B-group vitamin production by lactic acid bacteria--current knowledge and potential applications.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B(12) amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.

The resveratrol analogue 4,4'-dihydroxy-trans-stilbene inhibits cell proliferation with higher efficiency but different mechanism from resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antiproliferative activity. We previously found that 4-hydroxy group in the trans conformation was absolutely required for the inhibition of cell proliferation. In the present work we have synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene, which contains two OH in 4' and 4 positions, with the aim of developing a compound with an antiproliferative potential higher than that of resveratrol, on the basis of the correlation between structure and activity previously observed. In comparison with resveratrol, 4,4'-dihydroxy-trans-stilbene inhibited cell clonogenic efficiency of fibroblasts nine times more although with a different mechanism. First, 4,4'-dihydroxy-trans-stilbene induced predominantly an accumulation of cells in G1 phase, whereas resveratrol perturbed the G1/S phase transition. Second, although both compounds were able to inhibit DNA polymerase (pol) delta in an in vitro assay, 4, 4'-dihydroxy-trans-stilbene did not affect pol alpha activity. Finally, 4,4'-dihydroxy-trans-stilbene increased p21(CDKN1A) and p53 protein levels, whereas resveratrol led to phosphorylation of the S-phase checkpoint protein Chk1. Taken together, our results demonstrated for the first time that the two hydroxyl groups on 4- and 4'- positions of the stilbenic backbone enhance the antiproliferative effect and introduce additional targets in the mechanism of action of resveratrol. In conclusion, 4,4'-dihydroxy-trans-stilbene has potent antiproliferative activities that differ from the effect of resveratrol shown in this system, suggesting that it warrants further development as a potential chemopreventive or therapeutic agent.

Flavonoids uptake and their effect on cell cycle of human colon adenocarcinoma cells (Caco2).

Green tea, mainly through its constituents epigallocatechin gallate, epigallocatechin, epicatechin gallate and epicatechin, has demonstrated anticarcinogenic activity in several animal models, including those for skin, lung and gastro-intestinal tract cancer, although less is known about colorectal cancer. Quercetin, the major flavonoid present in vegetables and fruit, exerts potential anticarcinogenic effects in animal models and cell cultures, but less is known about quercetin glucosides. The objectives of this study were to investigate (i) the antioxidant activity of the phenolic compounds epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside; (ii) the cytotoxicity of different concentrations of epicatechin, epigallocatechin gallate, and gallic acid; (iii) the cellular uptake of epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside and (iv) their effect on the cell cycle. Human colon adenocarcinoma cells were used as experimental model. The results of this study indicate that all dietary flavonoids studied (epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside) show a significant antioxidant effect in a chemical model system, but only epigallocatechin gallate or gallic acid are able to interfere with the cell cycle in Caco2 cell lines. These data suggest that the antioxidant activity of flavonoids is not related to the inhibition of cellular growth. From a structural point of view, the galloyl moiety appears to be required for both the antioxidant and the antiproliferative effects.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisometry modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4' position is not the sole determinant for antioxidant activity. In contrast, the presence of 4'-OH together with stereoisometry in the trans-conformation (4'-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases alpha and delta.

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

Lipid oxidation deletes the nanodomain organization of artificial membranes.

Nanoscopic domains with different crystal structures have been induced in closed artificial membranes and have been directly imaged by atomic force microscopy at a spatial resolution better than 0.3 nm. These observations provide experimental evidence to the hydrophobic mismatching theory of lateral phase separation phenomena. Under oxidant conditions, the lipid-lipid assembly reorganizes into a new steady-state structure with disappearance of specific nanodomains. This finding may contribute to understanding the mechanism of peroxidative damage to membrane properties. In fact, alterations of specific modes of molecular conformation and packing may lead to perturbation of specific properties.

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

Hepatic subcellular storage of beta-carotene in rats following diet supplementation.

Beta-carotene (BC) storage was measured in liver and its subcellular fractions (plasma membranes, mitochondria, microsomes and nuclei) of rats fed BC added to diet. The BC supplementation dose was about 350 mg/week/rat. After 15 weeks of this supplementation, rats were killed and their livers were immediately excised and processed to obtain total liver tissue and its subcellular fractions. Their BC contents were measured by HPLC as pmols/mg. protein Intact BC was found to be stored in all the above subcellular fractions, thus showing that BC is probably taken up by liver cell lipid moiety. Interestingly, the mean BC concentrations in plasma membranes and mitochondria were significantly higher than that in total liver tissue. Our data confirmed that rodents are a good animal model for the study of BC metabolism and its effects on several pathologies, and cancer prevention and treatment in humans in spite of the fact that rodents are classified as white-fat animals because of their poor BC absorption and storage in fat and blood plasma, whereas humans are classified as yellow-fat organisms because of their opposite behavior in BC uptake and organ distribution.

Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts.

The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system.

Effect of beta-carotene on cell cycle progression of human fibroblasts.

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.

Effects of beta-carotene and alpha-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen.

The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage. Alpha-T was more protective than beta-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition. At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective. At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

Free radical production during metabolism of organic hydroperoxides by normal human keratinocytes.

Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.

Conjugated diene fatty acids in patients with chronic renal failure: evidence of increased lipid peroxidation?

Conjugated diene fatty acids (CDFA) were evaluated by second derivative spectrophotometry in the plasma and adipose tissue of 42 chronic renal failure (CFR) patients in conservative treatment, 40 patients treated by hemodialysis (HD) with cuprophane, cellulose acetate or hemophan, 29 treated by hemodiafiltration (HDF) with polysulfone, polyacrylonitrile or polyamide, and 28 healthy controls. Plasma CDFA were also evaluated at the beginning, at 30 min and at the end of the dialytic session. CDFA were unchanged in CRF patients with creatinine clearance (Ccr) > 10 ml/min respect to the controls, CRF patients with Ccr < 10 ml/min showed a higher level of CDFA both in plasma and adipose tissue (p < 0.02). HD patients showed values similar to those of the control group. The lowest level of CDFA was found in HDF patients (p < 0.01 for plasma, p < 0.05 for adipose tissue versus both control and any other group). A significant relationship between plasma and adipose tissue CDFA was found in all groups. In the group of CRF patients with Ccr < 10 ml/min, females exhibited a higher level of CDFA both in plasma and adipose tissue. No significant change was found during dialytic session, independently from the membrane used. CDFA are not only primary products of lipid peroxidation, but also have a dietary origin, primarily from dairy products. Taking into account the reduced dietary intake, the increase in end-stage CRF may be due to an enhanced oxidative stress and/or to abnormalities in CDFA metabolism. Uremic patients, particularly in the predialytic stage, should be considered at risk for increased oxidative stress. HDF treatment better corrects the abnormality compared to conventional HD.

Tert-butylhydroperoxide bioactivation to methyl radical in rat liver mitochondria and submitochondrial particles.

Electron paramagnetic resonance spectroscopy (EPR) coupled to the spin trapping technique was used to detect carbon-centered radicals in rat liver mitochondria and submitochondrial particles exposed to t-butyl-hydroperoxide (TBH), using the spin trapping agent 3,5-dibromo-4-nitroso-benzenesulfonic acid (DBNBS). The signal recorded was unambiguously assigned to the methyl radical adduct. DBNBS was added to isolated rat liver mitochondria energized with succinate, and the methyl radical adduct was observed. The addition of NADH, NADPH, inhibitors of the respiratory chain, and of monoaminoxidase (MAO) inhibitors did not cause any relevant modification in the yield of radical adduct formation. Boiling and the addition of a non-ionic detergent inhibited the formation of the radical adduct, while experiments carried out under hypoxic conditions generated a significant increase in methyl radical formation. Further experiments were carried out on sub-mitochondrial particles (SMP) giving rise to, basically, the same results. From the above results, we are proposing that haem prosthetic groups are the likely source of TBH bioactivation in mitochondria.

Detection and quantitation in rat tissues of the superparamagnetic magnetic resonance contrast agent dextran magnetite as demonstrated by electron spin resonance spectroscopy.

RATIONALE AND OBJECTIVES: The compound studies in this article is a superparamagnetic macromolecular complex of magnetite cores coated with hydrophilic dextran, which is under active investigation as a contrast agent for magnetic resonance imaging (MRI) in liver and spleen. The biodistribution of paramagnetic compounds is problematic and is usually studied by histochemical reactions or by radiolabeling the compound under study. The purpose of this article is to show how electron spin resonance (ESR) spectroscopy detects dextran magnetite (DM) particles in tissues. METHODS: DM injected intravenously in the experimental animal was detected in some reticulo-endothelial organs by ESR. The spectroscopic study was validated using electron microscopy and electron-probe microanalysis. RESULTS: DM exhibits an ESR spectrum; ESR delineated the distribution of DM distribution in liver, spleen, bone marrow, and blood as a function of time. The blood clearance was biphasic, dependent on the size of particles. CONCLUSIONS: ESR spectroscopy is a highly sensitive and reproducible method of studying DM distribution.

Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis.

Extracellular free radicals were detected in rat striatal perfusate samples by intracerebral microdialysis coupled to the spin trapping technique. Five Sprague-Dawley rats were subjected to 30 min of global ischemia followed by reperfusion; throughout the experimental period the intrastriatal dialysing probe was perfused with Ringer's solution containing the spin trap agent pyridyl-N-oxide-t-butylnitrone (100 mM) together with the iron chelating agent diethylentriaminepentacetic acid (100 microM). A radical adduct occurred during ischemia and early reperfusion, but not in basal conditions; the spin adduct was characterized as a carbon centered radical, consistent with the presence of an oxidative attack on membrane lipids. The direct evidence of the formation of free radicals supports the hypothesis that free radicals play a role in the pathogenesis of the histological damage during brain ischemia.

Blood clearance of dextran magnetite particles determined by a noninvasive in vivo ESR method.

Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes.

Enzymatic recycling of oxidized ascorbate in pig heart: one-electron vs two-electron pathway.

Enzymatic systems able to reduce either dehydroascorbate or ascorbyl radical back to ascorbate by "recycling" vitamin C may contribute to lowering the nutritional requirement of it and to increase tissue antioxidant capacity. The activities of two enzymatic activities, GSH-dehydroascorbate reductase (two-electron reduction pathway) and NADH-semidehydroascorbate reductase (one-electron reduction pathway) in pig tissues, have been investigated. The activity of glutathione-dependent reduction of dehydroascorbate, although measurable, appeared negligible taking into consideration the low physiological substrate concentration. On the other hand, the one-electron reduction of ascorbyl radical resulted fast enough to slow down the consumption of the antioxidant vitamin.

[Biochemical study of the dental pulp in the calf]. / Etude biochimique de la pulpe dentaire de veau.

A biochemical study of dental pulp of calves has been performed concerning: a) peroxydability b) A, E, C vitamins content c) glutation (GSH) content d) presence of paramagnetic compounds e) phosphorylation ratio The dental pulp from incisors of 5-months-old calves has been preserved. Immediately after decapitation the pulp was immersed in liquid nitrogen. Chromatographic (HPLC) and spectroscopic (NMR-ESR) techniques have been used. GSH in dental pulp are present and dosable (4.56 +/- 0.08 n moles/mg prot.) and GSSG (1.05 +/- 0.01 n moles/mg prot.). Because of blood traces in the extracted pulps, the AA. have determined the hemoglobin (Hb) dosage and GSH of erythrocytic derivation (Fig. 1). After deduction of GSH of erythrocytic derivation, the GSH really present in the pulp was 4.41 n moles/mg prot. and the GSSG was 0.90 n moles/mg prot. Peroxydability of the dental pulp has been evaluated with Lowry method with dental pulp homogenate and rat liver homogenate (see Table 1). The ESR spectre shows 4 resonances with the following values: g. 2.24-2.04-2.00-1.97; there are some free intermediary radicals (gr.-2.00) (Fig.2). The NMR spectre shows the presence of ATP (0.22 n moles/g) of inorganic phosphate (16.58 n moles g) (Fig.3). The pulp seems to have a lot of antioxidant factors. The next researches will be to study E, A and C vitamins concentrations. This high presence of GSH and GSSG may be an embryonic peculiarity.