Folate deficiency causes massive incorporation of uracil into human DNA (4 million per cell) and chromosome breaks. The likely mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into DNA by DNA polymerase. During repair of uracil in DNA, transient nicks are formed; two opposing nicks could lead to chromosome breaks. Both high DNA uracil levels and elevated micronucleus frequency (a measure of chromosome breaks) are reversed by folate administration. A significant proportion of the U.S. population has low folate levels, in the range associated with elevated uracil misincorporation and chromosome breaks. Such breaks could contribute to the increased risk of cancer and cognitive defects associated with folate deficiency in humans.

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a f luorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked f luorescent heterogeneity was observed in hepatocytes from old (20-28 months) but not young (3-5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 f luorescence pattern, with the largest population from old rats having significantly lower f luorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of ؊154 mV, in contrast to that of the three subpopulations from old rats of ؊70 mV (the largest population), ؊93 mV, and ؊154 mV. Production of oxidants was examined using 2,7di-chlorof luorescin, a dye that forms a f luorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an ageassociated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exdusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.Chronic inflammatory diseases affect millions of people world-wide and may be a primary factor in the development of up to one-third of all cancers (1). A major association between a persistent infection and cancer is evident in chronic active hepatitis B virus (HBV) infection (2). Epidemiological studies show that incidence of hepatocellular carcinoma (HCC) correlates strongly to geographical areas of endemic HBV infection (3). Furthermore, patients with chronic hepatitis have a much higher incidence of liver cirrhosis and HCC than individuals not infected with HBV (4). Also, HBV antigens in sera of patients are associated with HCC (5). Because the virus infects >300 million people throughout the world, HBV has been described as second only to tobacco as a known human carcinogen (6).Despite the apparent epidemiological association of chronic active hepatitis infection and cancer, the mechanisms involved are still not entirely understood. Research has been hampered due to the long latency period between the onset of infection and cancer development, and the lack of suitable animal models that mimic chronic active human hepatitis.HBV does not contain any known acutely transforming oncogenes, and only rarely do integrated HBV sequences activate cellular protooncogenes (7-10). In contrast, persistent inflammation associated with chro...

Mitochondrial function and ambulatory activity were monitored after feeding old rats acetyl-L-carnitine (ALCAR). Young (3-5 mo) and old (22-28 mo) rats were given a 1.5% (wt͞vol) solution of ALCAR in their drinking water for 1 mo, were sacrificed, and their liver parenchymal cells were isolated. ALCAR supplementation significantly reverses the age-associated decline of mitochondrial membrane potential, as assessed by rhodamine 123 staining. Cardiolipin, which declines significantly with age, is also restored. ALCAR increases cellular oxygen consumption, which declines with age, to the level of young rats. However, the oxidant production per oxygen consumed, as measured by 2,7-dichlorof luorescin f luorescence levels, is Ϸ30% higher than in untreated old rats. Cellular glutathione and ascorbate levels were nearly 30% and 50% lower, respectively, in cells from ALCARsupplemented old rats than in untreated old rats, further indicating that ALCAR supplementation might increase oxidative stress. Ambulatory activity in young and old rats was quantified as a general measure of metabolic activity. Ambulatory activity, defined as mean total distance traveled, in old rats is almost 3-fold lower than in young animals. ALCAR supplementation increases ambulatory activity significantly in both young and old rats, with the increase being larger in old rats. Thus, ALCAR supplementation to old rats markedly reverses the age-associated decline in many indices of mitochondrial function and general metabolic activity, but may increase oxidative stress.

Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-L-carnitine [ALCAR, 1.5% (wt͞vol) in the drinking water] and͞or (R)-␣-lipoic acid [LA, 0.5% (wt͞wt) in the chow] improved these endpoints, young (2-4 mo) and old (24 -28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR؉LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (P ‫؍‬ 0.02) hepatocellular O 2 consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR؉LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (P ‫؍‬ 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR؉LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (P ‫؍‬ 0.003) but was restored to the level seen in young rats when ALCAR؉LA was given. The level of malondialdehyde, which was significantly higher (P ‫؍‬ 0.0001) in old versus young rats, also declined after ALCAR؉LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone. H armon, Miguel, and others (1, 2) postulated that mitochondrial decay is a significant factor in aging, caused, in part, by the release of reactive oxygen species (ROS) as by-products of mitochondrial electron transport. Mitochondria are targets of their own oxidant by-products. The steady-state oxidative damage in mitochondria is high relative to other organelles, and the percentage of oxygen converted to superoxide increases with age (3-6). This leads to a vicious cycle of increasing mitochondrial damage, which adversely affects cell function (7), and results in a loss of ATP-generating capacity, especially in times of greater energy demand, thereby compromising vital ATP-dependent reactions. Cellular processes affected by mitochondrial decay include detoxification, repair systems, DNA replication, osmotic balance, and higher-order processes (7), such as cognitive function (7-9). Thus, preservation of mitochondrial function is important for maintaining overall health during aging (7). This theory is buttressed by the observation that caloric restriction, the only known regimen to increase mean life span in animals, maintains mitochondrial function and lowers oxidant production (7,8,(10)(11)(12). A spartan diet of calorie restriction appears to be too unappealing to be widely adopted in humans, and thus other alternative regimens to improve or maintain normal mitochondrial activities have been sought.Several dietary supplements, including the mitochondrial cofactor and antioxidant lipoi...

An immunoinity column is described that facilitates the analysis of oxidative damage products of DNA and RNA in urine, blood plasma, and medium isolated from cultures ofEscherichia coli. In intact animals, lesions (adducts) excised from DNA are transported from the cell through the circulation and excreted in urine. In bacteria, DNA adducts are excreted directly into the medium. In either case, the adducts can be assayed as a measure of oxidative damage to DNA. A monoclonal antibody that recognizes 8-oxo-7,8-dihydro-2'-deoxyguanosine(oxo dG; 8-hydroxy-2'-deoxyguanosine), abiomarker of oxidative damage to DNA, has been isolated, and its substrate binding properties have been characterized. The relative binding affinities of this monoclonal antibody for oxo8dG, unmodified nucleosides, or derivatives of Gua made it suitable for the preparation of immunoaffinity columns that greatly facilitate the isolation of oxo8dG, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroguanosine from various biological fluids. Quantitative analysis of these adducts in urine of rats fed a nucleic acid-free diet and in the medium from cultures of E. coli suggests that oxo8-7,8-dihydroguanine is the principal repair product from oxo8dG in DNA of both eukaryotes and prokaryotes. The results support our previous estimate of about 105 oxidative lesions to DNA being formed and excised in an average rat cell per day.

A diet supplemented with (R)-lipoic acid, a mitochondrial coenzyme, was fed to old rats to determine its efficacy in reversing the decline in metabolism seen with age. Young (3 to 5 months) and old (24 to 26 months) rats were fed an AIN-93M diet with or without (R)-lipoic acid (0.5% w/w) for 2 wk, killed, and their liver parenchymal cells were isolated. Hepatocytes from untreated old rats vs. young controls had significantly lower oxygen consumption (P<0. 03) and mitochondrial membrane potential. (R)-Lipoic acid supplementation reversed the age-related decline in O2 consumption and increased (P<0.03) mitochondrial membrane potential. Ambulatory activity, a measure of general metabolic activity, was almost threefold lower in untreated old rats vs. controls, but this decline was reversed (P<0.005) in old rats fed (R)-lipoic acid. The increase of oxidants with age, as measured by the fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly lowered in (R)-lipoic acid supplemented old rats (P<0.01). Malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were increased fivefold with age in cells from unsupplemented rats. Feeding rats the (R)-lipoic acid diet reduced MDA levels markedly (P<0.01). Both glutathione and ascorbic acid levels declined in hepatocytes with age, but their loss was completely reversed with (R)-lipoic acid supplementation. Thus, (R)-lipoic acid supplementation improves indices of metabolic activity as well as lowers oxidative stress and damage evident in aging.

Micronuclei induced in bone marrow erythroblasts by clastogenic chemicals are easily detected in peripheral blood. In mice treated with nitrogen mustard, 7,12 dimethylbenz(a)anthracene, or cyclophosphamide, the peak incidence of micronucleated polychromatic erythrocytes in peripheral blood was at least as great as the maximum incidence in bone marrow. In each case the peak incidence in blood occurred on the day following the peak incidence observed in bone marrow. Thus, for general genetic screening purposes, monitoring micronuclei in peripheral blood rather than in bone marrow smears provides at least equal sensitivity, offers greater simplicity in sample preparation and scoring, permits multiple sampling of treated animals, and may also facilitate automated scoring and human cytogenetic monitoring.