Effect of dietary boron on the aging process.

Total boron concentrations in Drosophila changed during development and aging. The highest concentration of boron was found during the egg stage, followed by a decline during the larval stages. Newly emerged flies contained 35.5 ppm boron. During the adult stage the boron concentration increased by 52% by 9 weeks of age. Adding excess dietary boron during the adult stage decreased the median life span by 69% at 0.01 M sodium borate and by 21% at 0.001 M sodium borate. Lower concentrations gave small but significant increases in life span. Supplementing a very low boron diet with 0.00025 M sodium borate improved life span by 9.5%. The boron contents of young and old mouse tissues were similar to those of Drosophila and human samples. Boron supplements of 4.3 and 21.6 ppm in the drinking water, however, did not significantly change the life span of old mice fed a diet containing 31.1 ppm boron.

Effect of vitamin A on longevity.

Increasing the dietary content of Vitamin A from inadequate to adequate during the developmental stages of Drosophila increased the median life span by as much as 17.5%. The optimum dietary range of concentrations of Vitamin A for increasing the life span of Drosophila was found to be between 4 and 8 IU/g food. The maximum life span was reduced as dietary concentrations of Vitamin A exceeded this value. Vitamin A palmitate and retinal inhibited the peroxidation of linolenic acid induced by the generation of superoxide radicals from acetaldehyde. Other forms of Vitamin A, such as retinol and retinoic acid, moderately inhibited lipid peroxidation at low concentrations but stimulated peroxidation considerably when present at high concentrations. Based upon the ability of these retinoids to inhibit the reduction of cytochrome c by superoxide radicals, we propose that retinoids can inhibit and stimulate lipid peroxidation depending upon their concentration by reacting with superoxide radicals. We suggest that this reaction is the basis for the apparent ability of Vitamin A to prolong and shorten life span depending upon the dietary intake.

Influence of photosensitizers and light on the life span of Drosophila.

The life span of adult Drosophila melanogaster fruit flies changed when they were fed two different photosensitizers. Methylene blue decreased the median life span by 49% when present in the food at a concentration of 0.001 M. Another photosensitizer, riboflavin, produced no changes in life span under the same conditions of a 12:12 h light/dark cycle at a daytime light intensity of 1000 lux. Flies exposed to constant darkness lived 43.2% longer than those exposed to constant light at a light intensity of 2000 lux. Under these conditions, riboflavin increased the life span of the flies exposed to constant light by as much as 25%. We conclude that riboflavin confers some degree of protection against the effects of constant light exposure. The completely different results obtained with riboflavin and methylene blue suggest a possible mechanism for photoageing involving photodynamic action mediated through the production of singlet oxygen.

Inhibition of iron absorption prolongs the life span of Drosophila.

The life span of Drosophila melanogaster (Oregon R) males was found to be proportional to the logarithm of the iron content of the diet. Life span was also shown to be proportional to the rate of iron accumulation for Drosophila, mice and man. The total body iron content was found to correlate with the total calcium content of adult Drosophila. Iron content during the developmental stages, however, remained relatively constant and did not change with changes in the calcium concentrations. Dietary tea (Camellia sinensis) extracts were found to inhibit the ageing-related accumulation of iron and to prolong the life span of Drosophila by as much as 21.4%. It is concluded that iron accumulation is a significant factor contributing to senescence.

The brain-to-liver mercury ratio increases with aging in mice.

There was no significant change in the total amount of mercury in organs (lung, heart, kidney, brain, and liver) from male C57BL/6J mice ranging in age from 133 to 904 days of age maintained under conventional conditions with no known source of mercury exposure other than background concentrations. The lowest values were found in the liver and the highest in the brain, with considerable variation in the mercury content between individual mice for all organs examined. The ratio of mercury in the brain to that in the liver, however, was found to significantly increase with aging in an exponential manner. A similar result was found for the ratio of brain to kidney mercury. We conclude that older mice are less able to maintain low brain-to-liver ratios of mercury regardless of the total body content of mercury. Dietary mercury ranging from 200 to 20,000 ppm Hg had little or no influence on the life span of Drosophila fruit flies, suggesting that the effect of mercury is probably not on life span itself but on other factors associated with the aging process such as neurological disfunction.

Changes with ageing in total dolichol and dolichol fractions in Drosophila.

Since it is unclear how the concentration of dolichol fractions change with ageing in mammals, we have examined the changes in another organism, Drosophila. Dolichol extracted from Drosophila melanogaster was found to consist of three fractions composed of 15, 16 and 17 isoprene units. The total dolichol content of female flies maintained at 25 degrees C increased with ageing between 0 and 64 days of adult age but the change was not significant. The total dolichol content of male flies decreased with ageing but the decrease was not significant. The relative amounts of the three different dolichol fractions in both male and female flies also failed to show any significant ageing-related change. The greatest amount of dolichol was found in the 16 isoprene unit fraction representing 67.2% of the total dolichol in male flies and 65.4% in female flies. Increased dietary dolichol had little or no influence on the life span of Drosophila when given either during the developmental or adult stages.

Elevated serum copper is associated with reduced immune response in aging mice.

Young mice were found to have serum copper concentrations ranging from a low of 0.291 to a high of 0.584 ppm. Old mice had serum copper concentrations ranging from 0.223 to 1.715 with 30.9% of the old animals having values greater than 0.6 ppm. The mitogen response of isolated lymphocytes from the spleens of aging mice was greatly reduced when these cells were taken from animals with naturally occurring serum copper levels in excess of 0.6 ng of copper/mg wet weight serum. The lymphocytes taken from young mice with higher serum copper concentrations, on the other hand, had increased response to mitogens. Addition of the copper protein, ceruloplasmin, to lymphocyte cultures in vitro reduced the mitogen response of purified splenic lymphocytes with the reduction being greater for cells from old animals. We suggest that excess serum copper and ceruloplasmin may be immunosuppressive, especially in older organisms.

Lead accumulation in the bones of aging male mice.

The lead content of mouse femurs increased by 83% between 76 and 958 days of age with values ranging from 0.192 to 1.78 ng Pb/mg dry weight. These values are remarkably lower than in previous reports for the lead content of bone. The lead content of mouse liver showed no aging-related trend with values ranging from 0.00823 to 0.0149 ng/mg dry weight. Bone density, calcium and collagen content were not related to the lead content. We conclude that while bone lead content is very low in mice, it increases with aging but does not appear to be related to the osteopenia which develops in the C57BL/6J male mouse.

Preliminary evidence for photochemical ageing in Drosophila.

Drosophila melanogaster (Oregon R) males were exposed to visible light intensities varying from 0.3 to 7300 lux at environmental temperatures of 30, 35 and 37 degrees C, on a 12-h light/dark diurnal rhythm. At 30 degrees C reducing the light exposure from 7300 to 4 lux increased the median life span by 141%. At 35 degrees C reducing the light exposure from 4650 to 0.3 lux increased the life span by 389%. At 37 degrees C a reduction from 6580 to 0.3 lux increased life span by 453%. Even dim light (65 lux) affected life span in a negative manner. Two phases of response to light were identified, with a slow change in life span occurring below 400 lux and a more rapid rate of change above 400 lux. We conclude that visible light may be a major factor in the ageing process for Drosophila and that photochemical effects may contribute to senescence in other organisms. Possible alternative reasons for the effect of light on the life span of Drosophila include changes in body temperature, physical activity and oxygen consumption.

Uric acid content of Drosophila decreases with aging.

Free uric acid concentrations declined with aging in male Oregon R Drosophila melanogaster by 59% or more between 0 and 50 days of adult age. Free xanthine concentrations increased between 0 and 5 days of age and declined by 75% between 5 and 50 days of age. Xanthine oxidase activity was maximal for newly emerged flies and then declined rapidly reaching a minimum at 9 days of age. After 9 days of age xanthine concentrations may be the limiting factor for the production of uric acid by xanthine oxidase in aging fruit flies. Declining uric acid concentrations may represent a loss of antioxidant potential in aging Drosophila.

Ascorbic acid in Drosophila and changes during aging.

The ascorbic acid content of Drosophila melanogaster was found to be high in the absence of a dietary source. The amount of ascorbic acid per fly declined with aging in both the Oregon R and Swedish C strains. The median life span at 25 degrees C was 45 days for Swedish C and 59 days for Oregon R. The amount of ascorbic acid in Swedish C flies (0.078 micrograms/fly) was higher than that for Oregon R (0.058 micrograms/fly) for newly emerged flies but the rate of decline with aging was greater for Swedish C than Oregon R. The decline in ascorbic acid content with aging was 70.4% for Swedish C versus 19.9% for Oregon R. A brief cold shock was found to significantly increase the amount of ascorbic acid in Oregon R flies. Feeding the precursor of ascorbic acid synthesis, L-gulonolactone, did not improve the life span. Life-time feeding of ascorbic acid did not improve the life span of either Swedish C or Oregon R flies.

Changes in boron concentration during development and ageing of Drosophila and effect of dietary boron on life span.

Total boron concentrations in Drosophila changed during development and ageing. The highest concentration of boron was found during the egg stage followed by a decline during the larval stages. Newly emerged flies contained 35.5 ppm boron. During the adult stage the boron concentration increased by 52% by 9 weeks of age. Adding excess dietary boron during the adult stage decreased the median life span by 69% at 0.01 M sodium borate and by 21% at 0.001 M sodium borate. Lower concentrations gave small but significant increases in life span. Supplementing a very low boron diet with 0.00025 M sodium borate improved life span by 9.5%. The boron contents of young and old mouse tissues were similar to those of Drosophila and human samples. We conclude that moderate levels of dietary boron may have a general protective effect in biological systems. The mechanism of this effect at present remains unknown.

Calcium, iron, copper, boron, collagen, and density changes in bone with aging in C57BL/6J male mice.

X-rays of old C57BL/6J male mice showed deformed vertebral columns. Bone density was found to increase between 76 and 517 days of age and to decrease after 685 days of age. The boron content of femurs declined by 9% with aging but the decrease was not significant. Calcium increased between 76 and 198 days of age but declined by 36% between 200 and 1000 days of age. Iron increased by 207% by 1000 days of age. Copper declined between 76 and 198 days of age but increased by 61% between 200 and 1000 days of age. Bone collagen as indicated by hydroxyproline and proline content decreased 17.4% by 1000 days of age. The largest single change with aging was, therefore, in the iron content of bone. Several correlations were found to be independent of the age of the animals. Bone density was correlated with bone calcium and collagen. Iron was negatively correlated with calcium and collagen. Calcium and collagen content were unrelated. Bone density and iron were also surprisingly unrelated. A possible explanation for this observation is given. Copper was negatively correlated with bone calcium, bone density, and collagen content. Excess copper was, therefore, the single most important factor associated with decreasing bone size and density.

Changes in taurine in aging fruit flies and mice.

The whole-body concentration of the amino acid taurine was found to be more than 1000% higher during the adult stage of Drosophila melanogaster than during the larval stage. Drosophila larvae were killed by adding taurine (0.01 to 0.10 M) to their food medium. Adult Drosophila failed to produce progeny when fed 0.2 M taurine for one week. Lifetime feeding of taurine (0.05 to 0.20 M) produced no change in life span. Feeding the taurine precursor, hypotaurine, and the taurine mobilizing agent, beta-alanine, to Drosophila did not change life span at low concentrations but both decreased life span at higher concentrations. Taurine concentration in male C57BL/6J mice increased with aging in the heart, decreased in leg muscle and remained unchanged in brain, liver, kidney, and blood. We suggest that an as yet undefined developmental process is altered in Drosophila by taurine and that this process may be unique to insects.

Calcium and calmodulin changes with ageing in C57BL/6J mice.

Male C57BL/6J mice ranging in age from 50 to 1186 days were used to measure total calcium and calmodulin concentrations. The increase in calcium between 0 and 1,000 days of age was 260% for kidney, followed by brain (189%), heart (173.5%), lung (106.5%) and liver (78.5%). Calcium in femur declined by 28.2%. The calmodulin content of liver increased with ageing. Both liver and kidney calmodulin concentrations declined early in life followed by ageing-related increases. Brain, lung and heart calmodulin concentrations did not change significantly with ageing. We conclude that changes in calcium homeostasis are not reflected in calmodulin changes. The loss of calcium in bone is consistent with the occurrence of osteoporosis in ageing C57 mice.

Aluminum in the organs and diet of ageing C57BL/6J mice.

Total aluminum concentrations increased with ageing in the liver and kidney of male C57BL/6J mice, remained unchanged in brain and heart, and decreased with ageing in femur and lung for mice ranging in age from 56 to 1186 days. Ligating one kidney did not significantly increase aluminum concentrations in the various organs. Feeding 1 X 10(-2) M aluminum chloride (270 ppm Al) in the drinking water beginning at 604 days of age decreased the average life span by 6.7%. We conclude that very little aluminum accumulation occurs with ageing in the organs tested in this study, in spite of a high dietary intake. Other organs might show a change. Only one aluminum concentration was used in this study which accelerated the rate of ageing as indicated by a change in the survival curve. The effect of higher or lower aluminum concentrations remains to be seen.

Influence of age on mitochondrial enzyme levels in Drosophila.

The specific activity and the activity per fly of four mitochondrial enzymes did not change with ageing in male Drosophila melanogaster (Oregon R). The enzymes assayed were rotenone-insensitive NADH-cytochrome c reductase, adenylate kinase, succinate cytochrome c reductase, and malate dehydrogenase, located in the outer membrane, inner membrane space, inner membrane and matrix, respectively. The specific activity of malate dehydrogenase showed no significant change for young and old head, thorax and abdomen. We conclude that there is no specific site for ageing damage in the mitochondrion, when the enzyme activities in this study are used as an indicator. It should be noted, however, that these enzymes represent only a small percentage of the total enzymes present in mitochondria.

Mitochondrial DNA and life span changes in normal and dewinged Drosophila at different temperatures.

At 11 and 30 degrees C there was no change in the amount or in the buoyant density of nuclear DNA of male Oregon R Drosophila melanogaster with aging. When normal flies were maintained at 11 degrees C (at which temperature they do not fly), mitochondrial DNA content declined gradually with aging and 39.4% of the original mitochondrial DNA was lost at the median survival time of 152 days of age. For normal flies maintained at 30 degrees C, 86.5% of the mitochondrial DNA was lost during aging at the median survival time of 25 days. In contrast only a 39.2% decrease in the mitochondrial DNA occurred in dewinged flies maintained at 30 degrees C. A slow phase of mitochondrial DNA loss appears to be related to aging and a fast phase to flight activity. Removing the wings of flies eliminates the fast phase of DNA loss but only slightly improves life span (by less than 10%). Lowering the environmental temperatures to 11 degrees C also eliminates fast phase DNA loss and decreases the rate of the slow phase DNA loss. We conclude that mitochondrial DNA loss is related to both physical activity and to the aging process itself.

Effect of dietary beta-carotene on the survival of young and old mice.

Feeding 0.5% beta-carotene in the diet for life beginning at 29 days of age improved the average life span of C57BL/6J male mice by 5.0% but decreased the life span of mice started at 608 days of age by 11.5%. Neither difference, however, proved to be statistically significant. Feeding beta-carotene increased the concentration of beta-carotene in the serum by 60% but did not change the beta-carotene content of heart, liver or kidney. We conclude that singlet oxygen, which is very efficiently quenched by beta-carotene, is an important factor in senescence only if it is produced at organ sites not accessible to serum beta-carotene. Since we have found that beta-carotene feeding is not a useful means for increasing tissue concentrations of beta-carotene, other more sophisticated means must be developed for accomplishing this purpose. It is also clear that while dietary beta-carotene is not an effective means for prolonging life span, it is nontoxic when fed continuously at high concentrations.