Biosynthesis of Gamma-Aminobutyric Acid (GABA) by Lactiplantibacillus plantarum in Fermented Food Production.

In recent decades, given the important role of gamma-aminobutyric acid (GABA) in human health, scientists have paid great attention to the enrichment of this chemical compound in food using various methods, including microbial fermentation. Moreover, GABA or GABA-rich products have been successfully commercialized as food additives or functional dietary supplements. Several microorganisms can produce GABA, including bacteria, fungi, and yeasts. Among GABA-producing microorganisms, lactic acid bacteria (LAB) are commonly used in the production of many fermented foods. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a LAB species that has a long history of natural occurrence and safe use in a wide variety of fermented foods and beverages. Within this species, some strains possess not only good pro-technological properties but also the ability to produce various bioactive compounds, including GABA. The present review aims, after a preliminary excursus on the function and biosynthesis of GABA, to provide an overview of the current uses of microorganisms and, in particular, of L. plantarum in the production of GABA, with a detailed focus on fermented foods. The results of the studies reported in this review highlight that the selection of new probiotic strains of L. plantarum with the ability to synthesize GABA may offer concrete opportunities for the design of new functional foods.

Activation of Kv7 Potassium Channels Inhibits Intracellular Ca2+ Increases Triggered By TRPV1-Mediated Pain-Inducing Stimuli in F11 Immortalized Sensory Neurons.

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.

The occurrence of l-lactate dehydrogenase in the inner mitochondrial compartment of pig liver.

Although pig represents a model species in biomedical research including studies dealing with liver patho-physiology, some aspects of liver metabolism need to be addressed. In particular, whether and how pig mitochondria can metabolize l-lactate remains to be established. We show here that pig liver mitochondria (PLM) possess their own l-lactate dehydrogenase (mL-LDH). This was shown both via immunological analysis and by assaying photometrically the L-LDH reaction in solubilised PLM. The mL-LDH reaction shows hyperbolic dependence on the substrate concentration, it is inhibited by oxamate and proves to differ from the cytosolic activity (cL-LDH), as revealed by the difference found in both pH profiles and temperature dependence of m- and cL-LDH. Titration experiments with digitonin show that mL-LDH is restricted in mitochondrial inner compartment. In agreement with the above findings, three genes in Sus scrofa genome encoded for L-LDH subunits which are predicted to have mitochondrial localization, as investigated by Target P 1.1 and PredSL analysis.

In boar sperm capacitation L-lactate and succinate, but not pyruvate and citrate, contribute to the mitochondrial membrane potential increase as monitored via safranine O fluorescence.

Having ascertained using JC-1 as a probe that, in distinction with the controls, during capacitation boar sperm maintains high mitochondrial membrane potential (ΔΨ), to gain some insight into the role of mitochondria in capacitation, we monitored ΔΨ generation due to externally added metabolites either in hypotonically-treated spermatozoa (HTS) or in intact cells by using safranine O as a probe. During capacitation, the addition to HTS of L-lactate and succinate but not those of pyruvate, citrate and ascorbate + TMPD resulted in increase of ΔΨ generation. Accordingly, the addition of L-lactate and succinate, but not that of citrate, to intact sperm resulted in ΔΨ generation increased in capacitation.

Production of Conjugated Linoleic Acid (CLA) by Lactiplantibacillus plantarum: A Review with Emphasis on Fermented Foods.

The term Conjugated Linoleic Acid (CLA) refers generically to a class of positional and geometric conjugated dienoic isomers of linoleic acid. Among the isomers of linoleic acid cis9, trans11-CLA (c9, t11-CLA) and trans10, cis12-CLA (t10, c12-CLA) are found to be biologically active isomers, and they occur naturally in milk, dairy products and meat from ruminants. In addition, some vegetables and some seafoods have also been reported to contain CLA. Although the CLA levels in these natural sources are insufficient to confer the essential health benefits, anti-carcinogenic or anti-cancer effects are of current interest. In the rumen, CLA is an intermediate of isomerization and the biohydrogenation process of linoleic acid to stearic acid conducted by ruminal microorganisms. In addition to rumen bacteria, some other bacteria, such as Propionibacterium, Bifidobacterium and some lactic acid bacteria (LAB) are also capable of producing CLA. In this regard, Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) has demonstrated the ability to produce CLA isomers from linoleic acid by multiple enzymatic activities, including hydration, dehydration, and isomerization. L. plantarum is one of the most versatile species of LAB and the bacterium is widely used in the food industry as a microbial food culture. Thus, in this review we critically analyzed the literature produced in the last ten years with the aim to highlight the potentiality as well as the optimal conditions for CLA production by L. plantarum. Evidence was provided suggesting that the use of appropriate strains of L. plantarum, as a starter or additional culture in the production of some fermented foods, can be considered a critical factor in the design of new CLA-enriched functional foods.

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

The Effect of Semen Cryopreservation Process on Metabolomic Profiles of Turkey Sperm as Assessed by NMR Analysis.

Semen cryopreservation represents the main tool for preservation of biodiversity; however, in avian species, the freezing−thawing process results in a sharp reduction in sperm quality and consequently fertility. Thus, to gain a first insight into the molecular basis of the cryopreservation of turkey sperm, the NMR-assessed metabolite profiles of fresh and frozen−thawed samples were herein investigated and compared with sperm qualitative parameters. Cryopreservation decreased the sperm viability, mobility, and osmotic tolerance of frozen−thawed samples. This decrease in sperm quality was associated with the variation in the levels of some metabolites in both aqueous and lipid sperm extracts, as investigated by NMR analysis. Higher amounts of the amino acids Ala, Ile, Leu, Phe, Tyr, and Val were found in fresh than in frozen−thawed sperm; on the contrary, Gly content increased after cryopreservation. A positive correlation (p < 0.01) between the amino acid levels and all qualitative parameters was found, except in the case of Gly, the levels of which were negatively correlated (p < 0.01) with sperm quality. Other water-soluble compounds, namely formate, lactate, AMP, creatine, and carnitine, turned out to be present at higher concentrations in fresh sperm, whereas cryopreserved samples showed increased levels of citrate and acetyl-carnitine. Frozen−thawed sperm also showed decreases in cholesterol and polyunsaturated fatty acids, whereas saturated fatty acids were found to be higher in cryopreserved than in fresh sperm. Interestingly, lactate, carnitine (p < 0.01), AMP, creatine, cholesterol, and phosphatidylcholine (p < 0.05) levels were positively correlated with all sperm quality parameters, whereas citrate (p < 0.01), fumarate, acetyl-carnitine, and saturated fatty acids (p < 0.05) showed negative correlations. A detailed discussion aimed at explaining these correlations in the sperm cell context is provided, returning a clearer scenario of metabolic changes occurring in turkey sperm cryopreservation.

Kv7.4 channels regulate potassium permeability in neuronal mitochondria.

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia-reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria.

Bioactivity of Wild Hop Extracts against the Granary Weevil, Sitophilus granarius (L.).

The use of bioinsecticides, rather than synthetic compounds, appears a goal to be pursued in pest control, especially for species such as Sitophilus granarius (L.) which attack stored products. Since Humulus lupulus (L.) is a remarkable source of bioactive compounds, this study investigated the bioactivity of hop flower extracts against S. granarius adults by evaluating toxic (contact, inhalation, and ingestion), repellent, antifeedant, and nutritional effects as well as their anticholinesterase activity and olfactory sensitivity. Hop extracts were obtained by soaking dried and ground hop cones in solvents of decreasing polarity: methanol, acetone, and n-hexane. Dried crude extracts were resuspended in each solvent, and used in topical application, ingestion, and fumigation toxicity assays, as well as in contact and short-range repellency tests, in vitro anticholinesterase activity evaluation, and electroantennographic tests. No inhalation toxicity for the extracts was found. On the contrary, all extracts showed adult contact toxicity 24 h after treatment (LD50/LD90 16.17/33.20, 25.77/42.64, and 31.07/49.48 µg/adult for acetone, n-hexane, and methanol extracts, respectively); negligible variations for these values at 48 h were found. The anticholinesterase activity shown by all extracts suggested that the inhibition of this enzyme was one of the mechanisms of action. Interestingly, flour disk bioassays revealed a significant ingestion toxicity for the acetone extract and a lower toxicity for the other two extracts. Moreover, all extracts affected insect nutritional parameters, at the highest dose checked. Filter paper and two-choice pitfall bioassays showed repellent activity and a strong reduction of insect orientation to a highly attractive food odor source, with minor differences among extracts, respectively. Finally, the presence of volatile compounds in the different extracts that are perceived by insect antennae was confirmed by electroantennography. All these findings strongly suggest a possible use of hop cone extracts against S. granarius, thus further confirming this plant as an interesting species for pest control.

Pyruvate kinase in pig liver mitochondria.

The existence of the pyruvate kinase (PK) in pig liver mitochondria was shown by monitoring photometrically the PK reaction in solubilised mitochondria with either phosphoenolpyruvate (PEP) or ADP used as a substrate. In distinction with the cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or ADP concentrations. The occurrence of the mitochondrial PK was confirmed by immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix. PEP addition to mitochondria resulted in reduction of the intramitochondrial NAD(P)+ inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of the pyruvate dehydrogenase complex. Citrate/oxaloacetate appearance outside mitochondria also occurred as result of PEP addition to PLM. Taken together these findings support a role for PEP itself in triggering fatty acid synthesis via its mitochondrial metabolism.

Biological Activity of Humulus Lupulus (L.) Essential Oil and Its Main Components Against Sitophilus granarius (L.).

Besides its use in the brewing industry, hop cones appear as a powerful source of biologically active compounds, already checked for their putative anticancer, antimicrobial, and other bioactivities. Conversely, hop use in pest control remains to date under-investigated. Therefore, the biological activity of hop essential oil (EO) and its main constituents was investigated here against Sitophilus granarius. Adult contact toxicity was found 24 h after treatment with hop EO (LD50/LD90 13.30/40.23 µg/adult), and its three most abundant components, α-humulene, ß-myrcene, and ß-caryophyllene (LD50/LD90 41.87/73.51, 75.91/126.05, and 138.51/241.27 µg/adult, respectively); negligible variations at 48 h, except for α-humulene (LD50/LD90 26.83/49.49 µg/adult), were found. The fumigant toxicity of the EO and terpenes was also checked: in the absence of wheat grains, ß-myrcene showed the highest inhalation toxicity (LC50/LC90 72.78/116.92 mg/L air), whereas α-humulene, ß-caryophyllene, and the EO induced similar values (LC50/LC90 about 130/200 mg/L air); with the exception for EO, the wheat presence increased (30-50%) LC50/LC90 values. Moreover, EO and terpenes were perceived by insect antennae and elicited repellent activity. Only ß-caryophyllene showed an anticholinesterase effect, this suggesting that different mechanisms of action should be responsible for hop EO toxicity. Therefore, hop EO appears suitable for developing control means against this pest.

Biological activity of Dittrichia viscosa (L.) Greuter extracts against adult Sitophilus granarius (L.) (Coleoptera, Curculionidae) and identification of active compounds.

Dittrichia viscosa (L.) Greuter, a perennial weed of the Mediterranean area, was reported to be source of active substances. Here, by means of both ingestion and contact assays, the biological activity of three different extracts (n-hexane, methanol, and distilled water) of D. viscosa aerial part has been evaluated against Sitophilus granarius (L.) adults, an important pest of stored grains. Ingestion assays showed negligible mortality and food deterrence for all the extracts, whereas only a slight reduction of some nutritional parameters (relative growth rate, relative consumption rate, food efficiency conversion) was recorded for water extract. High contact toxicity was found only for the n-hexane extract (24 h median lethal dose LD50 = 53.20 µg/adult). This extract was further subfractioned by silica gel column chromatography and then by thin layer chromatography. Further contact toxicity bioassays highlighted two active subfractions which were analyzed by GC-MS. This revealed the occurrence, in both subfractions, of two major peaks that were identified as α- and γ- costic acid isomers. Moreover, D. viscosa active subfractions, did not cause acetylcholinesterase (AChE) inhibition; therefore, in the light of progressive limitation of compounds acting by this mechanism of action, D. viscosa represents a promising eco-sustainable source of natural products for pest control.

Mitochondria and L-lactate metabolism.

Although mitochondria have been the object of intensive study over many decades, some aspects of their metabolism remain to be fully elucidated, including the L-lactate metabolism. We review here the novel insights arisen from investigations on L-lactate metabolism in mammalian, plant and yeast mitochondria. The presence of L-lactate dehydrogenases inside mitochondria, where L-lactate enters in a carrier-mediated fashion, suggests that mitochondria play an important role in L-lactate metabolism. Functional studies have demonstrated the occurrence of several L-lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.

Age-dependent changes in metabolic profile of turkey spermatozoa as assessed by NMR analysis.

Metabolic profile of fresh turkey spermatozoa at three different reproductive period ages, namely 32, 44 and 56 weeks, was monitored by Nuclear Magnetic Resonance (NMR) spectroscopy and correlated to sperm quality parameters. The age-related decrease in sperm quality as indicated by reduction of sperm concentration, sperm mobility and osmotic tolerance was associated to variation in the level of specific water-soluble and liposoluble metabolites. In particular, the highest levels of isoleucine, phenylalanine, leucine, tyrosine and valine were found at 32 weeks of age, whereas aspartate, lactate, creatine, carnitine, acetylcarnitine levels increased during the ageing. Lipid composition also changed during the ageing: diunsaturated fatty acids level increased from 32 to 56 weeks of age, whereas a reduction of polyunsaturated fatty acids content was observed at 56 weeks. The untargeted approach attempts to give a wider picture of metabolic changes occurring in ageing suggesting that the reduction of sperm quality could be due to a progressive deficiency in mitochondrial energy producing systems, as also prompted by the negative correlation found between sperm mobility and the increase in certain mitochondrial metabolites.

L-lactate metabolism in potato tuber mitochondria.

We investigated the metabolism of L-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti-lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of L-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an L-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium L-lactate in a stereospecific manner, thus showing that L-lactate enters mitochondria by a proton-compensated process. Externally added L-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on L-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent L-lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of L-lactate plays a previously unsuspected role in the response of potato to hypoxic stress.

Mitochondria isolated from local market potato tubers contain L-lactate dehydrogenase in an inactive state.

In order to gain some insight into metabolism of mitochondria isolated from materials subjected to storage treatments, we compared mitochondria isolated from potato tubers grown and stored in the post-harvest without any chemicals (N-PTM), and tubers, from local market, treated for commercial purpose (T-PTM) with respect to the L-lactate metabolism. Although no oxygen consumption due to L-lactate was found in T-PTM, L-lactate dehydrogenase existence was shown as immunologically investigated. Consistently, no L-lactate dehydrogenase activity was detected. Contrarily, N-PTM proved to metabolize externally added L-lactate, with oxygen consumption and intramitochondrial pyridine nucleotide reduction. All together these findings show that commercial treatments of foodstuffs could result in changes in their metabolism.

Is there a pyruvate kinase in pig liver mitochondria?

In order to ascertain whether mammalian mitochondria possess their own pyruvate kinase, we isolated mitochondria from liver of Large White pig and investigated pyruvate kinase occurrence both via immunological analysis and by assaying photometrically the pyruvate kinase reaction. We show that mitochondria contain pyruvate kinase located in the inner compartments; the pyruvate kinase reaction shows hyperbolic dependence on the substrate concentration, is inhibited by malonate and shows maximum activity at pH between 7-7.6 and Ea equal to 33 kJ/mol.

Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria.

We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.

Plant uncoupling protein in mitochondria from aged-dehydrated slices of Jerusalem artichoke tubers becomes sensitive to superoxide and to hydrogen peroxide without increase in protein level.

We investigated the occurrence of the plant Uncoupling Protein (UCP) in mitochondria isolated from both fresh (f-JAM) and aged-dehydrated (a-d-JAM) slices of Jerusalem artichoke tubers (Helianthus tuberosus L.). The presence of UCP was shown by immunological analysis and its function was investigated by measuring the decrease of the mitochondrial membrane potential due to linoleic acid (LA) and its inhibition by purine nucleotides under conditions in which the adenine nucleotide translocator (ANT) was inhibited by atractyloside (Atr). f-JAM and a-d-JAM had the same protein content, but differed from one another with respect to purine nucleotide inhibition, substrate specificity, and sensitivity to ROS. Hydrogen peroxide and superoxide anion, generated in situ by xanthine plus xanthine oxidase, caused a significant increase in the UCP function in a-d-JAM, but not in f-JAM. This occurred in a manner sensitive to ATP, but not to Atr, thus showing that ANT has no role in the process. The dependence of the rate of membrane potential decrease on increasing LA concentrations, either in the absence or the presence of ROS, showed a sigmoidal saturation both in f-JAM and a-d-JAM. However, addition of ROS in a-d-JAM resulted in about 40% increase of the Vmax value, with no change in the K0.5 (about 20 microM), whereas in f-JAM no effect on either the Vmax or K0.5 (about 28 microM) was found. Furthermore, a decreased ROS production as a result of LA addition was found in both f-JAM and a-d-JAM, the effect being more marked in a-d-JAM.

Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells.

We have investigated the ability of certain dietary flavonoids, known to exert beneficial effects on the central nervous system, to affect neuronal apoptosis. We used cerebellar granule cells undergoing apoptosis due to potassium deprivation in a serum-free medium in either the absence or presence of the flavonoids genistein and daidzein, which are present in soy, and of catechin and epicatechin, which are present in cocoa. These compounds were used in a blood dietary concentration range. We found that genistein and daidzein, but not catechin and epicatechin, prevented apoptosis, with cell survival measured 24h after the induction of apoptosis being higher than that of the same cells incubated in flavonoid free medium (80% and 40%, respectively); there was no effect in control cells. A detailed investigation of the effect of these compounds on certain mitochondrial events that occur in cells en route to apoptosis showed that genistein and daidzein prevented the impairment of glucose oxidation and mitochondrial coupling, reduced cytochrome c release, and prevented both impairment of the adenine nucleotide translocator and opening of the mitochondrial permeability transition pore. Interestingly, genistein and daidzein were found to reduce the levels of reactive oxygen species, which are elevated in cerebellar granule cell apoptosis. These findings strongly suggest that the prevention of apoptosis depends mainly on the antioxidant properties of genistein and daidzein. This could lead to the development of a flavonoid-based therapy in neuropathies.