Our results showed that NDGA and aspirin each increase survival in male UM-HET3 mice, but not in females. The body weight data (Miller et al., 2007
) suggest that these beneficial effects are not mediated by caloric restriction. Previous studies of agents reported to extend rodent lifespan when added in food (see Schneider & Miller, 1998
; Schneider & Reed, Jr., 1985
) for reviews), have been criticized frequently for small sample sizes, use of short-lived control groups, or the lack of information about a colony’s infectious status; and none, as far as we can determine, has ever been replicated either at the original test site or by a second laboratory group. Nearly all prior studies have employed isogenic animal groups, raising the possibility that positive results might reflect strain-specific idiosyncrasies that would not be reproducible in other genetic stocks. Our own results, using genetically heterogeneous mice, three specific pathogen-free vivaria, and a relatively long-lived set of control mice, may provide a better foundation for future replication.
We were only able to test each compound at a single dose, because of cost considerations, and the dose selected was in each case based on limited previous experience with that agent in rodents. It is possible, therefore, that different results, including a larger positive effect on survival, might have been seen at doses different from those used in this study. In planned Phase II testing of NDGA and aspirin, the survival studies will be repeated with multiple doses of each agent.
Our interim report (Miller et al., 2007
) found a positive effect of NDGA on male survival during the first half of the lifespan, but there are precedents in which an intervention, such as exercise (Holloszy, 1993
), has been shown to increase median lifespan or some other index of mortality risks in young and middle-aged adults, without any effect on maximum lifespan or an equivalent index of survival among the oldest test subjects. Therefore, we assessed the effect of NDGA and aspirin on the proportion of mice that live past the age at which 90% of the population has died using a quantile test (Wang et al., 2004
). Although the proportion of mice alive at 90% mortality was greater in the NDGA and aspirin groups as compared to the control group, the effects were not statistically significant (p = 0.12 and p = 0.16, respectively). Therefore, we are unable to conclude at this stage that either treatment had an effect on maximum lifespan at the dose used. If NDGA and/or aspirin do indeed modulate the rate of aging or the risks of specific lethal illnesses, it is entirely possible that the optimal benefit, including perhaps a change in probability of survival to the 90th percentile, might be seen at doses higher or lower than those used in this initial study.
Although our experimental design did not include sufficient animals to allow us to detect small intervention effects at each of the three test sites, we note from that the effects of aspirin and of NDGA were strongest at the two sites where control males had the shortest lifespan (see ). Significant differences among the three sites in survival of control males have also been seen in a separate cohort of control mice being used for comparison to other ITP interventions initiated in 2005 (“Cohort 2” mice; data not shown), with UM males again showing lowest mortality risks in the first half of the lifespan. The site-specific differences seem unlikely to reflect systematic effects on mouse health, in that there were no differences among sites in survival of control females in either Cohort 1 () or Cohort 2 (not shown). Median lifespan for females at all three ITP sites is similar to that found in previous lifespan studies of UM-HET3 mice (all conducted at UM; see Miller et al., 2007
, for citations). Median lifespan for ITP males at UM is slightly higher than in any of the three previous studies, but median lifespan for UT and TJL males is somewhat lower than in the historical controls conducted at UM. We suspect that these differences in control male life tables reflect differences among the sites in the specific dietary formulations administered to the breeders used to produce UM-HET3 test mice at each site, or to the formulations to which the test mice were exposed between weaning and transfer to the drug-containing test diets at age 4 months (aspirin, NFP, 4-OH-PBN, and control groups) or at 9 months (NDGA groups). The diets used for breeders and weanlings (prior to drug exposure) differ in fat content (4.5% to 6.5%), supplemental levels of thiamine and other heat-sensitive vitamins, protein content (18% – 24%) and source of protein (e.g. fish, beet, or pork meal). Control mice, and drug-exposed mice, at the UM site were routinely and significantly smaller than those at the other two sites throughout adult life, for both males and females, and these differences could well reflect lasting influences of differences in dietary regimen on breeders, weanlings, or both. It is also possible that other site-specific factors, such as minor differences in water quality, noise level, ventilation details, extraneous odors, cage-changing frequency, etc., might contribute to site-specific differences in survival of male (but not of female) mice. Starting with Cohort 4 (born in 2007), the three ITP sites have adopted a uniform protocol for diet composition at all stages of the test process, including diets for breeder mice and for test mice prior to drug administration. Replicate studies, at the ITP test sites and at other facilities, will be needed to determine whether the present findings on aspirin and NDGA are robust and reproducible in multiple colonies.
The observation that NDGA or aspirin increased survival in males, but not females, could be accounted for by gender differences in steady-state levels and/or in metabolizing the drugs. We conducted a four-week study in which male and female mice were fed diets containing two different doses of each compound; the original dose used in the survival studies reported here and a higher dose. The results for NDGA revealed that males in each dosage group had higher median plasma concentrations of NDGA than females; this difference was two-fold at the original dose used in the survival studies. Thus, it is plausible that NDGA may have extended the lifespan of male mice and not females because of differences between the sexes in peak or average NDGA serum concentrations. More definitive conclusions await Phase II studies in which the effects of a higher dose of NDGA on survival in male and female mice will be studied. Similarly, the results of feeding aspirin to male and female mice provided evidence for gender differences in aspirin metabolism. Thus, there was a 2- to 3-fold higher ratio of salicylic acid metabolite to acetylsalicylic acid in female mice fed either dose of aspirin, indicating that a greater proportion of the ingested aspirin is converted to salicylic acid in females as compared to males. Aspirin’s principal mechanism of action is to inhibit the activity of COX-1 and 2. ASA is 100-fold more potent than SA in inhibiting COX-1 activity and 2-fold more potent than SA in inhibiting COX-2 (Mitchell et al., 1994). Thus, a higher ratio of SA to ASA in females would be consistent with a reduced therapeutic effect of aspirin in females. This result is consistent with that of human studies of the effects of aspirin on the risk of myocardial infarction that indicate females are less responsive to aspirin (Yerman et al., 2007
). This may explain why aspirin increased survival in male mice and not female mice. More comprehensive pharmacokinetic studies and feeding a higher dose of aspirin, in addition to the original dose, in planned Phase II studies, will allow for more definitive conclusions.
The present results are consistent with previous reports that NDGA increases lifespan in both insects and mammals. Thus, NDGA has been reported to increase lifespan in fruit flies, mosquitoes, and rats (Buu-Hoi & Ratsimamang, 1959
; Miquel et al., 1982
; Richie et al., 1986
). Several properties of NDGA may contribute to its beneficial effects on lifespan. For example, NDGA may act to prevent deleterious effects of aging on the brain. Thus, it has been reported to extend the survival of a mouse model of amyotrophic lateral sclerosis, the G93A SOD1 mutant mouse (West et al., 2004
). Expression of 5-lipoxygenase is elevated with age and has been proposed to play a role in the pathobiology of aging-associated neurodegenerative diseases (Manev et al., 2000
; Qu et al., 2001
). Indeed, NDGA has been reported to be effective in preventing neuronal death and cognitive deficits resulting from forebrain ischemia-reperfusion injury (Shishido et al., 2001
). Moreover, it has been reported to enhance glucose clearance and insulin sensitivity in a rat diabetes model (Reed et al., 1999
). In the same study, it was reported to drastically reduce serum triglycerides (Reed et al., 1999
), which may relate in part to NDGA’s reported ability to block fatty acid synthesis in adipocytes through inhibition of fatty acid synthase (FAS) and to inhibit lipoprotein lipase (Li et al., 2005
; Park and Pariza, 2001
). In numerous studies, NDGA has been reported to have anti-cancer activity through its action as a 5-lipoxygenase inhibitor (e.g. McDonald et al, 2001
; Nony et al., 2005
; Hoferova et al, 2004
). It has also been reported to suppress growth of breast cancer cells through inhibition of the function of two receptor tyrosine kinases (RTKs), the insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu) receptor (Youngren et al., 2005
The effect of aspirin on lifespan in mice in the present study is consistent with reports of numerous epidemiological studies showing that aspirin use reduces the risk of mortality from a variety of diseases in humans including colon cancer, prostate cancer, and cardiovascular disease (e.g. Jacobs et al., 2005
; Chan et al., 2008
; Johnson et al., 2002
; Lim et al., 2007
). Recently, aspirin was reported to activate the NF-kappa B signaling pathway and induce apoptosis in two in vivo
models of human colorectal cancer (Stark et al., 2007
). Aspirin also normalized blood pressure in a rat model of hypertension (Tuttle et al., 1988
). Furthermore, it reportedly improved learning of a spatial memory task in old rats (Smith at al., 2002
). Interestingly aspirin use was reported to be associated with increased survival in extreme old age in humans. Aspirin use was associated with increased survival in a five-year follow-up study of subjects in the Finnish Centenarian Study (Aguero-Torres et al., 2001
). The authors concluded that the increased survival was probably not attributable simply to the anti-atherogenic effects of aspirin, because the aspirin group had significantly more cardiovascular disease than the non-aspirin users.
Both NDGA and aspirin have anti-oxidative effects and anti-inflammatory properties in addition to the other properties already discussed (Harper et al., 1999
; Shi et al., 1999
). NDGA, for example, is a potent anti-oxidant and has anti-inflammatory activity by inhibiting leukotriene synthesis (Harper et al., 1999
; West et al., 2004
). Aspirin potently inhibits oxidative damage, most likely through scavenging the hydroxyl radical and has anti-inflammatory actions by inhibiting prostaglandin synthesis (Shi et al., 1999
; Vane, 2000
). It is noteworthy that the compounds studied in Cohort I have either anti-inflammatory properties (nitroflurbiprophen), antioxidant properties (4OH-PBN), or both (NDGA and aspirin). Of the four compounds tested, only those with both anti-inflammatory action and antioxidant activity extended lifespan in males. Thus, the combination of antioxidant activity with anti-inflammatory effects may be important in the positive results seen from these two interventions in our initial Phase I study.
The effects of NDGA and aspirin on our measure of extreme longevity, i.e. proportion surviving at the start of the final survival decile, did not reach statistical significance (p = 0.12 and p = 0.16, respectively). It will be interesting to see if greater effects are seen in follow-up studies of these agents at higher or lower doses. Analysis of the effects of NDGA and aspirin on other indices of age-dependent change, including data on terminal pathology, incidence of non-lethal lesions (e.g. cataracts), and the pace of functional changes (e.g. loss of immune response and muscle strength) will all be needed to reach an inference as to whether the effects of NDGA and aspirin on survival reflect a general effect on aging and age-sensitive traits, or instead merely a deceleration of specific forms of lethal disease. Currently, we are planning follow-up (“Phase II”) studies that will include evaluations of the effects of additional doses of aspirin and NDGA on multiple markers of health in middle-aged mice, including analyses of CNS function, metabolic rate, insulin and IGF-1 sensitivity, IGF-I sensitivity, hematopoietic stem cells, DNA strand breaks, body composition, bone mineral density, measures of oxidative stress, T cell subsets, collagen cross-linking, lens turbidity, and early signs of neoplastic and degenerative illnesses. Evaluations of possible modes of action of NDGA and aspirin, including measurements of hormonal, oxidative, and metabolic indices could help to test specific hypotheses about NDGA- and aspirin-sensitive pathways relevant to the aging process and pathogenesis of late-life illnesses. Studies of other compounds with structures or pharmacologic profiles similar to NDGA and aspirin also could be informative. Our data also provide a rationale for evaluation of NDGA and aspirin effects on mortality risks in other varieties of mice and potentially for parallel studies using short-lived primates. Finally, the effects of NDGA and aspirin on long-lived mice, for example mice on calorically restricted diets, or dwarf mutants, could help to determine whether the pathways by which NDGA and aspirin affect survival may overlap with those involved in other models of lifespan extension in mice.