Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart.

Mitochondrial matrix proteins are sensitive to oxidative inactivation, and oxidized proteins are known to accumulate during ageing. The Lon protease is believed to play an important role in the degradation of oxidized matrix proteins such as oxidized aconitase. We reported previously that an age-related accumulation of altered proteins occurs in the liver matrix of rats and that the ATP-stimulated proteolytic activity, referred as to Lon-like protease activity, decreases considerably in 27 month-old rats, whereas no concomitant changes in the levels of Lon protein expression occur in the liver. Here, we report that this decline is associated with a decrease in the activity of aconitase, an essential Krebs' cycle enzyme. Contrary to what we observed in the liver, the ATP-stimulated protease activity was found to remain constant in the heart mitochondrial matrix during ageing, and the levels of expression of the Lon protease increased in the older animals in comparison with the younger ones. Although the ATP-stimulated protease activity remained practically the same in older animals as in younger ones, a decrease in the level of aconitase activity was still observed. Altogether, these results indicate that matrix proteins, such as the critical enzymes aconitase and Lon protease, are inactivated with ageing and that the effects of ageing vary from one organ to another.     

Increased level of glycoxidation product N(epsilon)-(carboxymethyl)lysine in rat serum and urine proteins with aging: link with glycoxidative damage accumulation in kidney

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43+/-0.14 pmol CML/micrograms protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50+/-0.14 pmol CML/micrograms protein vs 2.27+/-0.26 pmol CML/micrograms protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50+/-0.14 pmol CML/micrograms protein at 3 months and markedly increased from 10 months (2.99+/-0.24 pmol CML/micrograms protein) to 27 months (3.76+/-0.25 pmol CML/micrograms protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowman's capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85+/-28.95 pmol CML/microgrms OHPro at 3 months and significantly increased from 10 months (187.61+/-21.99 pmol CML/micrograms OHPro) to 27 months (334.55+/-62.21 pmol CML/micrograms OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.     

Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress

We compared Lon protease expression in murine skeletal muscle of young and old, wild-type and Sod2(-/+) heterozygous mice, and studied Lon involvement in the accumulation of damaged (oxidized) proteins. Lon protease protein levels were lower in old and oxidatively challenged animals, and this Lon deficiency was associated with increased levels of carbonylated proteins. We identified one of these proteins as aconitase, and another as an aconitase fragmentation product, which we can also generate in vitro by treating purified aconitase with H(2)O(2). These results imply that aging and oxidative stress down-regulate Lon protease expression which, in turn, may be responsible for the accumulation of damaged proteins, such as aconitase, within mitochondria.     

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000muM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

Downregulation of Na+-K+-ATPase pumps in skeletal muscle with training in normobaric hypoxia.

To investigate the effects of training in normoxia vs. training in normobaric hypoxia (fraction of inspired O2 = 20.9 vs. 13.5%, respectively) on the regulation of Na+-K+-ATPase pump concentration in skeletal muscle (vastus lateralis), 9 untrained men, ranging in age from 19 to 25 yr, underwent 8 wk of cycle training. The training consisted of both prolonged and intermittent single leg exercise for both normoxia (N) and hypoxia (H) during a single session (a similar work output for each leg) and was performed 3 times/wk. Na+-K+-ATPase concentration was 326 +/- 17 (SE) pmol/g wet wt before training (Control), increased by 14% with N (371 +/- 18 pmol/g wet wt; P < 0.05), and decreased by 14% with H (282 +/- 20 pmol/g wet wt; P < 0.05). The maximal activity of citrate synthase, selected as a measure of mitochondrial potential, showed greater increases (P < 0.05) with H (1.22 +/- 0.10 mmol x h-1 x g wet wt-1; 70%; P < 0.05) than with N (0.99 +/- 0.10 mmol x h-1 x g wet wt-1; 51%; P < 0.05) compared with pretraining (0.658 +/- 0.09 mmol x h-1 x g wet wt-1). These results demonstrate that normobaric hypoxia induced during exercise training represents a potent stimulus for the upregulation in mitochondrial potential while at the same time promoting a downregulation in Na+-K+-ATPase pump expression. In contrast, normoxic training stimulates increases in both mitochondrial potential and Na+-K+-ATPase concentration.     

Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism

Mitochondrial aconitase is sensitive to oxidative inactivation and can aggregate and accumulate in many age-related disorders. Here we report that Lon protease, an ATP-stimulated mitochondrial matrix protein, selectively recognizes and degrades the oxidized, hydrophobic form of aconitase after mild oxidative modification, but that severe oxidation results in aconitase aggregation, which makes it a poor substrate for Lon. Similarly, a morpholino oligodeoxynucleotide directed against the lon gene markedly decreases the amount of Lon protein, Lon activity and aconitase degradation in WI-38 VA-13 human lung fibroblasts and causes accumulation of oxidatively modified aconitase. The ATP-stimulated Lon protease may be an essential defence against the stress of life in an oxygen environment. By recognizing minor oxidative changes to protein structure and rapidly degrading the mildly modified protein, Lon protease may prevent extensive oxidation, aggregation and accumulation of aconitase, which could otherwise compromise mitochondrial function and cellular viability. Aconitase is probably only one of many mitochondrial matrix proteins that are preferentially degraded by Lon protease after oxidative modification.     

Mitochondrial protein quality control: implications in ageing

Mitochondria represent both a major source for reactive oxygen species (ROS) production and a target for oxidative macromolecular damage. Increased production of ROS and accumulation of oxidized proteins have been associated with cellular ageing. Protein quality control, also referred as protein maintenance, is very important for the elimination of oxidized proteins through degradation and repair. Chaperone proteins have been implicated in refolding of misfolded proteins while oxidized protein repair is limited to the catalyzed reduction of certain oxidation products of the sulfur-containing amino acids, cysteine and methionine, by specific enzymatic systems. In the mitochondria, oxidation of methionine residues within proteins can be catalytically reversed by the methionine sulfoxide reductases, an ubiquitous enzymatic system that has been implicated both in ageing and protection against oxidative stress. Irreversibly oxidized proteins are targeted to degradation by mitochondrial matrix proteolytic systems such as the Lon protease. The ATP-stimulated Lon protease is believed to play a crucial role in the degradation of oxidized proteins within the mitochondria and age-related declines in the activity and/or expression of this proteolytic system have been previously reported. Age-related impairment of mitochondrial protein maintenance may therefore contribute to the age-associated build-up of oxidized proteins and impairment of mitochondrial redox homeostasis.     

The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1

Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long‐ and branched‐chain fatty acids for subsequent β‐oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross‐linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging‐related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging‐related diseases indicating that peroxisome maintenance is a critical component of ‘healthy aging’. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age‐dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction.     

Beta-adrenoceptor and adenylate cyclase function in the infarct model of rat heart failure.

In order to determine the possible etiology for diminished inotropic responsiveness to catecholamines in the infarction model of chronic congestive heart failure in rats, we studied beta-adrenoceptor number and site-specific stimulated adenylate cyclase activity in noninfarcted left ventricular tissue of rats at 3 months after ligation of the left coronary artery. Rats were divided into sham, small infarct, and large infarct groups according to infarct size. The large infarct groups showed increased right ventricle to body weight ratio (0.93 +/- 0.07 mg/g for the large infarcts vs 0.52 +/- 0.02 and 0.54 +/- 0.02 mg/g for the shams and small infarcts, respectively). Beta-Adrenoceptor number among the groups was similar (shams, 27 +/- 1 fmol/mg; small infarcts, 26 +/- 1 fmol/mg; and large infarcts, 29 +/- 1 fmol/mg), as was Kd (20 +/- 1 pmol, 18 +/- 2 pmol, and 18 +/- 2 pmol, respectively). Site-specific stimulation of adenylate cyclase using isoproterenol, Gpp(NH)p, forskolin, and MnCl2 revealed no significant differences among the groups. We conclude that this system is not responsible for the altered inotropic responsiveness to catecholamines seen in this model.     

Sexual activity influences organ weight and androgen metabolism of rat prostate, bulbocavernosus/levator ani muscle and kidney

Previously we have shown that rats living under heterosexual conditions (HE-rats) have significantly higher weights of androgen target organs like prostate and bulbocavernosus/levator ani muscle (BCLA) than rats living under homosexual conditions (HO-rats). Knowing that androgen metabolism is an important regulator of androgenic action, we have measured in vitro by thin-layer chromatography the testosterone 5 alpha-reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase (3 alpha (beta)-HSDH) activity in prostate and BCLA of both groups. Furthermore, we looked for weight differences of the kidney from HE- and HO-rats. The main results are: (1) The mean apparent Michaelis constant (Km) of 5 alpha-reductase in prostate was identical in both groups, being 0.22 and 0.24 microM for HE- and HO-rats, respectively. (2) The mean 5 alpha-reductase activity was significantly (P less than 0.001; n = 18) lower in prostate of HE- (11.1 +/- 0.5 (SEM) pmol 5 alpha-reduced metabolites X mg protein-1 X h-1 1) than HO-rats (13.9 +/- 0.4). (3) The mean apparent Km of 3 alpha (beta)-HSDH was identical in HE- and HO-rats, being 3.7 and 4.3 microM, respectively. (4) The mean 3 alpha (beta)-HSDH activity was significantly (P less than 0.001; n = 20) lower in prostate of HE- (1.58 +/- 0.05 (SEM) nmol 3 alpha (beta)-reduced metabolites X mg protein-1 X h-1) than HO-rats (1.85 +/- 0.05). (5) The mean 3 alpha (beta)-HSDH activity was significantly (P less than 0.001; n = 24) lower in BCLA of HE- (284 +/- 9.6 (SEM) pmol 3 alpha (beta)-reduced metabolites X mg protein-1 X h-1 than HO-rats (422 +/- 18.7). (6) Besides prostate and BCLA, also the absolute as well as relative weights of the kidney were significantly higher in HE- than HO-rats. (7) It will be discussed that despite various significant differences in androgen metabolism, other factors might be responsible for the organ weight differences of prostate, BCLA and kidney between HE- and HO-rats.     

Deletion of the mitochondrial Pim1/Lon protease in yeast results in accelerated aging and impairment of the proteasome

The Saccharomyces cerevisiae homolog of the ATP-dependent Lon protease, Pim1p, is essential for mitochondrial protein quality control, DNA maintenance, and respiration. Here, we demonstrate that Pim1p activity declines in aging cells and that Pim1p deficiency shortens the replicative life span of yeast mother cells. This accelerated aging of pim1Δ cells is accompanied by elevated cytosolic levels of oxidized and aggregated proteins, as well as reduced proteasome activity. Overproduction of Hsp104p greatly diminishes aggregation of oxidized cytosolic proteins, rescues proteasome activity, and restores life span of pim1Δ cells to near wild-type levels. Our results show that defects in mitochondrial protein quality control have global intracellular effects leading to the increased generation of misfolded proteins and cytosolic protein aggregates, which are linked to a decline in replicative potential.     

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.     

Decrease of Choline Acetyltransferase Activity of Rat Cortex Capillaries with Aging

Choline acetyltransferase (ChAT) activity was estimated in brain cortex capillaries isolated from 3-, 12-, 18-, and 24-month-old rats. Maximum enzymatic activity was found at 12 months (55 +/- 0.3 pmol X mg-1 protein X min-1; mean +/- SEM) and then it decreased to reach a minimum at 24 months (34 +/- 3.1 pmol X mg-1 protein X min-1). A less marked decrease of enzymatic activity was also found in cortex homogenate and in a synaptosomal fraction obtained from the same groups of rats. Loss of ChAT of brain capillaries with aging could be related to a general phenomenon of cortical cholinergic deficit in that condition.     

Tonin as activator of renin

Tonin, a new serine protease, found in high concentration in rat submaxillary glands, leads to a significant activation of human amniotic fluid renin. The optimum pH on renin activation by tonin was found at pH 6.0. The reaction was time dependent and the initial rate of angiotensin I generation was constant up to 2 h. The two amniontic fluid samples studied showed an increase in renin activity after incubation with tonin to about five times the control level (268 to 1240 pmol x h-1 x mL-1 and 1490 to 7480 pmol x h-1 x mL-1).     

Aging does not contribute to the decline in insulin action on storage of muscle glycogen in rats

Increase in fat mass (FM) and changes in body composition may account for the age-associated impairment in insulin action on muscle glycogen storage. We wish to examine whether preventing the increase in FM abolishes this defect seen with aging. We studied the novel aging model of F1 hybrids of BN/F344 NIA rats fed ad libitum (AL) at 2 (weighing 259+/-17 g), 8 (459+/-17 g), and 20 (492+/-10 g) mo old. To prevent the age-dependent growth in FM, rats were caloric restricted (CR) at 2 mo by decreasing their daily caloric intake by 45% (weighing 292+/-5 g at 8 mo, 294+/-9 g at 20 mo). As designed, the lean body mass (LBM) and %FM remained unchanged through aging (8 and 20 mo old) in the CR rats and was similar to that of 2-mo-old AL rats. However, 8- and 20-mo-old AL-fed rats had three- to fourfold higher FM than both CR groups. Peripheral insulin action at physiological hyperinsulinemia was determined (by 3 mU x kg(-1). min(-1) insulin clamp). Prevention of fat accretion maintained glucose uptake (R(d); 29+/-2, 29+/-2, and 31+/-4 mg x kg LBM(-1) x min(-1)) and glycogen synthesis rates (GS, 12+/-1, 12 +/-1, and 14+/-2 mg x kg LBM(-1) x min(-1)) at youthful levels (2 mo AL) in 8- and 20-mo-old CR rats, respectively. These levels were significantly increased (P<0.001) compared with AL rats with higher %FM (R(d), 22+/-1 and 22+/-2 and GS, 7+/-1 and 8+/-2 mg x kg LBM(-1). min(-1) in 8- and 20-mo-old rats, respectively). The increase in whole body GS in age-matched CR rats was accompanied by approximately 40% increased accumulation of [(3)H] glucose into glycogen and a similar increase in insulin-induced muscle glycogen content. Furthermore, the activation of glycogen synthase increased, i.e., approximately 50% decrease in the Michaelis constant, in both CR groups (P<0.01). We conclude that chronic CR designed to prevent an increase in storage of energy in fat maintained peripheral insulin action at youthful levels, and aging per se does not result in a defect on the pathway of glycogen storage in skeletal muscle.     

Protein aging by carboxymethylation of lysines generates sites for divalent metal and redox active copper binding: relevance to diseases of glycoxidative stress.

Aging and age-related diseases are associated with the production of reactive oxygen species which modify lipids, proteins and DNA. Here we hypothesized the glyco- and lipoxidation product N(epsilon)-(carboxymethyl)lysine (CML) in proteins should bind divalent and redox active transition metal binding. CML-rich poly-L-lysine and bovine serum albumin (BSA) were chemically prepared and found to bind non-dialyzable Cu(2+), Zn(2+) and Ca(2+). CML-BSA-copper complexes oxidized ascorbate and depolymerized protein in the presence of H(2)O(2). CML-rich tail tendons implanted for 25 days into the peritoneal cavity of diabetic rats had a 150% increase in copper content and oxidized ascorbate three times faster than controls. CML-rich proteins immunoprecipitated from serum of uremic patients oxidized four times more ascorbate than control and generated spin adducts of DMPO in the presence of H(2)O(2). The chelator DTPA suppressed ascorbate oxidation thereby implicating transition metals in the process. In aging and disease, CML accumulation may result in a deleterious vicious cycle since CML formation itself is catalyzed by lipoxidation and glycoxidation.     

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.     

Effect of caloric restriction on Hprt lymphocyte mutation in aging rats

Caloric restriction (CR) reduces tumor incidence and retards aging in laboratory animals, including non-human primates. Because of the relationships among mutation, disease susceptibility, and aging, we investigated whether or not CR affects the accumulation of somatic cell mutations in aging animals. Starting at approximately 2 months of age, male CD rats (Harlan Sprague-Dawley-derived) were placed on different levels of dietary intake: ad libitum (AL) feeding, and 90% (10% CR), 75% (25% CR) and 60% (40% CR) of the total calories consumed by AL animals. At 3, 6, 12, and 24 months after the beginning of CR, Hprt mutant frequencies (MFs) were determined. The MFs measured in spleen lymphocytes from AL and CR rats sacrificed at 3 months of dietary restriction were similar for all dietary groups. However, the MFs at 6, 12, and 24 months of CR were significantly higher in AL-fed rats compared with animals on 40% CR: (4.5+/-0.4)x10(-6) versus (3.3+/-0.3)x10(-6) (P=0.032) in 6 months CR rats; (10.3+/-2.3)x10(-6) versus (7.3+/-1.2)x10(-6) in 12 months CR rats (P=0.04), and (18.3+/-3.2)x10(-6) versus (7.8+/-1.0)x10(-6) (P=0.001) in 24 months CR rats. In addition, rats receiving 25% CR for 24 months had a MF, (10.7+/-2.0)x10(-6), between the 40% CR and AL rats. Multiplex PCR of the Hprt gene in mutant clones from 12 and 24 months 40% CR rats and the corresponding AL rats detected deletions in 42% of CR mutants and 19% of AL mutants. Because of the difference in Hprt MF in the two groups, the estimated MF associated with deletions in CR rats was similar to the deletion MF in AL rats. This observation implies that the lower MF in CR rats is due to a reduction in smaller Hprt mutations (i.e. base substitutions and frameshifts). The pattern of smaller Hprt mutations from AL rats suggests that many were produced by reactive oxygen species (ROS). The results indicate that CR reduces the accumulation of spontaneous somatic cell mutation in aging rats, especially those caused by base substitutions and frameshifts.