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Keywords:

  • 4-hydroxy-2-nonenal (HNE);
  • aging;
  • biomarkers;
  • calorie restriction;
  • Drosophila melanogaster;
  • enzyme-linked immunosorbent assay (ELISA);
  • oxidative damage

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The oxidative stress hypothesis predicts that the accumulation of oxidative damage to a variety of macromolecules is the molecular trigger driving the process of aging. Although an inverse relationship between oxidative damage and lifespan has been established in several different species, the precise relationship between oxidative damage and aging is not fully understood. Drosophila melanogaster is a favored model organism for aging research. Environmental interventions such as ambient temperature and calorie restriction can alter adult lifespan to provide an excellent system to examine the relationship between oxidative damage, aging and lifespan. We have developed an enzyme-linked immunosorbent assay (ELISA) using commercially available reagents for measuring 4-hydroxy-2-nonenal (HNE) in proteins, a marker for oxidative damage to lipids, and present data in flies to show that HNE adducts accumulate in an age-dependent manner. With immunohistology, we also find the primary site of HNE accumulation is the pericerebral fat body, where induction of dFOXO was recently shown to retard aging. When subjected to environmental interventions that shorten lifespan, such as elevated ambient temperature, the chronological accumulation of HNE adduct is accelerated. Conversely, interventions that extend lifespan, such as lower ambient temperature or low calorie diets, slow the accumulation of HNE adduct. These studies associate damage from lipid peroxidation with aging and lifespan in Drosophila and show that calorie restriction in flies, as in mammals, slows the accumulation of lipid related oxidative damage.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The oxidative stress hypothesis provides one of the most widely accepted ways to explain aging (Sohal & Weindruch, 1996; Beckman & Ames, 1998; Finkel & Holbrook, 2000; Sohal et al., 2002). Highly reactive oxygen radicals are generated as byproducts of aerobic metabolism. An imbalance between the generation of these reactive oxygen species (ROS) and their detoxification permits various oxygen radicals to escape and damage a range of macromolecules. This damage is thought to induce further handicaps at a cellular level, resulting in accelerated decline in physiological functions and increased risk of death.

Many macromolecules are damaged by ROS, including mitochondrial and genomic DNA, lipids and proteins (Sohal & Weindruch, 1996; Beckman & Ames, 1998; Finkel & Holbrook, 2000). Oxidation of lipids generates lipid peroxides that break down to aldehydes, which can oxidize a variety of other macromolecules. Most notably, proteins modified by the primary oxidation products such as 4-hydroxy-2-nonenal (HNE) form Michael addition-type HNE-amino acid adducts (Esterbauer et al., 1991; Toyokuni et al., 1994; Uchida, 2003). HNE increases with age in mammals and is involved in age-associated neurodegenerative diseases (Zarkovic, 2003) and stress-mediated apoptosis signaling (Awasthi et al., 2003). Although a correlation between oxidative damage and aging has been shown in a number of systems (Sohal & Weindruch, 1996; Levine & Stadtman, 2001; Sohal et al., 2002), establishing a causal relationship between oxidative damage, aging and lifespan remains elusive (Stadtman, 2002).

Drosophila melanogaster is a powerful model organism with which to conduct mechanistic aging research (Helfand & Rogina, 2003). In addition to the fly's short lifespan and ease of maintenance and applicability of molecular genetic tools, lifespan can be manipulated through environmental and genetic methods, including changes in ambient temperatures, reproductive status and calorie restriction. Yet, to date, the relationship between oxidative damage and environmentally manipulated aging in Drosophila is poorly characterized, especially with regard to lipid oxidative damage. One obstacle to such data has been the quantity of tissue needed for standard assays. Here we adapt and present a simple assay to measure oxidative damage to lipids as they form HNE-oxidized proteins.

Earlier studies measuring HNE adducts presented a competitive enzyme-linked immunosorbent assay (ELISA) based on a novel antibody to HNE-oxidized proteins with limited availability (Uchida et al., 1993; Tsai et al., 1998). In this report we adapt this assay using commercially available reagents. Using this assay, we then describe how HNE adducts change with age in adult flies and demonstrate that interventions known to alter lifespan also affect the accumulation of HNE adducts.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Development of a competitive ELISA using commercially available reagents

The ELISA uses a conjugated HNE-containing protein and a commercially available anti-HNE antibody (Alpha Diagnostic, San Antonio, TX, USA). We generated the HNE-containing competitor by conjugating free HNE to purified GAPDH protein (CalBiochem, La Jolla, CA, USA) in an approximately one-to-one stochiometry (see Uchida & Stadtman, 1993; Ishii et al., 2003). The Alpha Diagnostic antibody recognized specifically the HNE–GAPDH conjugate in Western blot, without cross-reaction to GAPDH protein (Fig. 1A). Reactions with HNE–GAPDH were proportional to loaded protein and sensitive to 0.1 µg of HNE–GAPDH protein (0.68 pmol conjugated protein).

image

Figure 1. ELISA assay measuring HNE adducts conjugated to protein. (A) Western blot shows that the anti-HNE antibody (Alpha Diagnostic) specifically detects HNE adducts conjugated to GAPDH. Blot 1 shows that the anti-HNE antibody detects HNE conjugated to GAPDH (HNE–GAPDH) in a dose-dependent manner. Blot 3 shows that the anti-HNE antibody does not detect unconjugated GAPDH. Membranes 2 and 4 are replicates of membranes 1 and 3, respectively, stained with Ponceau S to reveal the amount of protein present. (B) The competitive ELISA assay detects HNE–GAPDH in a dose-dependent manner. The data from the dose–response fit a line y = 0.4211517 − 0.1535965 Ln(x) where R2 = 0.9506080 with optical density on the y-axis and HNE–GAPDH concentration on the x-axis. When samples of mitochondrial proteins from flies are examined, the most sensitive portion of the standard curve is found between OD 0.6 and 1.0. Each point represents the mean ± SD of three assays. Similar results were obtained from a minimum of three independent experiments.

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The conjugated HNE–GAPDH competes in a dose-dependent manner with HNE adducts as demonstrated by the ELISA assay (Fig. 1B). Detection was most sensitive when conjugated HNE–GAPDH was less than 0.3 µg of protein per assay. The assay gave highly reproducible results: intra-assay variation among at least three replicate trials was less than 8%, and the interassay variation was 5–14%.

Paraquat feeding increases HNE adducts in the adult fly

To determine that our competitive ELISA assay could measure oxidative damage in tissues of adult flies, paraquat-treated flies were examined. Paraquat is a free radical generator that reduces survival in D. melanogaster. The HNE ELISA readily detected oxidative damage in paraquat-fed flies (Fig. 2A). Ten-day-old Canton-S flies fed paraquat for 16 h had an increase in total HNE adducts from isolated mitochondria (females: control, 0.1 nmol mg−1 protein, treatment, 0.43 nmol mg−1 protein; P = 0.01; males: control, 0.04 nmol mg−1 protein, treatment, 0.35 nmol mg−1 protein; P = 0.006).

image

Figure 2. Paraquat increases the level of HNE adducts in adult flies. (A) The competitive ELISA assay shows that 10-day-old female (F) and male (M) flies fed 20 mm of paraquat in 5% sucrose for 16 h have increased levels of HNE adducts as compared with control flies fed only 5% sucrose. Each bar graph represents the mean ± SD of three assays of 30–50 flies each. Student's t-test shows P = 0.01 for females and P = 0.006 for males. (B,C). Paraquat-treated flies show an increase in anti-HNE antibody staining in pericerebral fat body. The photomicrograph of sections from heads of 10-day-old female flies fed 20 mm paraquat for 16 h (C) shows prominent staining in the pericerebral fat body (arrows point to pericerebral fat body) as compared with control (B). Scale bars = 20 µm.

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Paraquat feeding increases HNE adducts in fat body, brain and muscle

The commercial antibody was also useful in immunohistological studies. Ten-day-old flies fed 20 mm paraquat for 16 h showed elevated HNE staining in fat body, particularly in the pericerebral fat body of the head (Fig. 2B,C). Some positive HNE staining was also seen in neurons of the brain and the thoracic musculature. The increase of HNE adducts in the pericerebral fat body after paraquat treatment is interesting because activation of dFOXO specifically in this tissue increases male and female survival and stress resistance (Giannakou et al., 2004; Hwangbo et al., 2004).

HNE adducts accumulate throughout the lifespan of the fly

The relationship between oxidative damage and aging was examined by measuring the level of HNE adducts in mitochondrial extracts from tissues (head plus thorax) of wild-type Canton-S male and female flies cultured under normal conditions at 25 °C. Similar to what has been seen for the accumulation of carbonyls in a variety of invertebrate and mammalian species (Levine & Stadtman, 2001), the level of HNE adducts remains relatively unchanged during the first third to half of adult life before significantly increasing (Fig. 3C,D). When compared with the survivorship curve, the increase in HNE adducts is seen to occur just before the increase in mortality at median lifespan (Fig. 4A,B). After reaching a peak, the level of HNE adducts appears to decrease later in life. As with exposure to paraquat, HNE-positive staining became prominent in the pericerebral and abdominal fat bodies and to a lesser extent in neurons of the brain and muscle of the thorax of both old males and females, relative to young adults (data not shown).

image

Figure 3. HNE adduct levels increase during aging in the fly. HNE adduct levels are shown from heads and thoraces of adult female (A,C,E) and male (B,D,F) Canton-S flies living at 29 °C (A,B), 25 °C (C,D) and 18 °C (E,F). Each bar in the graph represents the mean ± SD of three assays of 20–30 flies each. All comparisons are made with the 3-day sample using Student's t-test. Asterix (*) shows a significant (P = 0.05) increase in HNE adduct level. At 29 °C, P = 0.05 and 0.002 on days 35 and 43, respectively, for females, while P = 0.03, 0.02 and 0.002 on days 30, 35 and 43, respectively, for males. At 25 °C, P = 0.05 on day 40 for females, while P = 0.03 and 0.01 on days 40 and 45, respectively, for males. At 18 °C, P = 0.01 on day 115 for females, while P = 0.04 and 0.0003 on days 75 and 100, respectively, for males.

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image

Figure 4. Changes in the level of HNE adducts scales with respect to lifespan when lifespan is altered using ambient temperature. Survivorship curves for female (A) and male (B) flies living at 29 °C (red filled triangles), 25 °C (blue open circles) and 18 °C (green filled boxes). Each survivorship curve represents over 200 flies as described (Helfand et al., 1995). Comparison of HNE adduct level in flies living at different temperatures with respect to physiological age is shown for female (C) and male (D) flies. Percentage lifespan for each HNE value was determined by dividing the age in days for each HNE measurement from Fig. 3 by the 5% maximum lifespan for that temperature condition derived from the survivorship curves in Fig. 4(A,B). The fold changes of HNE adducts were determined by dividing the HNE adduct level for each point (from Fig. 3) by the average of the HNE adduct levels over the first 10 days in each treatment.

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HNE adduct accumulation scales with relative age

Ambient temperature influences fly survival and lifespan, as illustrated in Fig. 4. To assess further the relationship between the accumulation of HNE adducts and lifespan, we examined the level of HNE adducts during adult life for Canton-S flies living at 29 and 18 °C. Figure 3 shows that the initial rise in HNE adduct levels occurs just prior to median lifespan for flies at 29 or 18 °C, similarly to what is seen at 25 °C. For flies living at 29 °C, an increase in HNE adducts is seen by 30 days in males and by 35 days in females (Fig. 3A,B), whereas at 18 °C a significant increase is delayed until after 75 days (Fig. 3E,F). To compare the age patterns of HNE accumulation across these temperature trials, we scaled age to represent the proportion of each cohort's maximum lifespan (demarcated by the age when 5% remain alive). In females, these rescaled data viewed together show an age trend for HNE accumulation because the elevated levels occur in all cases only after the cohorts have completed about 60% of their life course (Fig. 4C). In males, despite a nearly three-fold difference in lifespan, from 29 to 18 °C, the pattern of HNE adduct accumulation, especially their increase around mid-life, is similar among the cohorts when scaled for lifespan (Fig. 4D).

Calorie restriction delays the accumulation of HNE adducts

Reduced nutrient intake (calorie restriction, CR) increases lifespan in a variety of species, including yeast, nematodes, flies and mammals (Chippindale et al., 1993; Chapman & Partridge, 1996; Weindruch, 1996; Lakowski & Hekimi, 1998; Wanagat et al., 1999; Guarente, 2000; Lin et al., 2000; Good & Tatar, 2001; Kayo et al., 2001; Jiang et al., 2002; Lane et al., 2002). In mammals the lifespan extension with CR is associated with decreased accumulation of oxidative damage (Sohal & Weindruch, 1996; Bartke et al., 2001). Here, we manipulated the concentration of diet fed to wild-type adults to vary lifespan and test for associated change in HNE adduct accumulation. Trials were conducted at 25 °C with female flies. As expected, survival improved with decreasing dietary nutrient content (see Fig. 6A). In all treatments, levels of HNE adduct increased with age and the onset and rate of increase was positively associated with diet concentration (Fig. 5). In each trial it was also clear that HNE adduct levels declined in samples from the oldest surviving individuals. When we scale for age relative to the cohort maximum lifespan, as above for the temperature study, the age patterns of HNE adduct levels are remarkably consistent among the diet treatments (Fig. 6B). As in the temperature study, HNE adducts initially increased at 40–50% of cohort lifespan and peaked at approximately 60–70% of cohort lifespan.

image

Figure 6. Changes in the level of HNE adducts scale with respect to lifespan when lifespan is altered by calorie restriction. (A) Survivorship curves of female Canton-S flies fed with 3.0N (red filled triangles), 1.5N (blue open circles) and 0.5N (green filled boxes) food. Each curve was generated using over 200 females or males as described (Helfand et al., 1995). (B) Comparison of HNE adduct level in female flies living on different calorie foods 3.0N (red filled triangles), 1.5N (blue open circles) and 0.5N (green filled boxes) with respect to physiological age. Percentage lifespan for each HNE value was determined by dividing the age in days for each HNE measurement from Fig. 5 by the 5% maximum lifespan for that food condition derived from the survivorship curves in Fig. 6(A). The fold increase of HNE adducts was determined by dividing the HNE adduct level for each point (from Fig. 5) by the average of the HNE adduct levels over the first 10 days in each treatment.

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image

Figure 5. Alterations in calorie content of food changes the time of increase in HNE adduct levels in adult flies. HNE adduct levels for females living on (A) 3.0N, (B) 1.5N or (C) 0.5N food. Each bar graph represents the mean ± SD of three assays of 20–30 flies each. Asterix (*) shows a significant (P = 0.05) increase in HNE adduct level compared with the 3-day sample. For 3.0N food, P = 0.005, 0.0003, 0.03 and 0.006 on days 10, 17, 20 and 22, respectively; for 1.5N food, P = 0.004, 0.004, 0.0002, 0.0006 and 0.04 on days 30, 35, 40, 45 and 50, respectively; for 0.5N food, P = 0.05, 0.01, 0.05, 0.007, 0.03 and 0.002 on days 25, 35, 40, 45, 50 and 55, respectively.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

We describe a competitive ELISA to measure HNE-modified proteins in adult Drosophila that is efficient and sensitive. It requires only 3 µg of total mitochondrial protein and works at neutral pH in a buffer that reduces exposure to oxidation during the protocol. We validated this assay with wild-type adults fed paraquat, a known ROS generator. Animals fed paraquat for 16 h showed a significant increase in HNE adduct accumulation. Immunohistochemistry with the anti-HNE antibody confirmed that adducts accumulated in pericerebral fat body and to a lesser degree in other fat body depots, and also in neurons of the brain and in thoracic muscle. Physiologically, the fat body is thought to be equivalent to the mammalian liver (Sondergaard, 1993). HNE-positive staining was also observed in these tissues during the course of normal aging. It was recently reported that elevated expression of FOXO transcription factor (homolog of Caenorhabditis elegans daf-16) in adult pericerebral fat body (Hwangbo et al., 2004) or adult thoracic and abdominal fat body (Giannakou et al., 2004) increases lifespan.

In both male and female D. melanogaster, HNE adducts begin to accumulate at mid-life in every cohort. Studies of carbonyls in humans, rats and houseflies report a similar pattern: carbonyls initially rise in cohorts near the median lifespan and thereafter steadily increase (Levine & Stadtman, 2001). In D. melanogaster, however, we also find that after approximately 60–70% of the lifespan, the level of HNE adducts declines among the remaining individuals. As the assay precludes continuous assessment of the same individual during aging, a decrease in HNE at advanced age could arise from individual longitudinal change, such as improved HNE clearance or reduced HNE production, as well as from demographic selection acting upon cohort heterogeneity (Vaupel & Yashin, 1985).

Among experimentally manipulated cohorts, the relationship between lifespan and oxidative damage can help explain whether and how ROS affects aging. For poikilotherms, within bounds, the lifespan is inversely proportional to ambient temperature. It was previously reported that the amount of oxidative damage measured by carbonyl accumulation in mitochondrial aconitase is inversely related to ambient temperature in Drosophila (Das et al., 2001). Here we show that oxidative damage measured as HNE adducts accumulates at faster rates in flies aged at higher temperatures. At 29 °C HNE adducts rise as early as day 30 whereas at 18 °C they do not increase before day 70. When age is normalized relative to the cohort maximum lifespan, the age-dependent patterns among treatments are remarkably similar, especially in males. Thus, survival is proportional to the level of HNE adducts, suggesting that oxidative damage is a marker for biological age, and that it is either a causal determinant of mortality or correlated to the underlying process of senescence.

Diet restriction is a reliable way to extend lifespan in many animals, including D. melanogaster. Reducing dietary yeast is sufficient to increase survival (Chippindale et al., 1993), although adults maintained without yeast have elevated mortality (Good & Tatar, 2001). Switching adults between restricted and normal diets causes the mortality of cohorts rapidly to adopt the mortality trajectory characteristic of flies continuously maintained on the adopted food level (Good & Tatar, 2001; Mair et al., 2003). From such observations, Mair et al. (2003) suggest ‘DR [dietary restriction/calorie restriction] in Drosophila extends lifespan solely by reducing the short-term risk of death. DR and control flies accumulate irreversible, aging-related damage at the same rate.’ Here we show that long-lived CR flies accumulate damage in the form of HNE adducts at slower rates than control flies on high-concentration food. Whether or not the level of HNE adducts is reversible could help explain the mechanism by which CR mediates aging. According to Mair et al. dietary influences on oxidative damage, as indicated for example by HNE levels, may affect current mortality risk but not the underlying process of senescent degeneration. By contrast, based on our current observations, because the distribution of HNE adduct levels is similar among diet treatments when age is scaled by maximum lifespan, we conclude that, like temperature, calorie restriction influences the biological age of adults rather than their momentary mortality risk.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Fly cultures

All studies were carried out using Canton-S flies. Flies were cultured as previously described (Helfand et al., 1995). All flies were grown into adulthood life in a temperature-controlled environmental incubator at constant humidity and 12-h light–dark cycle. Within 2 h of eclosion, approximately 20 males and 20 females were placed in a plastic shell vial containing 10 mL of our standard food preparation and cultured in a temperature-controlled environmental incubator (constant humidity and 12-h light–dark cycle) set at 18, 25 or 29 °C. Flies were passed to new vials every other day.

Lifespan studies

A set of ten vials (250 males and 250 females) was used to make lifespan determinations (a larger number of additional vials was used for collecting animals for oxidative damage measurements). The number of dead male and female flies was counted every other day as described (Helfand et al., 1995). For CR experiments, 0.5N, 1.5N and 3.0N food media were used. The content of these three media included yeast, sucrose and agar as described (Chapman & Partridge, 1996). The amount of yeast and sucrose was 50, 150 and 300 g L−1 for 0.5N, 1.5N and 3.0N, respectively. The amount of agar was 20 g L−1 for all food levels. The media contained 2137, 1068 and 356 cal mL−1 for 3.0N, 1.5N and 0.5N, respectively, based on the information obtained from the supplier.

Paraquat assays

Twenty-five 10-day-old male or female flies were placed in a vial containing only water-soaked filter paper for 6 h, then transferred to a new vial containing a filter paper soaked with a 5% sucrose and 20 mm paraquat solution or 5% sucrose alone. After 16 h, flies were taken and examined for amount of mitochondrial HNE adducts. Experiments were performed in triplicate.

Competitive ELISA assay

Conjugation of free HNE (Alpha Diagnostic, San Antonio, TX, USA) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein (CalBiochem, La Jolla, CA) was carried out as described (Uchida & Stadtman, 1993). Briefly, 2 mm of HNE in 1 mL of 50 mm sodium phosphate buffer, pH 7.2, was incubated with 1 mg of GAPDH for 2 h at 37 °C. The reaction was quenched by addition of 100 µL of 10 mm N-acetylcystein followed by addition of 800 µL of 8 m guanidine hydrochloride/133 mm Tris/13 mm EDTA, pH 7.2. This procedure results in one to three HNE molecules conjugated per GAPDH (Uchida & Stadtman, 1993; Ishii et al., 2003). For our calculations we assumed one HNE molecule per GAPDH molecule. The competitive ELISA protocol was carried out as described (Satoh et al., 1999) by Alpha Diagnostic International. Briefly, wells in a 96-well plate were coated with 200 ng of HNE–GAPDH protein for 20 h at 4 °C. After washing with PBS-Tween 20 buffer, blocking buffer (1% BSA in PBS) was added to each well of the 96-well plate, and the plate was stored at 4 °C until use.

A standard dose–response curve was developed by making a dilution series using known amounts of HNE–GAPDH with polyclonal rabbit anti-4-HNE antibody (1 : 1000; Alpha Diagnostic) and then adding each dilution in triplicate to plates coated with HNE–GAPDH. For experimental samples, 3 µg protein from each lysate was mixed with polyclonal rabbit anti-4-HNE antibody (1 : 1000) and added to each well in triplicate. Plates were incubated for 1 h at room temperature with gentle shaking, the wells were then washed with buffer and a secondary anti-rabbit antibody conjugated to horseradish peroxidase (HRP) 1 : 5000 was added. Plates were then incubated for 1 h at room temperature and washed, and the detection buffer TMB (Alpha Diagnostic) was added. The plate was read using a Microplate Reader (Bio-Rad, Hercules, CA, USA) at OD450.

Preparation of mitochondrial protein

Preparation of mitochondrial proteins followed a published procedure (Yan & Sohal, 1998; personal communication with B. Sohal). Male and female adult flies were frozen separately in dry ice and stored in a −80 °C freezer. Heads and thoraces were removed on metal blocks kept at −20 °C, collected and immediately placed in homogenization buffer containing 0.25 m sucrose, 10 mm Tris, pH 7.4, 1 mm EGTA, and 1% bovine serum albumin (BSA). Heads and thoraces were gently homogenized using a plastic pestle by hand in Eppendorf tubes on ice. The lysates were then filtered through three layers of gauze and spun down at 150 g for 5 min. The supernatant was transferred to a new tube and spun down at 3000 g for 10 min. Pellets were collected, washed with isolation buffer (0.25 m sucrose, 10 mm Tris, pH 7.4, 1 mm EGTA), and collected again by centrifugation at 7000 g for 10 min. The pellets were stored at −80 °C in sonication buffer containing the antioxidant butylated hydroxyltoluene (BHT; 5 mm) in 50 mm phosphate buffer, pH 7.4, to prevent exogenous oxidation until use. In order to obtain mitochondrial proteins from matrix and inner membrane, mitochondria were sonicated on ice using a MISONIX sonicator 3000 on four separate occasions for 30 s, with 10 s between each sonication. Mitochondrial proteins were collected by centrifugation at 8400 g for 10 min. All manipulations were performed at 4 °C or on ice. In order to show that these conditions provide an optimal procedure for isolation of mitochondrial proteins, the quantity of the mitochondrial proteins in the pellet and the associated supernatant from each of the isolation steps above were assessed by Western blot analysis using an anti-OxPhos Complex IV subunit III antibody (Molecular Probes, Eugene, OR, USA) as a mitochondrial marker. Based upon these studies all subsequent experiments were carried out using pellets with the highest yield of mitochondrial proteins.

Immunohistology

Adult Canton-S flies were fixed in 4% paraformaldehyde in PBS for 20 min, dehydrated, embedded in paraffin and sectioned. Sections were incubated with an anti-HNE antibody (1 : 500) for 1 h at room temperature and washed three times with PBS containing 0.05% Triton X-100. A goat anti-rabbit secondary antibody (1 : 200) conjugated with biotin was applied for 1 h at room temperature followed by an avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA). DAB was added as substrate. Sections were counterstained with methyl green to visualize the nuclei.

Statistics

Statistical analysis included calculations of the mean and standard deviation of the mean (SD). Student's t-test P-values were determined. Significance is defined as P ≤ 0.05. Data are expressed as mean ± SD. Intra-assay variation is calculated as the percentage difference between the average values of identical samples repeated three times each in the same assay. The interassay variation is calculated as the percentage difference between the average values of identical samples (performed in triplicate) run on two separate occasions (different plates on different days). Each assay was performed in triplicate.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

We thank Joseph Jack for critical reading of the manuscript and Blanka Rogina and Tyson Bross for help with the calorie restriction studies. We thank Suzanne Kowalski and Dianna Schwarz for technical support. This work was supported by grants from the National Institute on Health (AG16667 to S.L.H.), the Ellison Medical Foundation and the Donaghue Foundation to S.L.H. S.L.H. is an Ellison Medical Research Foundation Senior Investigator and a member of the Scientific Advisory Board of Elixir Pharmaceuticals, Inc., Cambridge, MA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  • Awasthi YC, Sharma R, Cheng JZ, Yang Y, Sharma A, Singhal SS, Awasthi S (2003) Role of 4-hydroxynonenal in stress-mediated apoptosis signaling. Mol. Aspects Med. 24, 219230.
  • Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS (2001) Extending the lifespan of long-lived mice. Nature 414, 412.
  • Beckman KB, Ames BN (1998) The free radical theory of aging matures. Physiol. Rev. 78, 547581.
  • Chapman T, Partridge L (1996) Female fitness in Drosophila melanogaster: an interaction between the effect of nutrition and of encounter rate with males. Proc. R. Soc. Lond. B Biol. Sci. 263, 755759.
  • Chippindale AK, Leroi AM, Kim SB, Rose MR (1993) Phenotypic plasticity and selection in Drosophila life-history evolution. I. Nutrition and the cost of reproduction. J. Evol. Biol. 6, 171193.
  • Das N, Levine RL, Orr WC, Sohal RS (2001) Selectivity of protein oxidative damage during aging in Drosophila melanogaster. Biochem. J. 360, 209216.
  • Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81128.
  • Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239247.
  • Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ, Partridge L (2004) Long-lived Drosophila with overexpressed dFOXO in adult fat body. Science 305, 361.
  • Good TP, Tatar M (2001) Age-specific mortality and reproduction respond to adult dietary restriction in Drosophila melanogaster. J. Insect Physiol. 47, 14671473.
  • Guarente L (2000) Sir2 links chromatin silencing, metabolism, and aging. Genes Dev. 14, 10211026.
  • Helfand SL, Blake KJ, Rogina B, Stracks MD, Centurion A, Naptra B (1995) Temporal patterns of gene expression in the antenna of the adult Drosophila melanogaster. Genetics 140, 549555.
  • Helfand SL, Rogina B (2003) From genes to aging in the Drosophila. In Adv. Genet., vol. 49 (ed. TFriedmann), pp. 67109. Academic Press, San Diego.
  • Hwangbo DS, Gersham B, Tu MP, Palmer M, Tartar M (2004) Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429, 562566.
  • Ishii T, Tatsuda E, Kumazawa S, Nakayama T, Uchida K (2003) Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 42, 34743480.
  • Jiang JC, Wawryn J, Shantha Kumara HM, Jazwinski SM (2002) Distinct roles of processes modulated by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast. Exp. Gerontol. 37, 10231030.
  • Kayo T, Allison DB, Weindruch R, Prolla TA (2001) Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc. Natl Acad. Sci. USA 98, 50935098.
  • Lakowski B, Hekimi S (1998) The genetics of caloric restriction in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 95, 1309113096.
  • Lane MA, Mattison J, Ingram DK, Roth GS (2002) Caloric restriction and aging in primates: relevance to humans and possible CR mimetics. Microsc. Res. Tech. 59, 335338.
  • Levine RL, Stadtman ER (2001) Oxidative modification of proteins during aging. Exp. Gerontol. 36, 14951502.
  • Lin SJ, Defossez PA, Guarente L (2000) Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 21262128.
  • Mair W, Goymer P, Pletcher SD, Partridge L (2003) Demography of dietary restriction and death in Drosophila. Science 301, 17311733.
  • Satoh K, Yamada S, Koike Y, Igarashi Y, Toyokuni S, Kumano T, Takahata T, Hayakari M, Tsuchida S, Uchida K (1999) A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method. Anal. Biochem. 270, 323328.
  • Sohal RS, Mockett RJ, Orr WC (2002) Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic. Biol. Med. 33, 575586.
  • Sohal RS, Weindruch R (1996) Oxidative stress, caloric restriction, and aging. Science 273, 5963.
  • Sondergaard L (1993) Homology between the mammalian liver and the Drosophila fat body. Trends Genet. 9, 193.
  • Stadtman ER (2002) Importance of individuality in oxidative stress and aging. Free Radic. Biol. Med. 33, 597604.
  • Toyokuni S, Uchida K, Okamoto K, Hattori-Nakakuki Y, Hiai H, Stadtman ER (1994) Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc. Natl Acad. Sci. USA 91, 26162620.
  • Tsai L, Szweda PA, Vinogradova O, Szweda LI (1998) Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine. Proc. Natl Acad. Sci. USA 95, 79757980.
  • Uchida K (2003) 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 42, 318343.
  • Uchida K, Stadtman ER (1993) Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction. J. Biol. Chem. 268, 63886393.
  • Uchida K, Szweda LI, Chae HZ, Stadtman ER (1993) Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes. Proc. Natl Acad. Sci. USA 90, 87428746.
  • Vaupel JW, Yashin AI (1985) Heterogeneity's ruses: some surprising effects of selection on population dynamics. Am. Stat. 39, 176185.
  • Wanagat J, Allison DB, Weindruch R (1999) Caloric intake and aging: mechanisms in rodents and a study in nonhuman primates. Toxicol. Sci. 52 (Suppl. 2), 3540.
  • Weindruch R (1996) The retardation of aging by caloric restriction: studies in rodents and primates. Toxicol. Pathol. 24, 742745.
  • Yan LJ, Sohal RS (1998) Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proc. Natl Acad. Sci. USA 95, 1289612901.
  • Zarkovic K (2003) 4-hydroxynonenal and neurodegenerative diseases. Mol. Aspects Med. 24, 293303.