Oxidative stress and expression of chaperones in aging mollusks

Available online at www.sciencedirect.com

Comparative Biochemistry and Physiology, Part B 150 (2008) 53 – 61 www.elsevier.com/locate/cbpb

Oxidative stress and expression of chaperones in aging mollusks Anna V. Ivanina, Inna M. Sokolova, Alexey A. Sukhotin ⁎ Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte NC, 28223, USA Received 6 November 2007; received in revised form 22 January 2008; accepted 24 January 2008 Available online 6 February 2008

Abstract The mechanisms of aging are not well understood in animals with continuous growth such as fish, reptiles, amphibians and numerous invertebrates, including mollusks. We studied the effects of age on oxidative stress, cellular defense mechanisms (including two major antioxidant enzymes, superoxide dismutase (SOD) and catalase), and molecular chaperones in two mollusks — eastern oysters Crassostrea virginica and hard clams Mercenaria mercenaria. In order to detect the age-related changes in these parameters, correction for the effects of size was performed where appropriate to account for growth-related dilution. Fluorescent age pigments accumulated with age in both species. Protein carbonyls did not change with age or size indicating that they are not a good marker of aging in mollusks possibly due to the fast turnover and degradation of oxidized proteins in growing tissues. SOD did not show a compensatory increase with aging in either species, while catalase significantly decreased with age. Mitochondrial heat shock protein (HSP60) decreased with age in mollusks suggesting an age-related decline in mitochondrial chaperone protection. In contrast, changes in cytosolic chaperones were species-specific. HSP70 increased and HSP90 declined with age in clams, whereas in oysters HSP70 expression did not change, and HSP90 increased with aging. © 2008 Elsevier Inc. All rights reserved. Keywords: Aging; Oxidative stress; Heat shock proteins; Antioxidants; Mollusk

1. Introduction Energy production in aerobes is accompanied by generation of reactive oxygen species, driving cascades of chemical reactions, which can cause extensive cellular damage if run unchecked. According to numerous “stochastic” theories of aging, malfunction of damaged molecules and organelles as well as accumulation of end products of oxidative reactions accelerate (if not cause) aging and death in aerobic organisms (Medvedev, 1990; Cadenas and Packer, 1999; Kowald, 2001; Brunk and Terman, 2002; Yin and Chen, 2005). Cellular protective mechanisms against oxidative stress include specific antioxidant systems (e.g., antioxidant enzymes, glutathione, vitamins, etc.) and nonspecific molecules (e.g., molecular chaperones such as heat shock proteins). The degree and direction of change in antioxidant defenses during aging are tissue- and species-specific (Rikans and Hornbrook, 1997; Wei and Lee, 1998; Gianni et al., 2004; Fulle ⁎ Corresponding author. Tel.: +1 704 687 8508; fax: +1 704 687 3128. E-mail address: [email protected] (A.A. Sukhotin). 1096-4959/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2008.01.005

et al., 2005; Meng et al., 2007 and references therein), and functional significance of this variation is still debated. While increase in antioxidants with age is often interpreted as a compensatory defense against the escalating generation of free radicals, the age-related decline in antioxidant activity is considered a failure to synthesize sufficient antioxidants to prevent oxidative damage and is viewed as a cause of aging. Notably, in some organisms or tissues where production of free radicals may decline with age due to metabolic rate depression, a decrease in production of antioxidants may reflect reduced need rather than a failure to synthesize them. In addition to enzymatic and non-enzymatic antioxidants, an important aspect of aging process in animals involves functional changes in the molecular chaperone systems including heat shock proteins (HSPs) responsible for folding/refolding of newly synthesized and damaged proteins as well as for sequestering and degradation of proteins that are damaged beyond repair. Aging-related damage to the chaperone system may result in the impaired folding of the de novo synthesized cellular proteins and most importantly, in the faulty repair and

54

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

degradation of the proteins damaged by the senescence-related posttranslational modifications such as oxidation, glycation, deamidation, etc. Several recent studies on mammalian and insect models have addressed the changes in the constitutive levels of HSPs during aging as well as changes in their inducibility in response to stress (review in Sőti and Csermely, 2000, 2002). These studies show that similar to antioxidants, the degree and direction of changes in the constitutive levels of HSPs during senescence are tissue- and species-specific rising in some species and tissues and decreasing in others (Sőti and Csermely, 2000, 2002), whereas inducibility of HSPs is typically attenuated by senescence that may account for the impaired ability to respond to environmental stress in aged organisms. The diversity of aging-related changes in the cellular protective mechanisms makes it clear that more studies are needed, especially in the organisms with different types of metabolic physiology and rates of aging in order to gain a better insight into the physiological diversity and functional significance of antioxidant and chaperone response in aging. Currently, most studies on aging-related changes in cellular defense systems have been conducted on model vertebrates (such as mammals) and invertebrates (such as insects, or nematode Caenorhabditis elegans) which are characterized by definitive growth which stops after reaching maturity when senescence sets in. In contrast to these model organisms and similar to many other ectotherms including fish, amphibians, reptiles and numerous invertebrates, mollusks possess asymptotic growth (i.e., they grow during all their life although at a progressively declining rate). The aging processes and mechanisms including the effects of aging on cellular antioxidants and chaperones such as HSPs in these organisms are not well understood. Studies of agerelated physiological changes in continuously growing animals are further complicated by correlation between age and size. The influence of constantly increasing size may confound the effects of aging many physiological and cellular functions; therefore, in order to specifically detect age-related changes, allometric (size) effects should be tested and if needed, corrected for. The aim of this study was to determine the effects of age on oxidative stress markers (such as protein carbonyls and fluorescent age pigments (FAPs)) and cellular defense mechanisms including two major antioxidant enzymes — superoxide dismutase (cytosolic and mitochondrial SOD) and catalase, and molecular chaperones, including cytoplasmic HSP70 and 90 and predominantly mitochondrial HSP60 in two mollusk species — the eastern oyster Crassostrea virginica and the hard clam Mercenaria mercenaria. Oysters are short-lived fast growing mollusks, with a life span of 5–6 years in North Carolina and Virginia (Kirby and Miller, 2005; J. Swartzenberg, J&B AquaFood, personal communication). M. mercenaria has a longer life span (on average 25 years, Loesch and Haven, 1973) and grows at a relatively slow rate. Both species, however, reach sexual maturation at roughly the same age, during their second year of life (Stanley and de Witt, 1983; Eversole, 1987 for M. mercenaria; J. Swartzenberg, J&B Aquafood, personal communication for C. virginica in NC). Therefore, different rates of aging are expected in these species, with less pronounced aging manifestations in M. mercenaria compared

to C. virginica at the same chronological age. Characterization of key antioxidants and molecular chaperone systems from cytoplasm and mitochondria of slowly aging molluskan models will contribute to a comprehensive picture of aging in animals with different types of metabolic physiology and growth. 2. Materials and methods 2.1. Animals Wild-cultured eastern oysters C. virginica and hard clams M. mercenaria were purchased from a commercial shellfish grower (J&B AquaFood, Inc.) from Stump Sound, NC, USA. Exact age of all experimental mollusks was known because of the known time of settlement. Oysters of five age classes (0.58 (7 months), 0.83 (10 months), 1.5, 2, 2.5 and 4 years old), and clams of three age classes (1, 2 and 4 years old) were studied. Mollusks were placed in temperature-controlled recirculating water tanks with biological filtration systems and protein skimmers and maintained at 20 °C and 30 ppt salinity for three days. They were fed ad libitum with commercial algal blend (Premium Reef Blend, DT's Plankton Farm, Sycamore, IL, USA). Then animals were sacrificed, gills were extracted, weighed and frozen in liquid nitrogen for further measurements. The rest of the soft tissues was blotted dry on a tissue paper and weighed. 2.2. Analyses of metabolites and enzyme activities Fluorescent age pigments (FAPs) were determined by an extraction method (Nicol, 1987; Vernet et al., 1988). Gill tissue was ground under liquid nitrogen and homogenized (1:20 w/v) in a chloroform–methanol solution (2:1 v/v). After 10 min of centrifugation at 2000 ×g the FAPs levels were analyzed in the chloroform phase using a fluorescence spectrophotometer (F2500, Hitachi). An emission spectrum between 350 and 550 nm was obtained at an excitation wavelength of 350 nm. The luminescence of the sample was determined at an emission maximum of 432 nm. FAPs concentrations were expressed as relative fluorescence intensity (RFI) according to Hill and Womersley (1991), using 0.1 μg quinine sulfate per mL of 1 N H2SO4 as a standard. Protein carbonyl groups were measured spectrophotometrically as described in Levine et al. (1990) and Philipp et al. (2005). Tissues were ground under liquid nitrogen and homogenized in the buffer containing 50 mM HEPES, 125 mM KCl, 1.1 EDTA and 0.6 mM MgSO4 (pH 7.4) and protease inhibitors [leupeptin (0.5 μg mL− 1), pepstatin (0.7 μg mL− 1), phenylmethylsulfonyl fluoride (40 μg mL− 1) and aprotinin (0.5 μg mL− 1)]. Samples were centrifuged at 100,000 ×g for 15 min, supernatant was collected and incubated at room temperature with 10 mM 2,4dinitrophenylhydrazine (DNP) in 2 M HCl. The blanks were incubated with HCl without DNP. After incubation, proteins were precipitated by adding 100% TCA and centrifuged at 11,000 ×g for 10 min. The pellet was collected, washed with ethanol ethylacetate (1:1) and resuspended in 6 M guanidine hydrochloride in 20 mM in KH2PO4 (pH 2.5) until dissolved. The absorbance was measured at 360 nm on a spectrophotometer (Cary 50, Varian)

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

using guanidine HCl solution as reference. The amount of carbonyls was estimated as a difference in absorbance between samples and blanks using a molar extinction coefficient of carbonyls ɛ = 22,000 cm− 1 M− 1. Amount of carbonyls was expressed per mg total protein measured in the same samples using Bradford method. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by a colorimetric method using an SOD Assay KitWST (Fluka) according to the manufacturer's protocol. The assay is based on inhibition by SOD of the reduction of O2 and accompanying production of formazan dye recorded at 450 nm. Catalase (EC 1.11.1.6) activity (CAT) was measured using 0.05 M potassium phosphate as a homogenization buffer (pH = 7.0) and 10.5 mM H2O2 as a substrate as described in Abele-Oeschger et al. (1994). The degradation of peroxide was monitored at 240 nm and 25 °C. One unit of CAT decomposes 1 μmol H2O2 min− 1. 2.3. Immunoblotting of heat shock proteins Gill tissues from oysters and clams of different ages were homogenized in ice-cold buffer containing 100 mM Tris, pH = 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X, 10% glycerol, 0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 0.5 μg mL− 1 leupeptin, 0.7 μg mL− 1 pepstatin, 40 μg mL− 1 phenylmethylsulfonyl fluoride (PMSF), and 0.5 μg mL− 1 aprotinin. HSPs were detected using standard immunoblotting methods using antibodies known to crossreact with molluskan HSPs (Snyder et al., 2001; Hamdoun et al., 2003; Franco et al., 2006). Briefly, the homogenate was sonicated three times for 10 s each (output 69 W, Sonicator 3000, Misonix, Farmingdale, NY, USA) and centrifuged at 14,000 ×g for 5 min at 4 °C. Protein content was measured in the supernatant using Bio-Rad Protein Assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). 30 μg of sample protein per lane was loaded onto 8% polyacrylamide gels and run at 100 mA for 2 h at room temperature. The resolved proteins were transferred onto a nitrocellulose membrane in 96 mM glycine, 12 mM Tris and 20% methanol (v/v) using a Trans-Blot semi-dry cell (Thermo Fisher Scientific Inc, Portsmouth, NH, USA). To verify equal protein loading, membranes were stained with Amido Black Stain Solution (1 g L− 1 Amido Black in 10% methanol, 10% glacial acetic acid) for 30 s. The membranes were then destained by washing in several changes of water and blocked overnight in 5% nonfat milk in Tris-buffered saline, pH = 7.6 (TBST). Blots were probed with primary monoclonal antibodies against HSP70, HSP90 and HSP60, respectively (MA3-007, Affinity Bioreagents, Golden, CO, USA; SPA-835 & SPA-805, Stressgen Bioreagents, Ann Arbor, MI, USA). After washings off the primary antibody, membranes were probed with the respective polyclonal secondary antibodies conjugated with horseradish peroxidase (Jackson Immunoresearch, West Grove, PA, USA) and proteins detected by enhanced chemiluminescence according to the manufacturer's instructions (Pierce, Rockford, IL, USA). Densitometric analysis of the signal was performed by GelDoc 2000™ System with Quantity One 1-D Analysis

55

Software (Bio-Rad Laboratories Inc., Hercules, CA, USA). Each blot included a sample from the youngest age group as an internal standard, and expression of HSPs was expressed as % of the youngest age group of the respective species. 2.4. Statistics The effect of size of mollusk size on FAPs, protein carbonyls levels and SOD and catalase activities was tested by power regressions of the respective parameter on individual wet tissue mass. One-way ANOVA was used to analyze the effects of the factors Age and Species on FAPs, protein carbonyls levels and enzyme activities, followed by post-hoc comparisons by Tukey's HSD test for unequal N. Effect of age on HSPs expression was tested using repeated measures ANOVA with individual immunoblotting gels used as repeated variable in order to account for gel-to-gel variability; the test was followed by post-hoc comparisons between individual age groups. Although ANOVA was performed on original densitometry data for immunoblots, data on the graphs are represented as % of expression in the youngest age group for the sake of clarity and ease of

Fig. 1. Size-related changes in FAPs content in 7-month old C. virginica (A). Age effect on FAPs concentration in C. virginica and M. mercenaria (B). FAPs levels were standardized to the average mean size of mollusks (8 g tissue wet mass) using the power function (see graph 1A). A trend line on B refers to oysters only; since FAPs content in M. mercenaria changed non-monotonically with age, no trend line is given. Vertical bars represent S.E.M. N = 3–15 for different age groups.

56

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

comparisons. All data are presented as mean values ± standard errors of means (S.E.M.) unless specified otherwise. 3. Results 3.1. Oxidative stress markers Variation of size in 7-month old oysters (0.22–1.97 g wet tissue mass) allowed us to estimate the age-independent effects of size on tissue FAPs levels. As expected, within the 7-month old age group, FAPs progressively decreased with increase of tissue wet mass (Fig. 1A). This relationship between size and FAPs concentration (independent of age) can be described with a power function (regression analysis, n = 10, P b 0.001) (Fig. 1A). Due to the small size range within each studied age group of M. mercenaria, the regression of FAPs concentration on size could not be obtained. However, similarity of power coefficients for FAPs concentration vs size in oysters (this study) and mussels Mytilus edulis (Sukhotin et al., 2002) suggests that they are conserved in bivalves; therefore, the same regression coefficient (− 0.52) was used to correct for size effect on FAPs concentration in C. virginica and M. mercenaria.

Fig. 3. Age-dependent expression of mitochondrial HSP60 in C. virginica (A) and M. mercenaria (B). Horizontal lines connect values, which do not significantly differ (P N 0.05 according to the post-hoc comparisons). Vertical bars represent S.E.M. N = 5–6.

Fig. 2. Superoxide dismutase (A) and catalase (B) activity in gills of C. virginica and M. mercenaria as a function of age. Size had no significant effect on SOD or CAT activities (P N 0.05); therefore, no correction for size was performed. Vertical bars represent S.E.M. N = 3–15 for different age groups.

To analyze the effects of age on FAPs, FAPs levels were standardized to the common mean size of mollusks (8 g tissue wet mass) using the above described power function (Fig. 1A). Age of mollusks significantly affected FAPs content in gills of both species (ANOVA, F5,44 = 13.8, P b 0.0001 for C. virginica and F2,13 = 4.9, P b 0.05 for M. mercenaria). Age-related FAPs accumulation followed somewhat different patterns in the two studied species. The rate of accumulation was similar in younger oysters and clams (up to 2 years of age). Later, FAPs concentration continued to increase significantly in oysters although at a slower rate than between 0.58 and 2 years of age. In contrast, in hard clams it did not increase between 2 and 4 years of age (Fig. 1B). Thus, 4-year old hard clams accumulated 1.75 times less FAPs per gram gill tissues than oysters of the same chronological age (t-test, t = 2.83, df = 18, P = 0.011), while younger mollusks did not show interspecific differences in this parameter. Protein carbonyls concentration was three times lower in oysters (2.85 ± 0.24 nmol mg− 1 protein) than in hard clams (10.51 ± 0.69 nmol mg− 1 protein) (ANOVA, F1,29 = 123.3, P b 0.001). No significant effects of size on protein carbonyl content were found; therefore, size correction was not needed for this parameter. Age had no significant effect on protein carbonyl content in clams or oysters.

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

3.2. Activity of antioxidant enzymes Neither SOD nor CAT activity showed significant dependence on size of mollusks at any of the studied ages. Thus, no correction for size effects was needed for these parameters. SOD activity was higher in M. mercenaria than in C. virginica of the respective chronological age (Tukey's HSD, P b 0.05) (Fig. 2A). In oysters, age significantly affected SOD activity (ANOVA, F5,39 = 3.1, P = 0.018), although the change was not monotonic. Thus, high SOD values were recorded in 7-month, 1.5- and 2.5-year old oysters, while relatively low activity was found in 10-month and 4-year old ones. In hard clams age effect was not significant. As opposed to SOD, catalase activity was 3.5–4 times higher in oysters than in hard clams of the respective chronological age (Fig. 2B). CAT levels progressively decreased with age in oysters (from about 1300 in the youngest to 560 U g− 1 wet tissues in the oldest) (ANOVA, F5,41 = 4.6, P = 0.002). In contrast, while a similar tendency of CAT decrease with age was found in M. mercenaria, it was not significant. 3.3. Heat shock protein expression Mitochondrial HSP60 demonstrated a significant decrease with age in both studied species (Fig. 3). HSP60 expression

57

decreased by 50–72% in the oldest studied individuals (4– 5 years old) compared to the respective youngest age groups (7–12 months old). In both species, a decline in HSP60 started at around 2 years of age (bearing in mind that the age resolution was 0.5–1 year in this study). In contrast, age-associated changes in expression of cytosolic heat shock proteins HSP70 and HSP90 differed in oysters and hard clams. In M. mercenaria, there was a nearly 30% decrease in HSP90 and 50% increase in HSP70 expression in the oldest animals compared to the young ones (Fig. 4B, D). In contrast, in C. virginica HSP70 levels did not change with age whereas HSP90 levels increased by approximately 50% in older animals (Fig. 4A, C). When present, the age-related changes in HSP70 and/or HSP90 became apparent around 2 years of age in both studied species (but see the caveat about the age resolution above). 4. Discussion Applying size correction to a physiological or biochemical parameter affected by growth is critical for determination of the true age effects on this parameter in continuously growing animals. Accumulation of FAPs with age is an excellent example of the efficiency of this approach. FAPs, including lipofuscin are end products of lipid and protein oxidation which are only slowly removed from cells. They accumulate in non-

Fig. 4. Age-dependent expression of cytosolic HSP70 (A in C. virginica, B in M. mercenaria) and HSP90 (C in C. virginica, D in M. mercenaria). Horizontal lines connect values, which do not significantly differ (P N 0.05 according to the post-hoc comparisons). Vertical bars represent S.E.M. N = 5–6.

58

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

proliferating tissues like neural tissue, retina, myocardium, etc., and thus can serve as a good biomarker of physiological aging. However, in a growing organism or in proliferating tissues or cell culture, accumulation of FAPs with age goes in parallel with its “dilution” due to active cell division (Sitte et al., 2001). An approach correcting for size effects used in our study allowed us to separate these two processes: FAPs accumulation and growth-related dilution and to detect clear trends for age-related accumulation of FAPs in both species. Both hard clams and oysters showed rapid age-related FAPs accumulation during the first 2 years followed by a somewhat slower increase at older ages in oysters and essential absence of further accumulation between ages 2 and 4 in hard clams (Fig. 1). It is worth noting that unlike oysters, oldest hard clams used in the study have not approached their potential maximal life span; thus, it is possible that further accumulation of FAPs may occur later in life in clams. This suggests progressive cellular lipid peroxidation by free radicals with aging, with especially fast rate in early life. The increase in FAPs levels with age found in this study agrees with the patterns earlier described in other mollusks (Sepia lolligunculus, Zielinski and Pörtner, 2000; Eurhomalea exalbida, Lomovasky et al., 2002; M. edulis, Sukhotin et al., 2002; Laternula elliptica, Philipp et al., 2005; Aequipecten opercularis and Adamussium colbecki, Philipp et al., 2006), although in some species this pattern is more complex with initial decrease and increase at more advanced ages (digestive gland of Monodonta lineata, Clarke et al., 1990; Mya arenaria, Philipp et al., 2005), and in rare cases, even a decrease with age can be found (e.g. in ganglia of M. lineata, Clarke et al., 1990). Notably, a decrease of FAPs concentration with age has been reported for some fish species (e.g., Mullin and Brooks, 1988; Vernet et al., 1988) and reptiles (Majhi et al., 2000), but not in animals with no post-maturational growth like insects (Sheldahl and Tappel, 1974; Sohal and Donato, 1979; Ettershank et al., 1983) or nematodes (Davis et al., 1982). Likely, the age-related decrease in FAPs content recorded in some of the earlier studies on continuously growing organisms may be due to the fact that no size correction was applied and thus steady-state FAPs levels reflected age-related accumulation, break-down (which is generally slow but likely to be faster in actively growing tissues) and growth “dilution” of FAPs due to the fact that cell growth and division rates outstrip the age-related FAPs accumulation. The latter phenomenon has been clearly shown in fibroblast cell culture in vitro (Sitte et al., 2001). Thus, if not corrected for size (or growth rate), agerelated FAPs accumulation per unit mass may be underestimated in growing tissues. This emphasizes the importance of allometric correction for age-related parameters in aging studies of continuously growing organisms. Slower FAPs accumulation at old ages can in principle indicate a decreasing rate of free radicals production and/or enhanced levels of antioxidant protection with age in the studied species. However, this explanation unlikely applies to the studies species in this research, at least as far as two key antioxidant enzymes (CAT and SOD) are concerned, since they showed no increase in activity (and in fact a decline in the case of CAT) at older ages in oysters or hard clams. Another possible

explanation for slowing down the rate of lipid peroxidation at advanced ages in oysters and hard clams may involve an effective decline in metabolic rate accompanied by a reduction in free radicals generation. Indeed, the allometric decrease in mass-specific metabolic rate within and across species is typical for virtually all animals (review in: West et al., 2002; Hochachka et al., 2003). However, allometry alone cannot fully explain the observed reduced rate of tissue oxidation in mollusks. For example, average size of M. mercenaria of 2 and 4 years was similar implying similar mass-specific metabolic rates, whereas no additional accumulation of FAPs was found between ages 2 and 4 suggesting slower-than-expected rate of lipid peroxidation in 4-year old mollusks. Possibly, the older mollusks were able to further decrease metabolic rate beyond the levels dictated by their body size, thus lowering free radical generation and thus reducing the need for energetically costly production of antioxidant enzymes and chaperones. Such agedependent metabolic rate depression (not explained by the effects of larger body size) has been recorded in other continuously growing species including bivalve mollusks M. edulis (Sukhotin and Pörtner, 2001; Sukhotin et al., 2006), Crenomytilus grayanus (Zolotarev and Ryabushko, 1977), Argopecten irradians irradians (Bricelj et al., 1987), and fish Cichlasoma nigrofasciatum (Fidhiany and Winckler, 1998). It would be interesting to determine whether such age-related metabolic depression (irrespective of and additional to the mass-related metabolic decline) is typical of continuously growing animals and whether it may contribute to the slower rates of lipid peroxidation at older ages. Another marker of oxidative damage, protein carbonyls, represents one of the several types of protein oxidation products (reviewed in Stadtman and Levine, 2000), which can accumulate with age and facilitate irreversible changes associated with aging (Yin and Brunk, 1995; Yin and Chen, 2005). Age-related changes in protein carbonyl content vary greatly between different studied species. In species with definitive growth, such as mammals and insects (flies), protein carbonyls content tends to increase with age although they can be modulated by factors affecting metabolic activity and/or oxidative stress (Sohal, 2002; Yin and Chen, 2005). In contrast, in mollusks age-related changes in protein carbonyls do not show a consistent trend. Thus, in an Antarctic bivalve L. elliptica protein carbonyls accumulated with age of mollusks, while in a temperate clam M. arenaria the highest levels of carbonylated proteins were recorded in young animals (Philipp et al., 2005). Recent studies on very old (up to 192 years old) ocean quahog Arctica islandica showed very low levels of protein carbonyls (0.5–1 nmol per mg proteins) with no trend of age-associated increase, indicating an efficient autophagic removal (Strahl et al., 2007). In our study neither age nor size of mollusks had a significant effect on protein carbonyls content in hard clams or oysters. Notably, the absolute amount of protein carbonyls was significantly lower in gill tissues of C. virginica (2.5–3 nmol carbonyls per mg proteins, close to the levels recorded in some bivalves — cf. Philipp et al., 2005, 2006), than in hard clams (10.5 nmol carbonyls per mg proteins). This may reflect decreased protein turnover and/or elevated oxidative stress in

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

hard clams, or simply a basal difference in protein composition between the two species. In this context, it is interesting that hard clams had four times lower catalase activity than oysters of the respective ages, which may indicate their higher susceptibility to protein oxidation. It is worth noting that unlike FAPs, which are metabolized very slowly, oxidized (including carbonylated) proteins can be rapidly degraded by proteolytic activity of proteasome (Sitte et al., 1998; Stadtman and Levine, 2000), and therefore eliminated. Thus, it is likely that the absence of consistent accumulation of protein carbonyls with age in continuously growing species such as mollusks reflects effective degradation and removal of protein oxidation products preventing their accumulation in growing tissues. Two important antioxidant enzymes (CAT and SOD) showed discordant age-related trends in hard clams and oysters, with a decrease in CAT activity and no change in SOD activity with age. Notably, the age-dependent decline in CAT activity was more pronounced in oysters than in clams (and only statistically significant in the former). This may reflect the fact that the oldest clams used in this study have far not reached their maximum life span (around 25 years) unlike oysters that at 4 years of age were close to their longevity limit (5–6 years). Earlier studies show similar discrepancy in age-associated changes in the activity of SOD and CAT. Thus, elevated or constant levels of SOD and a decline in catalase with aging were recorded in mice (Andziak et al., 2005), bivalve mollusks M. edulis (Viarengo et al., 1991) and C. virginica (this study), the cephalopod Sepia officinalis (Zielinski and Pörtner, 2000) and the marine shrimp Aristeus antennatus (Mourente and DiazSalvago, 1999). In contrast, decrease in SOD and concomitant increase or no change in catalase activity with age have been shown in rats (Cand and Verdetti, 1989; Meng et al., 2007), fish larvae (Mourente et al., 1999), amphipod crustaceans Gammarus locusta (Correia et al., 2003), and an amphibian Rana perezi (López-Torrez et al., 1991). Other studies report no age-related variations in SOD and catalase activity (e.g., in bivalve mollusks M. edulis or M. mercenaria, Sukhotin et al., 2002; L. elliptica and M. arenaria, Philipp et al., 2005 or naked mole-rates, Andziak et al., 2005). This indicates that age-related responses of different antioxidant enzymes are species-specific and may be somewhat uncoupled possibly reflecting differences in regulation of different antioxidant enzymes, redundancy of cellular antioxidant defenses or some unidentified factors such as shifts in steady-state levels of different ROS with aging; the reasons for this are not well understood and require further investigation. This study for the first time showed significant age-related changes in molecular chaperones (cytosolic and mitochondrial HSPs) in continuously growing animals — oysters and hard clams. Interestingly, the most consistent pattern between the two studied species was a decline in the levels of mitochondrial HSP60 with age. This may have important implications for mitochondrial function because mitochondria are the major ROS producing site in eukaryotic cells, and mitochondrial proteins are among the earliest and most sensitive targets of oxidative damage during senescence. Currently, a few studies have addressed changes in HSP60 expression during senes-

59

cence. A strong decrease in HSP60 with aging was reported in hearts while an increase in HSP60 was found in the skeletal muscle of aging rats indicating that response may be tissuespecific (Colotti et al., 2005; Chung and Ng, 2006). Notably, studies on mitochondrial proteolysis also indicate that aging impairs mitochondrial protein degradation systems (Bota et al., 2002; Bakala et al., 2003; Delaval et al., 2004; Bulteau et al., 2006). In conjunction with a decreased expression of HSP60 this may result in accumulation and aggregation of damaged cellular proteins ultimately leading to the dysfunction and loss of the damaged mitochondrion. A similar decrease in the levels of HSP60 in the two studied species of mollusks which have different life spans may indicate that a decrease in mitochondrial HSP60 is an early sign of aging in mollusks. Alternatively, an age-dependent decrease in HSP60 may reflect a developmental change in mitochondrial function in mollusks unrelated to senescence such as a decrease in mitochondrial density with age or compensatory increase in other mitochondrial protection mechanisms such as antioxidants or protein degradation systems. Interestingly, the significant decrease in HSP60 levels occurs around 2 years of age in both studied species coinciding with sexual maturation (Stanley and de Witt, 1983; Eversole, 1987; J. Swartzenberg, J&B Aquafood, personal communication) indicating that this change may be due to the onset of gametogenesis and associated hormonal and metabolic shifts. Currently, very little is known about age-related changes in mitochondrial protective mechanisms or mitochondrial quantity and quality in mollusks, and this question must await further studies. In contrast to the mitochondrial HSP60, response of the cytoplasmic chaperones was less uniform and differed between the two studied species. In C. virginica, HSP90 levels increased with age whereas HSP70 levels remained unchanged. In contrast, in M. mercenaria HSP90 levels decreased while HSP70 expression increased with aging. Previous studies in other organisms including Drosophila and mammals indicate similar variability in cytoplasmic HSP response with increase (Wheeler et al., 1995; Maiello et al., 1998; Razzague et al., 1999; Cuervo and Dice, 2000), unchanged levels (Wu et al., 1993) or a decrease (Krawczyk and Szymic, 1989; Colotti et al., 2005) with aging. Increased levels of some cytoplasmic HSP isoforms during aging may indicate an attempt of the cell to counteract the increase in levels of modified proteins in the cytoplasm and/or to partially compensate for a decline in the quality and quantity of other cellular chaperones. Indeed, the fact that no accumulation of carbonylated proteins in aging mollusks was observed in the present study indirectly supports the suggestion about effective compensation of oxidative damage to proteins by cytosolic HSP isoforms. Since carbonylated proteins in this study were measured in the whole tissue extract, the bulk of extracted proteins were cytosolic. An increase in at least some isoforms of HSPs in cytoplasm during aging suggests that cytoplasmic chaperone systems may have more flexibility and capability to compensate for aging-related proteotoxicity than mitochondrial chaperones thus supporting the notion that mitochondria are the critical and most sensitive target in aging. Irrespective of the exact molecular mechanisms of age-related

60

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61

changes in chaperones, the observed expression pattern suggests that mitochondrial but not cytoplasmic HSPs can serve as a marker of aging in continuously growing organisms. In conclusion, this study shows that animals with continuous growth and slow aging, such as bivalve mollusks experience age-related oxidative stress. FAPs accumulation can serve as a good marker of aging and age-related oxidative damage in mollusks, especially those with a relatively short life span and faster aging. However, in order to detect the age-related changes in FAPs, size correction must be performed to account for growth effects. In contrast, protein carbonyls are not a good marker of aging in mollusks possibly due to their fast turnover and degradation. Cellular protection systems responded differently to aging in oysters in clams. Some parameters showed similar patterns of decrease (CAT activity, HSP60 expression) or no change (SOD) in both species, whereas others (HSP70 and 90) showed discordant patterns in oysters and hard clams indicating that these responses may be species-specific. Further studies addressing age-dependent changes in metabolic rates, and rates of generation and steady-state levels of different reactive species are needed in order to resolve these apparent controversies of age-related changes in cellular protective mechanisms in mollusks and shed new light onto the mechanisms of aging in continuously growing animals. Acknowledgements We thank Thuy-Anh Ngo, Tuong Vy Ngo, Calvin Wilson and Brian DeGuzman for technical assistance in the lab. This work was supported by the Faculty Research Grant to A.A.S. (UNCC, #1-11247), Research Development Initiative of the UNC General Administration grant to T.-A.N. and A.A.S. and by funds provided the National Science Foundation CAREER award to I.M.S. (IBN-0347238). References Abele-Oeschger, D., Oeschger, R., Theede, H., 1994. Biochemical adaptations of Nereis diversicolor (Polychaeta) to temporarily increased hydrogen peroxide levels in intertidal sandflats. Mar. Ecol. Prog. Ser. 106, 101–110. Andziak, B., O'Connor, T.P., Buffenstein, R., 2005. Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat. Mech. Ageing Dev. 126, 1206–1212. Bakala, H., Delaval, E., Hamelin, M., Bismuth, J., Borot-Aloi, C., Corman, B., Friguet, B., 2003. Changes in rat liver mitochondria with aging. Lon protease-like reactivity and N(epsilon)-carboxymethyllysine accumulation in the matrix. Eur. J. Biochem. 270, 2295–2302. Bota, D.A., Van Remmen, H., Davies, K.J., 2002. Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress. FEBS Lett. 532, 103–106. Bricelj, V.M., Epp, J., Malouf, R.E., 1987. Comparative physiology of young and old cohorts of bay scallop Agropecten irradians irradians (Lamarck): mortality, growth and oxygen consumption. J. Exp. Mar. Biol. Ecol. 112, 73–91. Brunk, U.T., Terman, A., 2002. The mitochondrial–lysosomal axis theory of aging. Accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur. J. Biochem. 269, 1996–2002. Bulteau, A.L., Szweda, L.I., Friguet, B., 2006. Mitochondrial protein oxidation and degradation in response to oxidative stress and aging. Exp. Gerontol. 41, 653–657.

Cadenas, E., Packer, L. (Eds.), 1999. Understanding the Process of Aging. Marcel Dekker Inc., New York, Basel. Cand, F., Verdetti, J., 1989. Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats. Free Radical Biol. Med. 7, 59–63. Chung, L., Ng, Y.C., 2006. Age-related alterations in expression of apoptosis regulatory proteins and heat shock proteins in rat skeletal muscle. Biochim. Biophys. Acta 1762, 103–109. Clarke, A., Kendall, M.A., Gore, D.J., 1990. The accumulation of fluorescent age pigments in the trochid gastropod Monodonta lineata. J. Exp. Mar. Biol. Ecol. 144, 185–204. Colotti, C., Cavallini, G., Vitale, R.L., Donati, A., Maltinti, M., Del Ry, S., Bergamini, E., Giannessi, D., 2005. Effects of aging and anti-aging caloric restrictions on carbonyl and heat shock protein levels and expression. Biogerontology 6, 397–406. Correia, A.D., Costa, M.H., Luis, O.J., Livingstone, D.R., 2003. Age-related changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in whole body Gammarus locusta (Crustacea: Amphipoda). J. Exp. Mar. Biol. Ecol. 289, 83–101. Cuervo, A.M., Dice, J.F., 2000. Age-related decline in chaperone-mediated autophagy. J. Biol. Chem. 275, 31505–31513. Davis, B.O., Anderson, G.L., Dusenbery, D.B., 1982. Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans. Biochemistry 21, 4089–4095. Delaval, E., Perichon, M., Friguet, B., 2004. Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart. Eur. J. Biochem. 271, 4559–4564. Etterhank, G., 1983. Age structure and cyclical annual size change in the Antarctic krill Euphausia superba. Polar Biol. 2, 189–193. Eversole, A.G., 1987. Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (South Atlantic) — Hard Clam. U.S. Fish Wildl. Serv. Biol. Rep., vol. 82(11.75). U.S. Army Corps of Engineers. TR EL-82-4. 33 pp. Fidhiany, L., Winckler, K., 1998. Influence of body mass, age, and maturation on specific oxygen consumption in a freshwater cichlid fish, Cichlasoma nigrofasciatum (Guenther, 1869). Comp. Biochem. Physiol. 119A, 613–619. Franco, J.F., Trivella, D.B.B., Trevisan, R., Dinslaken, D.F., Marques, M.R.F., Bainy, A.C.D., Dafre, A.L., 2006. Antioxidant status and stress proteins in the gills of the brown mussel Perna perna exposed to zinc. Chem.Biol. Interac. 160, 232–240. Fulle, S., Di Donna, S., Puglielli, C., Pietrangelo, T., Beccafico, S., Bellomo, R., Protasi, F., Fano, G., 2005. Age-dependent imbalance of the antioxidative system in human satellite cells. Exp. Gerontol. 40, 189–197. Gianni, P., Jan, K.J., Douglas, M.J., Stuart, P.M., Tarnopolsky, M.A., 2004. Oxidative stress and the mitochondrial theory of aging in human skeletal muscle. Exp. Gerontol. 39, 1391–1400. Hamdoun, A.M., Cheney, D.P., Cherr, G.N., 2003. Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oysters (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance. Biol.Bull. 205, 160–169. Hill, K.T., Womersley, C., 1991. Critical aspects of fluorescent age-pigment methodologies: modification for accurate analysis and age assessments in aquatic organisms. Mar. Biol. 109, 1–11. Hochachka, P.W., Darveau, C.A., Andrews, R.D., Suarez, R.K., 2003. Allometric cascade: a model for resolving body mass effects on metabolism. Comp. Biochem. Physiol. A 134, 675–691. Kirby, M.X., Miller, H.M., 2005. Response of a benthic suspension feeder (Crassostrea virginica Gmelin) to three centuries of anthropogenic eutrophication in Chesapeake Bay. Estuarine, Coastal Shelf Sci. 62, 679–689. Kowald, A., 2001. The mitochondrial theory of aging. Biol. Signal. Recept. 10, 162–175. Krawczyk, Z., Szymic, N., 1989. Effect of age and busulphan treatment of the hsp 70 gene-related transcript level in rat testes. Int. J. Androl. 12, 72–79. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.-G., Ahn, B.-W., Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Meths Enzymol. 186, 464–478.

A.V. Ivanina et al. / Comparative Biochemistry and Physiology, Part B 150 (2008) 53–61 Loesch, J.G., Haven, D.S., 1973. Estimated growth functions and size–age relationships of the hard clam, Mercenaria mercenaria, in the York River, Virginia. The Veliger 16, 76–81. Lomovasky, B.J., Morriconi, E., Brey, T., Calvo, J., 2002. Individual age and connective tissue lipofuscin in the hard clam Eurhomalea exalbida. J. Exp. Mar. Biol. Ecol. 276, 83–94. López-Torrez, M., Pérez-Campo, R., Barja de Quiroga, G., 1991. Effect of natural ageing and antioxidant inhibition on liver antioxidant enzymes, glutathione system, peroxidation, and oxygen consumption in Rana perezi. J. Comp. Physiol. 160B, 655–661. Maiello, M., Boeri, D., Sampietro, L., Pronzato, M.A., Odetti, P., Marinari, U.M., 1998. Basal synthesis of heat shock protein 70 increases with age in rat kidneys. Gerontology 44, 15–20. Majhi, S., Jena, B.S., Patnaik, B.K., 2000. Effect of age on lipid peroxides, lipofuscin and ascorbic acid contents of the lungs of male garden lizard. Comp. Biochem. Physiol. C 126, 293–298. Medvedev, Z.H.A., 1990. An attempt at a rational classification of theories of ageing. Biol. Rev. 65, 375–398. Meng, Q., Wong, Y.T., Chen, J., Ruan, R., 2007. Age-related changes in mitochondrial function and antioxidative enzyme activity in fisher 344 rats. Mech. Ageing Dev. 128, 286–292. Mourente, G., Diaz-Salvago, E., 1999. Characterisation of antioxidant systems, oxidation status and lipids in brain of wild-caught size-class distributed Aristeus antennatus (Risso, 1816) Crustacea, Decapoda. Comp. Biochem. Physiol. B 124, 405–416. Mourente, G., Tocher, D.R., Diaz, E., Grau, A., Pastor, E., 1999. Relationships between antioxidants, antioxidant enzyme activities and lipid peroxidation products during early development in Dentex dentex eggs and larvae. Aquaculture 179, 309–324. Mullin, M.M., Brooks, E.R., 1988. Extractable lipofuscin in larval marine fish. Fish. Bull. 86, 407–415. Nicol, S., 1987. Some limitations on the use of the lipofuscin ageing techniques. Mar. Biol. 93, 609–614. Philipp, E., Brey, T., Pörtner, H.-O., Abele, D., 2005. Chronological and physiological ageing in a polar and a temperate mud clam. Mech. Ageing Dev. 126, 598–609. Philipp, E., Heilmayer, O., Brey, T., Abele, D., Pörtner, H.-O., 2006. Physiological ageing in a polar and a temperate swimming scallop. Mar. Ecol. Prog. Ser. 307, 187–198. Razzaque, M.S., Shimakawa, I., Nazneen, A., Lui, D., Naito, T., Higami, Y., Taguchi, T., 1999. Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fischer 344 rat kidney. Histochem. J. 32, 123–132. Rikans, L.E., Hornbrook, K.R., 1997. Lipid peroxidation, antioxidant protection and aging. Biochim. Biophys. Acta 1362, 116–127. Sheldahl, J.A., Tappel, A.L., 1974. Fluorescent products from aging Drosophila melanogaster: an indicator of free radical lipid peroxidation damage. Exp. Gerontol. 9, 33–41. Sitte, N., Merker, K., Grune, T., 1998. Proteasome-dependent degradation of oxidized proteins in MRC-5 fibrobroblasts. FEBS Lett. 440, 399–402. Sitte, N., Merker, K., Grune, T., von Zglinicki, T., 2001. Lipofuscin accumulation in proliferating fibroblasts in vitro: an indicator of oxidative stress. Exp. Gerontol. 36, 475–486. Snyder, M.J., Girvetz, E., Mulder, E.P., 2001. Induction of marine mollusk stress proteins by chemical or physical stress. Arch. Environ. Contam. Toxicol. 41, 22–29.

61

Sohal, R.S., 2002. Role of oxidative stress and protein oxidation in the aging process. Free Radical Biol. Med. 33, 37–44. Sohal, R.S., Donato, H., 1979. Effects of experimentally altered life spans on the accumulation of the fluorescent age pigment in the housefly Musca domestica. Exp. Gerontol. 13, 341–355. Sőti, C., Csermely, P., 2000. Molecular chaperones and the aging process. Biogerontology 1, 225–233. Sőti, C., Csermely, P., 2002. Chaperones come of age. Cell Stress Chaperon 7, 186–190. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. N.Y. Acad. Sci. 899, 191–208. Stanley, J.G., de Witt, R., 1983. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (North Atlantic). Hard Clam. U.S. Fish Wildl. Serv. FWS/OBS-82/11.18. U.S. Army Corps of Engineers. TR EL-82-4. 19 pp. Strahl, J., Philipp, E., Brey, T., Broeg, K., Abele, D., 2007. Physiological aging in the Icelandic population of the ocean quahog Arctica islandica. Aquat Biol. 1, 77–83. Sukhotin, A.A., Abele, D., Pörtner, H.-O., 2002. Growth, metabolism and lipid peroxidation in Mytilus edulis L.: age and size effects. Mar. Ecol. Prog. Ser. 226, 223–234. Sukhotin, A.A., Abele, D., Pörtner, H.-O., 2006. Ageing and metabolism of Mytilus edulis: populations from various climate regimes. J. Shellfish Res. 25, 893–899. Sukhotin, A.A., Pörtner, H.O., 2001. Age-dependence of metabolism in mussels Mytilus edulis L. from the White Sea. J. Exp. Mar. Biol. Ecol. 257, 53–72. Vernet, M., Hunter, J.R., Vetter, R.D., 1988. Accumulation of age-pigments (lipofuscin) in two cold-water fishes. Fish. Bull. 86, 401–407. Viarengo, A., Canesi, L., Petrica, M., Livingstone, D.R., Orunesu, M., 1991. Age-related lipid peroxidation in the digestive gland of mussels: the role of the antioxidant defense systems. Experientia 47, 454–457. Wei, Y.H., Lee, H.C., 1998. Oxidative stress, mitochondrial DNA mutation and impairment of antioxidant enzymes in aging. Proc. Soc. Exp. Biol. Med. 217, 53–63. West, G.B., Woodruff, W.H., Brown, J.H., 2002. Allometric scaling of metabolic rate from molecules and mitochondria to cells and mammals. Proc. Natl. Acad. Sci. USA 99, 2473–2478. Wheeler, J.C., Bieschke, E.T., Tower, J., 1995. Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress. Proc. Natl. Acad. Sci. USA 92, 10408–10412. Wu, B., Gu, M.J., Heydari, A.R., Richardson, A., 1993. The effect of age on the synthesis of two heat shock proteins in the hsp70 family. J. Gerontol. 48, B50–B56. Yin, D., Brunk, U.F., 1995. Carbonyl toxification hypothesis of biological aging. In: Macieira-Coelho, A. (Ed.), Molecular Basis of Aging. CRC Press, Inc., New York, pp. 421–436. Yin, D., Chen, K., 2005. The essential mechanisms of aging: irreparable damage accumulation of biochemical side-reactions. Exp. Gerontol. 40, 455–465. Zielinski, S., Pörtner, H.-O., 2000. Oxidative stress and antioxidative defense in cephalopods: a function of metabolic rate or age? Comp. Biochem. Physiol. B 125, 147–160. Zolotarev, V.N., Ryabushko, V.I., 1977. Age changes of energy metabolism in Crenomytilus grayanus Dunker. Zhurnal Obshchey Biologii 38, 923–928 (In Russian).