabolic activity. Diminished activity of mitochondria may also contribute to resistance to the toxicity of paraquat; the mechanism of paraquat-mediated cell death is most often attributed to its induction of superoxide formation in the mitochondria (50). Investigation into the nature of mitochondria in these animals may prove useful in understanding the relationship between the resistance (or sensitivity) to these agents and longevity.

Phylogenetic history may also contribute to the unexpected sensitivity of NMR cells to H2O2 and UV light. NMRs are members of the rodent suborder Hystricognathi and are more closely related to other rock rats, cane rats, guinea pigs, and porcupines than to mice, rats, and other rodents in other suborders (5). Our previous comparison of cells from multiple rodent species included only a single species of hystricognath, the North American porcupine (23). Although the data set as a whole showed a correlation between peroxide resistance and longevity, the average resistance of cells from the long-lived porcupine (mean ± standard error = 41 µM ± 15) was not different from those from short-lived DC mice (47 µM ± 12, t test p =.8) or from cells derived from wild-caught mice (55 µM ± 14, t test p =.5). Furthermore, cells from porcupines (47 J/m2 ± 34) tended to be sensitive to UV light compared to cells from DC mice (57 J/m2 ± 12, t test p =.8) and were significantly more sensitive than cells from wild-caught mice (147 J/m2 ± 28, t test p =.04). Glutathione peroxidase activity has been shown to be low in tissues from hystricognath guinea pigs and NMRs compared to mice and rats (29,73–76); this low activity might contribute to a relative sensitivity to peroxide among the members of the suborder. It is plausible that these two suborders may have diverged, early in their history, in the biochemical factors by which they respond to various forms of cell injury. It would thus be of interest to evaluate stress resistance of cells derived from hystricognaths with maximal life spans shorter than those of NMRs and porcupines.

As part of this study, the compounds thapsigargin and tunicamycin were used to examine the effect of ER stress on fibroblasts from dwarf mice and NMRs. Both of these agents act to hamper the transport of proteins from the ER, thapsigargin by disrupting ER Ca2+ homeostasis and tunicamycin through abolishing N-linked glycosylation of proteins (40,77,78). To our surprise, fibroblasts from both dwarf mice and NMRs proved to be more sensitive than their respective controls to the induction of ER stress. The accumulation of unfolded or misfolded proteins in the ER causes ER stress, and instigates a concerted adaptive program known as the unfolded protein response (UPR). The UPR alleviates ER stress by down-regulating the synthesis of secreted proteins, up-regulating ER chaperone and foldase expression levels, and activating ER-associated degradation (ERAD), thereby easing the burden on the stressed ER by reducing its protein load, increasing its folding capacity, and eliminating irreparably misfolded proteins (40,77,78). In higher eukaryotes, PERK (a double-stranded RNA-activated protein kinase-like ER kinase) is the proximal transducer of protein synthesis inhibition, whereas ATF6 (a basic leucine zipper transcription factor) and IRE1 (a protein kinase/endoribonuclease) orchestrate the up-regulation of chaperones and foldases and activation of ERAD. If these pro-survival efforts are overwhelmed, ER stress-related apoptosis ensues.

Recent studies tend to implicate ER stress in the pathogenesis of many late-life illnesses (40,79). It has also been contended that the ER stress response might affect the aging process itself (79). So far, the C. elegans model provides the most compelling evidence in this respect (81). Treatment of C. elegans with the small molecule resveratrol extends life span in a daf-16–independent manner. Resveratrol induces the transcription of abu-11 (Formulactivated in Formulalocked FormulaPR), a member of a gene family with a role in the ER stress response. RNA interference data have shown that the resveratrol effect requires abu-11, and that worms overexpressing abu-11 displayed long life span. Furthermore, age has been shown to modulate certain effectors of the UPR. A decline with age in eIF2{alpha} phosphorylation together with an increase in GADD34 levels (82), an increase in C/EBP-homologous protein (CHOP) and phosphorylation of un N-terminal kinase (phospho-JNK) levels (82,83) in rat livers, and the carbonylation of immunoglobulin binding protein (BiP), protein disulfide isomerase (PDI), and calreticulin in livers of aged mice (84) are cases in point. Our data show that dwarf and NMR fibroblasts are sensitive to the lethal effects of ER stress. It is possible that this sensitivity, if it occurred in appropriate cells in vivo, might help to rid the organism of defective cells and thus postpone pathological outcomes. It is also possible that heightened sensitivity to ER stress might induce compensatory increases in pathways that protect against the lethal effects of some of the other agents tested in our in vitro analyses. More information on the molecular basis for sensitivity of dwarf and NMR cells to thapsigargin and tunicamycin, and on the relationship of the UPR to cellular responses to other forms of stress, is thus likely to be informative.

Understanding what mechanisms nature uses to create long-lived species is a fundamental problem in biogerontology. Overall, the data presented here contribute to the growing evidence associating unusual longevity with resistance to cellular injury, both within and across species. Disparities among the models shown in Table 2, however, suggest that the specific pattern of cellular resistance may well vary from clade to clade. Developing methods to extend these analyses to other cell types, in vitro and in intact animals, is likely to provide additional insight into these relationships. Our data further illustrate the value of using nontraditional models, such as the NMR, for tests of specific hypotheses about the control of aging.


   Acknowledgments
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This work was supported by National Institute on Aging grants R01-AG022891, T32-AG000114, AG023122, and NIGMS S06-GM08168.

We thank Maggie Lauderdale and Jessica Sewald for development of the DC mouse stock and the Snell dwarf colony, James Harper for allowing us to use his unpublished data on Ames dwarf and GHR-KO cells in Table 2, and Andrzej Bartke, Michal Masternak, and John Kopchick for providing Ames and GHR-KO skin samples. Yael Edrey, Mario Pinto, Roxanne Cheung, Ting Yang, and the animal care staff at CCNY are sincerely thanked for their assistance with the NMR husbandry.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received December 15, 2007

Accepted February 7, 2008


   References
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  1. Comfort A. The Biology of Senescence. New York: Elsevier; 1979.
  2. Finch CE. Longevity, Senescence and the Genome. Chicago, IL: University of Chicago Press; 1990.
  3. Carey JR, Judge DS. Longevity Records: Life Spans of Mammals, Birds, Amphibians, Reptiles, and Fish. Odense, Denmark: Odense University Press; 2000.
  4. Miller RA, Austad SN. Growth and aging: why do big dogs die young? In: Masoro EJ, Austad SN, eds. Handbook of the Biology of Aging. 6th Ed. New York: Academic Press; 2006:512–533.
  5. Buffenstein R. The naked mole-rat: a new long-living model for human aging research. J Gerontol A Biol Sci Med Sci. 2005;60:1369-1377.[Abstract/Free Full Text]
  6. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11:298-300.
  7. Kirkwood TB. Evolution of ageing. Nature. 1977;270:301-304.[Medline]
  8. Murakami S, Johnson TE. A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics. 1995;143:1207-1218.
  9. Lithgow GJ, White TM, Melov S, Johnson TE. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc Natl Acad Sci U S A. 1995;92:7540-7544.[Abstract/Free Full Text]
  10. Johnson TE, Cypser J, de Castro E, et al. Gerontogenes mediate health and longevity in nematodes through increasing resistance to environmental toxins and stressors. Exp Gerontol. 2000;35:687-694.[Medline]
  11. Johnson TE, Henderson S, Murakami S, et al. Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease. J Inherit Metab Dis. 2002;25:197-206.[Medline]
  12. Migliaccio E, Giorgi M, Mele S, et al. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature. 1999;402:309-313.[Medline]
  13. Holzenberger M, Dupont J, Ducos B, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003;421:182-187.[Medline]
  14. Yamamoto M, Clark JD, Pastor JV, et al. Regulation of oxidative stress by the anti-aging hormone Klotho. J Biol Chem. 2005;280:38029-38034.[Abstract/Free Full Text]
  15. Murakami S, Salmon A, Miller RA. Multiplex stress resistance in cells from long-lived dwarf mice. FASEB J. 2003;17:1565-1566.[Abstract/Free Full Text]
  16. Salmon AB, Murakami S, Bartke A, Kopchick J, Yasumura K, Miller RA. Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress. Am J Physiol Endocrinol Metab. 2005;289:E23-E29.[Abstract/Free Full Text]
  17. Brown-Borg HM, Borg KE, Meliska CJ, Bartke A. Dwarf mice and the ageing process. Nature. 1996;384:33.[Medline]
  18. Flurkey K, Papaconstantinou J, Miller RA, Harrison DE. Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc Natl Acad Sci U S A. 2001;98:6736-6741.[Abstract/Free Full Text]
  19. Coschigano K, Holland AN, Riders ME, List EO, Flyvbjerg A, Kopchick JJ. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased life span. Endocrinology. 2003;144:3799-3810.[Abstract/Free Full Text]
  20. Leiser SF, Salmon AB, Miller RA. Correlated resistance to glucose deprivation and cytotoxic agents in fibroblast cell lines from long-lived pituitary dwarf mice. Mech Ageing Dev. 2006;127:821-829.[Medline]
  21. Silberberg R. Articular aging and osteoarthritis in dwarf mice. Pathol Microbiol (Basel). 1972;38:417-430.[Medline]
  22. Vergara M, Smith-Wheelock M, Harper JM, Sigler R, Miller RA. Hormone-treated Snell dwarf mice regain fertility but remain long-lived and disease resistant. J Gerontol A Biol Sci Med Sci. 2004;59:1244-1250.[Abstract/Free Full Text]
  23. Harper JM, Salmon AB, Leiser SF, Galecki AT, Miller RA. Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone. Aging Cell. 2007;6:1-13.[Medline]
  24. Buffenstein R, Jarvis R. The naked mole-rat–a new record for the oldest rodent. Science's SAGE KE 2002 May 29 Available from: http://sageke.sciencemag.org/cgi/content/full/sageke;2002/21/pe7. Last accessed February 2008.
  25. Prothero J, Jurgens KD. Scaling of maximal lifespan in mammals: a review. Basic Life Sci. 1987;42:49-74.[Medline]
  26. Austad SN, Fischer KE. Mammalian aging, metabolism, and ecology: evidence from the bats and marsupials. J Gerontol. 1991;46:B47-B53.
  27. O'Connor TP, Lee A, Jarvis JUM, Buffenstein R. Prolonged longevity in naked mole-rats: age-related changes in metabolism, body composition and gastrointestinal function. Comp Biochem Physiol A Mol Integr Physiol. 2002;133:835-842.[Medline]
  28. Labinskyy N, Csiszar A, Orosz Z, et al. Comparison of endothelial function, O2• and H2O2 production, and vascular oxidative stress resistance between the longest-living rodent, the naked mole rat, and mice. Am J Physiol Heart Circ Physiol. 2006;291:H2698-H2704.[Abstract/Free Full Text]
  29. Andziak B, O'Connor TP, Buffenstein R. Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat. Mech Ageing Dev. 2005;126:1206-1212.[Medline]
  30. Andziak B, O'Connor TP, Qi W, et al. High oxidative damage levels in the longest-living rodent, the naked mole-rat. Aging Cell. 2006;5:463-471.[Medline]
  31. Andziak B, Buffenstein R. Disparate patterns of age-related changes in lipid peroxidation in long-lived naked mole-rats and shorter-lived mice. Aging Cell. 2006;5:525-532.[Medline]
  32. Hulbert AJ, Faulks SC, Buffenstein R. Oxidation-resistant membrane phospholipids can explain longevity differences among the longest-living rodents and similarly-sized mice. J Gerontol A Biol Sci Med Sci. 2006;61:1009-1018.[Abstract/Free Full Text]
  33. Miller RA, Nadon NL. Principles of animal use for gerontological research. J Gerontol Biol Sci. 2000;55A:B117-B123.[Abstract/Free Full Text]
  34. Lorenzini A, Tresini M, Austad SN, Cristofalo VJ. Cellular replicative capacity correlates primarily with species body mass not longevity. Mech Ageing Dev. 2005;126:1130-1133.[Medline]
  35. Buffenstein R, Yahav S. Is the naked mole-rat Heterocephalus glaber an endothermic yet poikilothermic mammal? J Therm Biol 1991 Jul;16:(4): 227-232.
  36. Harper JM, Salmon AB, Chang Y, Bonkowski M, Bartke A, Miller RA. Stress resistance and aging: influence of genes and nutrition. Mech Ageing Dev. 2006;127:687-694.[Medline]
  37. Urano F, Calfon M, Yoneda T, et al. A survival pathway for Caenorhabditis elegans with a blocked unfolded protein response. J Cell Biol. 2002;158:639-646.[Abstract/Free Full Text]
  38. Wu S, Hu Y, Wang JL, Chatterjee M, Shi Y, Kaufman RJ. Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum. J Biol Chem. 2002;277:18077-18083.[Abstract/Free Full Text]
  39. Liu F, Inageda K, Nishitai G, Matsuoka M. Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells. Environ Health Perspect. 2006;114:859-864.[Medline]
  40. Schröder M, Kaufman RJ. The mammalian unfolded protein response. Ann Rev Biochem. 2005;74:739-789.[Medline]
  41. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990;87:2466-2470.[Abstract/Free Full Text]
  42. Burdakov D, Petersen OH, Verkhratsky A. Intraluminal calcium as a primary regulator of endoplasmic reticulum function. Cell Calcium. 2005;38:303-310.[Medline]
  43. Tkacz JS, Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun. 1975;65:248-257.[Medline]
  44. Ericson MC, Gafford JT, Elbein AD. Tunicamycin inhibits GlcNAc-lipid formation in plants. J Biol Chem. 1977;252:7431-7433.[Abstract/Free Full Text]
  45. Speakman JR. Correlations between physiology and lifespan–two widely ignored problems with comparative studies. Aging Cell. 2005;4:167-175.[Medline]
  46. Wheatley DN. Convergence of metabolic rate of cultured cells from animals of different sizes. Am J Physiol Regul Integr Comp Physiol. 2007;292:R2113-R2114.[Free Full Text]
  47. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239-247.[Medline]
  48. Barja G. Rate of generation of oxidative stress-related damage and animal longevity. Free Radic Biol Med. 2002;33:1167-1172.[Medline]
  49. Kapahi P, Boulton ME, Kirkwood TB. Positive correlation between mammalian life span and cellular resistance to stress. Free Radic Biol Med. 1999;26:495-500.[Medline]
  50. Bus JS, Cagen SZ, Olgaard M, Gibson JE. A mechanism of paraquat toxicity in mice and rats. Toxicol Appl Pharmacol. 1976;35:501-513.[Medline]
  51. Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology. 2002;180:65-77.[Medline]
  52. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 2nd Ed. Oxford, U.K.: Oxford University Press; 1989.
  53. Barroso MP, Gomez-Diaz C, Villalba JM, Buron MI, Lopez-Lluch G, Navas P. Plasma membrane ubiquinone controls ceramide production and prevents cell death induced by serum withdrawal. J Bioenerg Biomembr. 1997;29:259-267.[Medline]
  54. Cantin AM, Paqueete B, Richter M, LaRivee P. Albumin-mediated regulation of cellular glutathione and nuclear factor kappa B activation. Am J Respir Crit Care Med. 2000;162:1539-1546.[Abstract/Free Full Text]
  55. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989;49:6449-6665.[Abstract/Free Full Text]
  56. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol. 2003;5:741-747.[Medline]
  57. Busuttil RA, Rubio M, Dolle ME, Campisi J, Vijg J. Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture. Aging Cell. 2003;2:287-294.[Medline]
  58. Lambert AJ, Boysen HM, Buckingham JA, et al. Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms. Aging Cell. 2007;6:607-618.[Medline]
  59. Gille JJP, Joenje H. Cell culture models for oxidative stress: superoxide and hydrogen peroxide versus normobaric hyperoxia. Mutat Res. 1992;275:405-414.[Medline]
  60. de Haan JB, Crack PJ, Flentjar N, Iannello RC, Hertzog PJ, Kola I. An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse. Redox Rep. 2003;8:69-79.[Medline]
  61. Buffenstein R. Ecophysiological responses of subterranean rodents to underground habitats. In: Lacey EA, Patton JL, Cameron GN, eds. Life Underground: The Biology of Subterranean Rodents. Chicago, IL: University of Chicago Press; 2000:62–110.
  62. Lavocat R. Rodentia and Lagomorpha. In: Magio VJ, Cooke HBS, eds. Evolution of African Mammals. Cambridge, MA: Harvard University Press; 1978:69–89.
  63. Daly TJM, Buffenstein R. Skin morphology and its role in thermoregulation in mole-rats, Heterocephalus glaber and Cryptomys hottentotus. J Anat. 1998;193:495-502.[Medline]
  64. Contreras LC, McNab BK. Thermoregulation and energetics in subterranean mammals. Prog Clin Biol Res. 1990;335:231-250.[Medline]
  65. Buffenstein R. Ecophysiological responses to a subterranean habitat; a Bathyergid perspective. Mammalia. 1996;60:591-605.
  66. Salmon AB, Ljungman M, Miller RA. Cells from long-lived mutant mice exhibit enhanced repair of ultraviolet lesions. J Gerontol A Biol Sci Med Sci. 2008;63A:219-231.[Abstract/Free Full Text]
  67. Sancar A. DNA excision repair. Ann Rev Biochem. 1996;65:43-81.[Medline]
  68. Reardon JT, Sancar A. Recognition and repair of the cyclobutane thymine dimer, a major cause of skin cancers, by the human excision nuclease. Genes Dev. 2003;17:2539-2551.[Abstract/Free Full Text]
  69. Huang J, Hsu DS, Kazantsev A, Sancar A. Substrate spectrum of human excinuclease: repair of abasic sites, methylated bases, mismatches, and bulky adducts. Proc Natl Acad Sci U S A. 1994;91:12213-12217.[Abstract/Free Full Text]
  70. Mitchell JR, Hoeijmakers JH, Niedernhofer LJ. Divide and conquer: nucleotide excision repair battles cancer and ageing. Curr Opin Cell Biol. 2003;15:232-240.[Medline]
  71. Else PL, Hulbert AJ. An allometric comparison of the mitochondria of mammalian and reptilian tissues: the implications for the evolution of endothermy. J Comp Physiol B Biochem System Environ Physiol. 1985;156:3-11.
  72. Else PL, Hulbert AJ. Evolution of mammalian endothermic metabolism: "leaky" membranes as a source of heat. Am J Physiol Regul Integr Comp Physiol. 1987;253:R1-R7.[Abstract/Free Full Text]
  73. Sohal RS, Sohal BH, Brunk UT. Relationship between anti-oxidant defenses and longevity in different mammalian species. Mech Ageing Dev. 1990;53:217-227.[Medline]
  74. Janssen M, van der Meer P, de Jong JW. Antioxidant defenses in rat, pig, guinea pig, and human hearts: comparison with xanthine oxidoreductase activity. Cardiovasc Res. 1993;27:2052-2057.[Abstract/Free Full Text]
  75. Himeno S, Takekawa A, Imura N. Species difference in hydroperoxide-scavenging enzymes with special reference to glutathione peroxidase in guinea-pigs. J Comp Physiol B Biochem System Environ Physiol. 1993;104:27-31.
  76. Floreani M, Napoli E, Palatini P. Role of antioxidant defences in the species-specific response of isolated atria to menadione. Comp Biochem Physiol C Toxicol Pharmacol. 2002;132:143-151.[Medline]
  77. Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ. 2006;13:363-373.[Medline]
  78. Wu J, Kaufman RJ. From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ. 2006;13:374-384.[Medline]
  79. Yoshida H. ER stress and diseases. Eur J Biochem. 2007;274:630-658.[Medline]
  80. Sierra F. Is (your cellular response to) stress killing you? J Gerontol A Biol Sci Med Sci 2006;61:557-561.[Abstract/Free Full Text]
  81. Viswanathan M, Kim SK, Berdichevsky A, Guarente L. A role for SIR-2.1 regulation of ER stress response genes in determining C. elegans life span. Dev Cell. 2005;9:605-615.[Medline]
  82. Hussain SG, Ramaiah KV. Reduced eIF2alpha phosphorylation and increased proapoptotic proteins in aging. Biochem Biophys Res Commun. 2007;355:365-370.[Medline]
  83. Li J, Holbrook NJ. Elevated gadd153/chop expression and enhanced c-Jun N-terminal protein kinase activation sensitizes aged cells to ER stress. Exp Gerontol. 2004;39:735-744.[Medline]
  84. Rabek JP, Boylston WH, III, Papaconstantinou J. Carbonylation of ER chaperone proteins in aged mouse liver. Biochem Biophys Res Commun. 2003;305:566-572.[Medline]



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