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Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX(®) Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation.

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.

Females live longer than males. Work from our laboratory has shown that this may be due to the up-regulation of longevity-associated genes by estrogens. Estrogens bind to the estrogen receptors and subsequently activate the mitogen activated protein kinase and nuclear factor kappa B signalling pathways, resulting in an up-regulation of antioxidant enzymes. Estrogen administration, however, has serious undesirable effects and of course, cannot be administered to males because of its powerful feminizing effects. Thus, we tested the effect of genistein, a phytoestrogen of high nutritional importance whose structure is similar to estradiol, on the regulation of the expression of antioxidant, longevity-related genes and consequently on oxidant levels in mammary gland tumour cells in culture. Phytoestrogens mimic the protective effect of oestradiol using the same signalling pathway. The critical importance of up-regulating antioxidant genes, by hormonal and dietary manipulations, to increase longevity is discussed.

Moderate exercise is a healthy practice. However, exhaustive exercise generates free radicals. This can be evidenced by increases in lipid peroxidation, glutathione oxidation, and oxidative protein damage. It is well known that activity of cytosolic enzymes in blood plasma is increased after exhaustive exercise. This may be taken as a sign of damage to muscle cells. The degree of oxidative stress and of muscle damage does not depend on the absolute intensity of exercise but on the degree of exhaustion of the person who performs exercise. Training partially prevents free radical-formation in exhaustive exercise. Treatment with antioxidants such as vitamins C or E protects in part against free radical-mediated damage in exercise. Xanthine oxidase is involved in free-radical formation in exercise in humans and inhibition of this enzyme with allopurinol decreases oxidative stress and muscle damage associated with exhaustive exercise. Knowledge of the mechanism of free-radical formation in exercise is important because it will be useful to prevent oxidative stress and damage associated with exhaustive physical activity.

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver. Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.

(1999). The IX Biennial Meeting of the International Society for Free Radical Research, Sao Paulo, Brazil, September 7–11, 1998. Free Radical Research: Vol. 30, No. 5, pp. 329-339.