Stimulation of macroautophagy can rescue older cells from 8-OHdG mtDNA accumulation: a safe and easy way to meet goals in the SENS agenda.

Reduction of oxidative stress within mitochondria is a major focus and important part in the SENS agenda. The age-related accumulation of mitochondria rich in oxidatively altered DNA may be a biomarker of malfunctioning and increased oxidative stress. Macroautophagy is the cell repair mechanism responsible for the disposal of excess or altered mitochondria under the inhibitory control of nutrition and insulin, and may mediate the antiaging effects of caloric restriction. The authors investigated the effects of stimulation of macroautophagy by the injection of an antilipolytic agent on the age-related accumulation of oxidatively altered mitochondrial DNA (mtDNA) in rat liver cells. Results showed that treatment rescued older cells from the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the mtDNA in less than 6 hours. It is concluded that the age-related changes in mtDNA and function are likely to be the consequence of a failure of macroautophagy in the recognition and disposal of a small number of severely injured mitochondria, and that easy and safe ways are available to counteract this change.

An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

Influence of piracetam and oxiracetam on the content of high-energy phosphates and morphometry of astrocytes in vitro.

The nootropic drugs, including piracetam (PIR) and oxiracetam (OXI) are used in the adjunctive treatment of dementia. They are thought to directly influence energetic processes in the brain and, therefore, they are supposed to improve memory and cognition. The content of adenosine triphosphate (ATP) and phosphocreatine (PCr) and 3H-valine incorporation into proteins were measured, and the morphometry was performed after PIR and OXI treatment of astrocytes cultured in vitro with or without dibutyryl 3',5'-cyclic adenosine monophosphate (dBcAMP). Nootropics were added into the culture medium for 2 weeks at a final concentration of 10(-7) M. It was shown that OXI increased ATP content in astrocytes cultured with or without dBcAMP. The increase in 3H-valine incorporation into astrocytes after PIR and OXI together with dBcAMP treatment was found. These results indicate that the presented research model allows to study energetic processes in cultured astrocytes. However, nootropic drugs changed morphometric parameters (cell area, perimeter and form factor) of cultured astrocytes as well. It can be concluded that PIR and OXI as nootropics have an opposing effect on the content of high-energy phosphates and shape of astrocytes in vitro.

Mechanisms contributing to muscle-wasting in acute uremia: activation of amino acid catabolism.

Acute uremia (ARF) causes metabolic defects in glucose and protein metabolism that contribute to muscle wasting. To examine whether there are also defects in the metabolism of essential amino acids in ARF, we measured the activity of the rate-limiting enzyme for branched-chain amino acid catabolism, branched-chain ketoacid dehydrogenase (BCKAD), in rat muscles. Because chronic acidosis activates muscle BCKAD, we also evaluated the influence of acidosis by studying ARF rats given either NaCl (ARF-NaCl) or NaHCO3 (ARF-HCO3) to prevent acidosis, and sham-operated, control rats given NaHCO3. ARF-NaCl rats became progressively acidemic (serum [HCO3] = 21.3 +/- 0.7 mM within 18 h and 14.7 +/- 0.8 mM after 44 h; mean +/- SEM), but this was corrected with NaHCO3. Plasma valine was low in ARF-NaCl and ARF-HCO3 rats. Plasma isoleucine, but not leucine, was low in ARF-NaCl rats, and isoleucine tended to be lower in ARF-HCO3 rats. Basal BCKAD activity (a measure of active BCKAD in muscle) was increased more than 17-fold (P < 0.01) in ARF-NaCl rat muscles, and this response was partially suppressed by NaHCO3. Maximal BCKAD activity (an estimate of BCKAD content), subunit mRNA levels, and BCKAD protein content were not different in ARF and control rat muscles. Thus, ARF increases branched-chain amino acid catabolism by activating BCKAD by a mechanism that includes acidosis. Moreover, in a muscle-wasting condition such as ARF, there is a coordinated increase in protein and essential amino acid catabolism.

Monitoring exposure to acrylonitrile using adducts with N-terminal valine in hemoglobin.

Human exposure to acrylonitrile (ACN), a carcinogen in rats, may occur in industrial settings, through waste water and tobacco smoke. ACN is an electrophilic compound and binds covalently to nucleophilic sites in macromolecules. Measurements of adducts with hemoglobin could be utilized for improved exposure assessments. In this study, a method for quantification of N-(2-cyanoethyl)valine (CEVal), the product of reaction of ACN with N-terminal valine in hemoglobin has been developed. The method is based on the N-alkyl Edman procedure, which involves derivatization of the globin with pentafluorophenyl isothiocyanate and gas chromatographic-mass spectrometric analysis of the resulting thiohydantoin. An internal standard was prepared by reacting valylglycylglycine with [2H3]ACN, spiked with [14C]ACN to a known sp. act. Levels of CEVal were measured in globin from rats exposed to 3-300 p.p.m. ACN in drinking water for 105 days and from humans (four smokers and four non-smokers). CEVal was detected at all exposure levels in the drinking water study. The relationship between adduct level and water concentration was linear at concentrations of 10 p.p.m. (corresponding to an average daily uptake of c. 0.74 mg ACN/kg body wt during the 65 days prior to sacrifice) and below, with a slope of 37.7 pmol CEVal/g globin/p.p.m. At higher concentrations, adduct levels increased sublinearly, indicating saturation of a metabolic process for elimination of ACN. Comparison of adduct formation with the estimated dose (mg/kg/day) of ACN indicated that at low dose (0-10 p.p.m.) CEVal = 0.508 x ACN dose + 0.048 and at high dose (35-300 p.p.m.) CEVal = 1.142 x ACN dose - 1.098. Globin from the smokers (10-20 cigarettes/day) contained about 90 pmol CEVal/g, whereas the adduct levels in globin from non-smokers were below the detection limit. The analytical sensitivity should be sufficient to allow monitoring of occupationally exposed workers at levels well below the current Occupational Safety and Health Administration standard of 2 p.p.m.

Effect of ursodeoxycholic acid on in vivo and in vitro toxic liver injury in rats.

BACKGROUND: Benefits of ursodeoxycholic acid (UDCA) in cholestatic disorders have been well documented. However, the therapeutic potential of UDCA in parenchymal liver disease is unclear. METHODS: We tested UDCA in rat models of hepatotoxicity: (a) in subacute liver injury induced by repetitive CCl4 and dietary ethyl alcohol (ETH) over seven weeks while receiving oral UDCA; and, (b) in liver slides incubated with CCl4, ETH or p-acetaminophen (APAP) when UDCA was added to the incubating solution. RESULTS: Experiment 1: CCl4 combined with ETH reduced the body weights and resulted in 43% mortality. There was a significant rise in serum ALT, alkaline phosphatase, lipoperoxides (LPO) and in hepatic weight, triglycerides, LPO and histological scores of liver injury. Experiment 2: When liver slides were incubated with hepatotoxins there was an increased transfer of AST and LPO from the tissue into the incubate and a reduction in the valine and thymidine incorporation into the liver proteins or DNA. In none of these situations, whether the liver damage was severe or mild, in vivo or in vitro, UDCA did abolish these hepatotoxic effects. CONCLUSION: In contrast to clinical cholestatic disorders where the reported benefits of UDCA depend on replacement of the accumulated hydrophobic bile acids, these bile acids have a less prominent role in toxic liver injury and UDCA is ineffective.

Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I.

Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus? Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy? To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats.