Carol M. Wehr

(R)-α-Lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate

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′‐dichlorofluo‐rescin, 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.—Hagen, T. M., Ingersoll, R. T., Lykkesfeldt, J., Liu, J., Wehr, C. M., Vinarsky, V., Bartholomew, J. C., Ames, B. N. (R)‐α‐Lipoic acid‐supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. FASEB J. 13, 411–418 (1999)

Feeding acetyl-l-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing 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% (wt/vol) in the drinking water] and/or (R)-α-lipoic acid [LA, 0.5% (wt/wt) 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 O2 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.

Mitochondrial Decay in Aging: Reversal through Supplementation of Acetyl-l-Carnitine and N-tert-Butyl-α-phenyl-nitronea

ABSTRACT: We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo) is significantly impaired. Mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption decline, and the level of oxidants increases. To examine whether dietary supplementation of micronutrients that may have become essential with age could reverse the decline in mitochondrial function, we supplemented the diet of old rats with 1% (w/v) acetyl‐l‐carnitine (ALCAR) in drinking water. ALCAR supplementation (1 month) resulted in significant increases in cellular respiration, mitochondrial membrane potential, and cardiolipin values. However, supplementation also increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. To counteract the potential increase in oxidative stress, animals were administered N‐tert‐butyl‐a‐phenyl‐nitrone (30 mg/kg) (PBN) with or without ALCAR. Results showed that PBN significantly lowered oxidant production as measured by 2,7′‐dichlorofluorescin diacetate (DCFH), even when ALCAR was coadministered to the animals. Thus, dietary supplementation with ALCAR, particularly in combination with PBN, improves mitochondrial function without a significant increase in oxidative stress.

(R)-α-Lipoic Acid Reverses the Age-Associated Increase in Susceptibility of Hepatocytes to tert-Butylhydroperoxide both In Vitro and In Vivo

Hepatocytes were isolated from young (3-5 months) and old (24-28 months) rats and incubated with various concentrations of tert-butylhydroperoxide (t-BuOOH). The t-BuOOH concentration that killed 50% of cells (LC50) in 2 hr declined nearly two-fold from 721 ± 32 μM in cells from young rats to 391 ± 31 μM in cells from old rats. This increased sensitivity of hepatocytes from old rats may be due, in part, to changes in glutathione (GSH) levels, because total cellular and mitochondrial GSH were 37.7% and 58.3% lower, respectively, compared to cells from young rats. Cells from old animals were incubated with either (R)- or (S)-lipoic acid (100 μM) for 30 min prior to the addition of 300 μM t-BuOOH. The physiologically relevant (R)-form, a coenzyme in mitochondria, as opposed to the (S)-form significantly protected hepatocytes against t-BuOOH toxicity. Dietary supplementation of (R)-lipoic acid [0.5% (wt/wt)] for 2 weeks also completely reversed the age-related decline in hepatocellular GSH levels and the incr...

Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short‐term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short‐term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short‐term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short‐term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN‐PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30‐day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity. Environ. Mol. Mutagen. 36:163–194, 2000

A simple fluorescent staining procedure for micronuclei and RNA in erythrocytes using Hoechst 33258 and pyronin Y.

Micronuclei in bone-marrow or peripheral blood erythrocytes provide a useful index of chromosomal breakage or anaphase lag in erythroblasts (Heddle et al., in press; MacGregor et al., 1980; Schmid, 1976; Schlegel and MacGregor, 1982, 1983). We describe below a simple fluorescent staining procedure which facilitates rapid manual scoring of micronuclei in erythrocytes, and which possesses a number of characteristics required for automated scoring. The procedure utilizes two fluorescent stains: Hoechst 33258, which is a specific DNA stain that emits blue fluorescence under UV illumination (Labarca and Paigen, 1980; Latt and Wohlleb, 1975); and pyronin Y, which stains both RNA and DNA and emits red-orange fluorescence under green illumination (Pollack et al., 1982). Advantages of this technique over conventional Giemsa-based procedures include: (1) The double fluorescent labeling of true micronuclei clearly differentiates micronuclei from basophilic granules, stain artifacts, and RNA-containing structures which are produced by certain chemicals (Maier and Schmid, 1975). (2) Selective visualization of DNA-containing objects, RNAand DNAcontaining objects, and/or total cells is possible by varying excitation wavelengths and the intensity of transillumination. For example, in peripheral blood only erythrocytes containing RNA, micronuclei, and leukocyte nuclei are visible with green excitation, greatly facilitating scoring of the incidence of micronuclei in RNAcontaining erythrocytes (the parameter of interest in a typical acute micronucleus assay). (3) The entire procedure consists of immersion of the fixed cells in the stain solution. Rinsing or further manipulation is not required.

Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei.

Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344/N rats and B6C3F1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased. Exposure to the same level of benzene for an additional 2 weeks did not further increase the frequency of micronuclei in PCEs. These results indicate that repeated exposures to benzene, such as might be encountered by humans as a result of occupational or environmental exposures, are not likely to change or increase benzene metabolism.

Inactivation of aflatoxin b1 mutagenicity by thiols

The mutagenicity of aflatoxin B1 to Salmonella typhimurium strain TA98 decreased rapidly upon exposure of aflatoxin B1 to various thiols in aqueous solution. Mutagenic activity was reduced to control values within 24 hr with N-acetyl-L-cysteine (NAC), N-2-mercaptopropionylglycine (MPG), mercaptoethanol, reduced glutathione or mercaptopropionic acid at pH values near 4. Mercaptoacetic acid, mercaptosuccinic acid, cysteine, acetyl-D,L-homocysteine thiolactone, cysteine methyl ester, D-penicillamine and beta-mercaptoethylamine were less effective. Relatively high thiol concentrations (greater than or equal to 0 . 25 M) were required to achieve complete inactivation within 24 hr with the thiols tested. The inactivation rate was strongly dependent on thiol concentration and pH, but was relatively independent of the aflatoxin concentration under the conditions examined. With MPG and NAC reaction rates were much slower at neutral pH values than at pHs between 3 and 4. HPLC and thin-layer chromatographic examination of aflatoxin B1 solutions partially inactivated with NAC revealed the formation of a new product at a rate that correlated with the disappearance of aflatoxin B1 and the loss of mutagenic activity. This reaction product has not yet been identified, but the evidence suggests that it is the product of an addition of the thiol at the difuran region of the aflatoxin.

Zinc deficiency, erythrocyte production, and chromosomal damage in pregnant rats and their fetuses

Abstract During periods of rapid growth and development, chromosomal and DNA damage has been observed in hepatic tissue samples of zinc-deprived animals. In this study, the erythrocyte micronucleus assay was used to estimate cytogenetic damage due to zinc deprivation in pregnant rat dams and their fetuses. Rat dams were assigned to one of three dietary groups on day 0 of gestation: zinc deficient (