ZhongMao Guo

Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline

Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.

Dietary restriction reduces atherosclerosis and oxidative stress in the aorta of apolipoprotein E-deficient mice

Dietary restriction (DR) has been shown to inhibit almost all the age-related diseases, e.g. cardiomyopathy and cancers, in rodents. However, there is little information for the effect of DR on atherosclerosis. In the present study, we examined the effect of DR on the development of atherosclerosis in mice homozygous knockout for apolipoprotein E gene (ApoE(-/-)). The ApoE(-/-) mice were fed either ad libitum (AL) or 60% of the diet consumed by the mice fed AL. Atherosclerotic lesions in the proximal aorta of these mice were measured. Our results showed that ApoE(-/-) mice fed the calorie-restricted diet had smaller and relatively early stages of atherosclerotic lesions (e.g. foam cells and free lipids) when compared to ApoE(-/-) mice fed AL, who developed more advanced lesions (e.g. fibrous caps and acellular areas). In addition, ApoE(-/-) mice fed the calorie-restricted diet showed a significant decrease in the level of lipid hydroperoxides and the production of superoxide and hydrogen peroxide in the aorta as compared to ApoE(-/-) mice fed AL. These observations suggest that reduction of oxidative stress in the arterial wall may contribute to the anti-atherogenic effect of DR in ApoE(-/-) mice.

Attenuation of DNA polymerase β-dependent base excision repair and increased DMS-induced mutagenicity in aged mice

The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.

Effects of age and food restriction on oxidative DNA damage and antioxidant enzyme activities in the mouse aorta

In this study, DNA damage in mouse aortic cells was measured using the comet assay. The tail moment of the comet assay in aortic cells obtained from 26-month-old mice fed ad libitum (O-AL) was significantly increased as compared to 6-month-old mice fed ad libitum (Y-AL) after the cells were incubated with formamidopyrimidine-DNA glycosylase (Fpg), which specifically recognizes oxidized purines, endonuclease III (Endo III), which specifically recognizes oxidized pyrimidines, or the combination of Endo III and Fpg. The tail moment in aortic cells obtained from 26-month-old mice fed a food-restricted diet (O-FR) was significantly reduced as compared to O-AL mice after the cells were incubated with the combination of Endo III and Fpg. These results indicate that oxidative DNA lesions, i.e. the Endo III- and Fpg-sensitive sites, increase with age in mouse aortic cells and that FR attenuates the age-related increase in oxidative DNA damage. To determine if the changes in oxidative DNA damage in mouse aortic cells are related to the antioxidant status in these cells, we measured the activities of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, extracellular-SOD, catalase and glutathione peroxidase-1 in the mouse aorta. We observed that the activities of all antioxidant enzymes studied were significantly increased with age and that FR attenuated the age-related increase. These data indicate that the age-related increase and FR-induced decrease in oxidative DNA damage, i.e. the Endo III- and Fpg-sensitive sites, in mouse aortic cells is not due to alteration of the antioxidant defense system.

Characterization of gene‐specific DNA repair by primary cultures of rat hepatocytes

At present, almost all the information on gene‐specific DNA repair in mammals comes from studies with transformed cell lines and proliferating primary cells obtained from rodents and humans. In the present study, we measured the repair of specific DNA regions in primary cultures of nondividing rat hepatocytes (parenchymal cells). DNA damage was induced by irradiating the primary cultures of hepatocytes with ultraviolet (UV) light, and the presence of cyclobutane pyrimidine dimers (CPDs) was measured by using T4 endonuclease V in the following: a 21‐kb BamHI fragment containing the albumin gene, a 14‐kb BamHI fragment containing the H‐ras gene, and the genome overall. The frequency of CPDs in the two BamHI fragments and the genome overall were similar and ranged from 0.5 to 1.3 CPDs per 10 kb for UV doses of 5–30 J/m2. However, the removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than from that of the genome overall and the DNA fragment containing the H‐ras gene. Within 24 hr, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene versus less than 40% for the genome overall and the DNA fragment containing the H‐ras gene. The lower repair observed for the 14‐kb fragment containing the H‐ras gene is probably indicative of repair of the nontranscribed region of this fragment because the H‐ras gene makes up only 2.4 kb of the 14‐kb fragment. Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the nontranscribed strand; however, no differences were observed in the repair of the two strands of the fragment containing the H‐ras gene. These results demonstrate that primary cultures of nondividing rat hepatocytes show differential repair of UV‐induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines. J. Cell. Physiol. 176:314–322, 1998.

Effect of cAMP-induced transcription on the repair of the phosphoenolpyruvate carboxykinase gene by hepatocytes isolated from young and old rats.

The repair of UV-induced DNA damage in the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied in primary cultures of hepatocytes isolated from young (6-month-old) and old (24-month-old) rats fed ad libitum and old rats fed a calorie-restricted diet. Incubation of the hepatocytes with cAMP rapidly induced PEPCK transcription and mRNA levels 4- to 5-fold. In absence of cAMP, the repair of the PEPCK fragment was similar in cultured hepatocytes isolated from young and old rats fed ad libitum. However, cAMP significantly increased the percentage of cyclobutane pyrimidine dimers (CPDs) removed from the PEPCK fragment 12 h after UV-irradiation in cultured hepatocytes isolated from young rats fed ad libitum. This increase was due to an increase in the repair of the transcribed strand of the PEPCK fragment. In contrast, cAMP did not increase the repair of the PEPCK fragment in cultured hepatocytes isolated from old rats fed ad libitum in spite of an increase in PEPCK transcription. Thus, it appears that the coupling of transcription and DNA repair is compromised in cultured hepatocytes isolated from old rats fed ad libitum. However, cultured hepatocytes isolated from old rats fed a calorie-restricted diet showed an induction in the rate of repair of the transcribed strand of the PEPCK fragment by cAMP that was similar to hepatocytes isolated from young rats fed ad libitum.

Normal immune function in young and old DNA polymerase-β deficient mice

Abstract The effect of the DNA polymerase-β (β-pol) deficiency on mitogenic response and cytokine production was studied in spleen lymphocytes from 4–5- and 20–22-month-old β-pol −/+ mice and their age-matched wild-type littermates. The proliferative response of lymphocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [ 3 H]thymidine incorporation, and the induction of cytokine production (interleukin (IL)-2, IL-4, and interferon necrosis factor (IFN)-γ) was assessed by enzyme-linked immunosorbent assay. There was no significant difference in Con A- or LPS-induced proliferation or cytokine production in young β-pol −/+ mice compared with young wild-type littermates or in old β-pol −/+ mice compared with old wild-type littermates. However, mitogen-induced proliferation and cytokine production changed significantly with age. The proliferative response to Con A and to LPS, and the IL-2 production was significantly lower, and IL-4 and IFN-γ levels were significantly higher in lymphocytes from old β-pol −/+ mice and old wild-type mice than in lymphocytes from young β-pol −/+ mice and young wild-type littermates. In addition, flow cytometric analysis showed no significant differences between young β-pol −/+ mice and young wild-type littermates or between old β-pol −/+ mice and old wild-type littermates in the proportion of B- and T-cell populations, and T-cell subsets. However, the number of lymphocytes expressing CD4 + phenotype slightly decreased and the proportion of lymphocytes expressing CD44/Pgp-1 (memory) phenotype increased with age. Thus, we found no evidence for alteration in immune function in DNA polymerase-β deficient mice, although they exhibit a decline in immunologic function with age.

Effects of aging on gene specific repair

Publisher Summary DNA in cells is constantly exposed to insults from both endogenous and exogenous factors that may cause breaks in the phosphodiester backbone, cleavage of N -glycosylic bonds, alteration of structure of the purine or pyrimidine bases, intrastrand or interstrand crosslinks, and distortion of helix by intercalation. The age-related decrease in the ability of the cells to repair non-transcribed, silent regions of the DNA could play a role in the age-related accumulation of DNA damage and mutations, which occur with increasing age. The results from several studies suggest that chromatin becomes more condensed with age because of an increase in protein-DNA cross-links and internucleosome interaction. The chromatin template activity for α and β-DNA polymerases declines with age. This age-related change is due to an increased chromosomal condensation. Increased chromosomal condensation could also reduce the access of DNA repair proteins to the DNA and thereby decrease the repair of the lesions. The potential importance of the age-related decrease in preferential repair in aging is also illustrated by a genetic disease, Cockaynes syndrome. This disease is associated with a loss of preferential repair of the transcribed strand of transcriptionally active genes.