Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the "identify (OS) and treat (by low molecular weight antioxidants, LMWA)" approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different "antioxidants" are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the "identify and treat" approach is questionable.

Adiponectin: possible link between metabolic stress and oxidative stress in the elderly.

OBJECTIVE: The aim of this study was to evaluate the relationships between the serum levels of adiponectin and systemic oxidative stress exerted on lipids, proteins, as well as endothelial function and cardiovascular diseases (CVD) risk markers, in elderly subjects with metabolic syndrome (MS). METHODS: The serum advanced glycation and oxidation protein products, low-density lipoprotein susceptibility to oxidation (oxLDL), nitric oxide metabolic pathway products (NOx), serum lipid peroxidation, as well as total antioxidant/oxidative capacity (TAC/TOC), were analyzed in elderly subjects with MS (n = 44), compared to aged-matched control (n = 39). RESULTS: We pointed out significantly lower levels of adiponectin in elderly MS subjects concomitantly with significantly higher levels of oxidative stress and CVD risk markers. Significant positive correlations were found between serum adiponectin levels and HDL-cholesterol (p < 0.05) and the total cholesterol/LDL-cholesterol ratio (p < 0.01). Additionally, adiponectin levels were significantly inversely associated with insulin resistance index (HOMA-IR, r = -0.348; p < 0.05) and serum lipid peroxidation (r = -0.337; p < 0.05), and significantly positively with the antioxidant capacity (TAC, r = 0.339; p < 0.05). Conversely, adiponectin levels were significantly negatively (r = -0.310; p < 0.05) associated with serum uric acid concentration. CONCLUSIONS: The major protective role of adiponectin versus stress related to an impaired glucose and lipid metabolism suggests that adiponectin plays a critical role in adiposity-related metabolic stress and redox homeostasis.

In vitro effects of prolonged exposure to quercetin and epigallocatechin gallate of the peripheral blood mononuclear cell membrane.

The study aimed to assess biophysical changes that take place in the peripheral blood mononuclear cell (PBMC) membranes when exposed in vitro to 10 µM quercetin or epigallocatechin gallate (EGCG) for 24 and 48 h. PBMCs isolated from hypercholesterolemia patients were compared to those from normocholesterolemia subjects. The membrane fluidity and transmembrane potential were evaluated and the results were correlated with biochemical parameters relevant to oxidative stress, assessed in the patients' plasma. The baseline value of PBMC membrane anisotropy for the hypercholesterolemia patients was lower than that of the control group. These results correlated with the plasma levels of advanced glycation end products, which were significantly higher in the hypercholesterolemia group, and the total plasma antioxidant status, which was significantly higher in normocholesterolemia subjects. In the case of normocholesterolemia cells in vitro, polyphenols induced a decrease in membrane anisotropy (7.25-11.88% at 24 h, 1.82-2.26% at 48 h) and a hyperpolarizing effect (8.30-8.90% at 24 h and 4.58-13.00% at 48 h). The same effect was induced in hypercholesterolemia cells, but only after 48 h exposure to the polyphenols: the decrease in membrane anisotropy was 5.70% for quercetin and 2.33% for EGCG. After 48 h of in vitro incubation with the polyphenols, PBMCs isolated from hypercholesterolemia patients exhibited the effects that had been registered in cells from normocholesterolemia subjects after 24 h exposure. These results outlined the beneficial action of the studied polyphenols, quercetin and EGCG, as dietary supplements in normocholesterolemia and hypercholesterolemia patients.

Circadian Rhythms, Chrononutrition, Physical Training, and Redox Homeostasis-Molecular Mechanisms in Human Health.

A multitude of physiological processes, human behavioral patterns, and social interactions are intricately governed by the complex interplay between external circumstances and endogenous circadian rhythms. This multidimensional regulatory framework is susceptible to disruptions, and in contemporary society, there is a prevalent occurrence of misalignments between the circadian system and environmental cues, a phenomenon frequently associated with adverse health consequences. The onset of most prevalent current chronic diseases is intimately connected with alterations in human lifestyle practices under various facets, including the following: reduced physical activity, the exposure to artificial light, also acknowledged as light pollution, sedentary behavior coupled with consuming energy-dense nutriments, irregular eating frameworks, disruptions in sleep patterns (inadequate quality and duration), engagement in shift work, and the phenomenon known as social jetlag. The rapid evolution of contemporary life and domestic routines has significantly outpaced the rate of genetic adaptation. Consequently, the underlying circadian rhythms are exposed to multiple shifts, thereby elevating the susceptibility to disease predisposition. This comprehensive review endeavors to synthesize existing empirical evidence that substantiates the conceptual integration of the circadian clock, biochemical molecular homeostasis, oxidative stress, and the stimuli imparted by physical exercise, sleep, and nutrition.

Development and validation of cardiometabolic risk predictive models based on LDL oxidation and candidate geromarkers from the MARK-AGE data.

The predictive value of the susceptibility to oxidation of LDL particles (LDLox) in cardiometabolic risk assessment is incompletely understood. The main objective of the current study was to assess its relationship with other relevant biomarkers and cardiometabolic risk factors from MARK-AGE data. A cross-sectional observational study was carried out on 1089 subjects (528 men and 561 women), aged 40-75 years old, randomly recruited age- and sex-stratified individuals from the general population. A correlation analysis exploring the relationships between LDLox and relevant biomarkers was undertaken, as well as the development and validation of several machine learning algorithms, for estimating the risk of the combined status of high blood pressure and obesity for the MARK-AGE subjects. The machine learning models yielded Area Under the Receiver Operating Characteristic Curve Score ranging 0.783-0.839 for the internal validation, while the external validation resulted in an Under the Receiver Operating Characteristic Curve Score between 0.648 and 0.787, with the variables based on LDLox reaching significant importance within the obtained predictions. The current study offers novel insights regarding the combined effects of LDL oxidation and other ageing markers on cardiometabolic risk. Future studies might be extended on larger patient cohorts, in order to obtain reproducible clinical assessment models.

Relationships between Serum Biomarkers of Oxidative Stress and Tobacco Smoke Exposure in Patients with Mental Disorders.

The role of cigarette smoking as an aggravating factor of systemic oxidative stress in patients with mental disorders has not been extensively investigated, although significantly higher rates of smoking are recorded in these subjects in comparison with the general population. In the present study, we tested the hypothesis that smoking might be an exacerbator of systemic oxidative stress, being directly correlated with the degree of exposure to tobacco smoke. We analyzed, in 76 adult subjects from a public health care unit, the relationships between serum cotinine levels as a marker of tobacco smoke exposure, and three biomarkers of oxidative stress: the serum glutathione (GSH), the advanced oxidation protein products (AOPPs), and the total serum antioxidant status (FRAP). The results indicate that the degree of tobacco smoke exposure was inversely associated with GSH levels in both passive and active smokers, suggesting that smoke particulate components' toxicity is associated with a systemic GSH depletion. Paradoxically, the lowest AOPP levels which were positively associated with GSH, were recorded in active smoking patients whereas in passive smokers individual values of AOPPs decreased along with the increase in GSH levels. Our data suggest that an enhanced inhalation of particulate constituents of cigarette smoke could induce critical changes in systemic redox homeostasis and GSH can no longer exert its antioxidant role.

Bacterial DNAemia in Older Participants and Nonagenarian Offspring and Association With Redox Biomarkers: Results From MARK-AGE Study.

Aging and age-related diseases have been linked to microbial dysbiosis with changes in blood bacterial DNA concentration. This condition may promote chronic low-grade inflammation, which can be further aggravated by antioxidant nutrient deficiency. Low plasma carotenoids are associated with an increased risk of inflammation and cellular damage and predict mortality. However, no evidence is yet available on the relationship between antioxidants and the blood bacterial DNA (BB-DNA). Therefore, this study aimed to compare BB-DNA from (a) GO (nonagenarian offspring), (b) age-matched controls (Randomly recruited Age-Stratified Individuals from the General population [RASIG]), and (c) spouses of GO (SGO) recruited in the MARK-AGE project, as well as to investigate the association between BB-DNA, behavior habits, Charlson Comorbidity Index (CCI), leucocyte subsets, and the circulating levels of some antioxidants and oxidative stress markers. BB-DNA was higher in RASIG than GO and SGO, whereas GO and SGO participants showed similar values. BB-DNA increased in smokers and males with CCI ≥ 2 compared with those with CCI ≤ 1 within RASIG. Moreover, BB-DNA was positively associated with lymphocyte, neutrophil, and monocyte counts, but not with self-reported dietary habits. Higher quartiles of BB-DNA were associated with low lutein and zeaxanthin and elevated malondialdehyde plasma concentrations in RASIG. BB-DNA was also positively correlated with nitric oxide levels. Herein, we provide evidence of a reduced BB-DNA in individuals from long-living families and their spouses, suggesting a decreased microbial dysbiosis and bacterial systemic translocation. BB-DNA was also associated with smoking, CCI, leukocyte subsets, and some redox biomarkers in older participants.

Circulating cell-free DNA in health and disease - the relationship to health behaviours, ageing phenotypes and metabolomics.

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17-82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

Vitamin D status and oxidative stress markers in the elderly with impaired fasting glucose and type 2 diabetes mellitus.

BACKGROUND AND AIMS: Vitamin D deficiency has been identified in the elderly as a potential risk factor for cardiovascular disease development, possibly through its association with other risk factors, such as type 2 diabetes mellitus (T2DM), hypercholesterolemia and hypertension. The aim of this cross-sectional study was to evaluate the vitamin D status in elderly subjects with impaired fasting glucose (IFG) or T2DM, and to examine its relationships to systemic oxidative stress and biochemical markers of endothelial dysfunction. METHODS: Serum 25-hydroxyvitamin D [25(OH)D], fasting glucose, insulin, lipid profile, advanced glycation end products (AGEs), advanced oxidation protein products (AOPPs), low-density lipoprotein susceptibility to oxidation (oxLDL) and nitric oxide metabolic pathway products (NOx) were analyzed in elderly subjects with IFG (n=30) and T2DM (n=35) compared with aged-matched controls (n=25). RESULTS: 25(OH)D levels in the IFG and T2DM groups were significantly lower than in controls (31.9±1.9 and 28.5±1.9 vs 39.4±2.4 ng/mL, p<0.001), and associated with significantly (p<0.001) higher levels of the oxidative stress parameters AGEs, AOPPs, oxLDL and NOx. Hypovitaminosis D [25(OH)D)<30 ng/ml] markedly enhanced the oxidative stress and cardiovascular risk in hyperglycemic subjects compared with sufficient vitamin D [25(OH)D)≥30 ng/mL] status subjects. In subjects with IFG and T2DM (n=65), the vitamin D status was significantly inversely correlated both with oxLDL (r=-0.413, p=0.001) and AOPPs (r=-0.475, p<0.001), and strongly positively associated with highdensity lipoprotein cholesterol (r=0.609, p<0.001). CONCLUSIONS: In the elderly with impaired glucose metabolism the vitamin D status is inversely associated with levels of circulating markers of oxidative stress and endothelial dysfunction, especially in subjects with hypovitaminosis D.

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential.

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.

Quercetin and epigallocatechin gallate induce in vitro a dose-dependent stiffening and hyperpolarizing effect on the cell membrane of human mononuclear blood cells.

The bioactivity of polyphenols is closely linked to their ability to interact with biological membranes. The study evaluates the in vitro effect of quercetin and epigallocatechin on the membrane anisotropy and transmembrane potential of peripheral blood mononuclear cells (PBMCs) isolated from 26 type 2 diabetes mellitus patients compared to 25 age matched controls. The in vitro assays were analyzed in correlation with the biochemical and inflammatory profile of the subjects and with insulin resistance parameters (HOMA-IR, plasma resistin) as well. For type 2 diabetes patients, the increase of HOMA-IR and resistin concentration was associated with a significant decrease of the PBMCs membrane anisotropy. The two tested polyphenols induced a dose-dependent hyperpolarizing effect and stiffening of the cell membranes for all tested subjects. Physiological levels of quercetin and epigallocatechin gallate had the tendency to normalize the PBMCs membrane anisotropy of the cells isolated from diabetes patients, bringing it to the level of cells isolated from normoglycemic ones. Epigallocatechin gallate induced higher effects compared to quercetin on the membranes isolated from subjects with higher cardiovascular risk. The decrease of membrane fluidity and the hyperpolarizing effect could explain the cardiovascular protective action of the tested compounds.

Interleukins and redox impairment in type 2 diabetes mellitus: mini-review and pilot study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) represents a leading cause of morbidity and premature mortality, low-grade inflammation being acknowledged as a key contributor to its development and progression. A tailored therapeutic approach, based on sensitive and specific biomarkers, could allow a more accurate analysis of disease susceptibility/prognostic and of the response to treatment. OBJECTIVES: This mini-review and pilot study had two main goals: (1) reviewing the most recent literature encompassing the use of interleukins as inflammatory markers influenced by the redox imbalances in T2DM and (2) assessing parameters that conjunctly evaluate the redox impairment and inflammatory burden of T2DM patients, taking into consideration smoking status, as such group-specific biomarkers are scarcely reported in literature. METHODS: Firstly, PubMed database was surveyed to select and review the relevant studies employing interleukins as T2DM biomarkers and to assess if studies using combined inflammatory-redox indices were reported. Then, routine biochemical parameters were assessed in a pilot study -T2DM patients with 3 subgroups: non-smokers, smokers and ex-smokers, were compared to a control group of non-diabetic, apparently healthy non-smokers. Protein (AOPPs, AGEs), lipid/HDL (Amplex Red-based method) oxidative damage and inflammatory status (CRP, IL-1ß, IL-6, IL-10) biomarkers were assessed. Cytokine ratios and 2 oxidative-inflammatory status indices were developed (IH1 and IH2) and evaluated. RESULTS: We observed significant differences in terms of serum redox and inflammatory status (AOPPs, AGEs, CRP, CRP/HDL, CRP/IL-6, IL-10/IL-6, IH1) between T2DM patients compared to control and, moreover, between the subgroups formed considering smoking status (CRP, CRP/HDL, IH1). Glycemic control strongly influenced inflammatory status biomarkers: glycemia was positively correlated with the inflammatory parameters (CRP/IL-10) and inversely with the anti-inflammatory ones (IL-10, IL-10/IL-1ß ratio). CONCLUSIONS: Several of the assessed parameters may possess prognostic value for diabetics, especially when comparing subgroups with a different smoking history and could prove useful in clinical practice for assessing disease progress and therapeutic efficacy.

Discovery of New Microbial Collagenase Inhibitors.

Bacterial virulence factors are mediating bacterial pathogenesis and infectivity. Collagenases are virulence factors secreted by several bacterial stains, such as Clostridium, Bacillus, Vibrio and Pseudomonas. These enzymes are among the most efficient degraders of collagen, playing a crucial role in host colonization. Thus, they are an important target for developing new anti-infective agents because of their pivotal roles in the infection process. A primary screening using a fluorescence resonance energy-transfer assay was used to experimentally evaluate the inhibitory activity of 77 compounds on collagenase A. Based on their inhibitory activity and chemical diversity, a small number of compounds was selected to determine the corresponding half maximal inhibitory con-centration (IC50). Additionally, we used molecular docking to get a better understanding of the enzyme-compound interaction. Several natural compounds (capsaicin, 4',5-dihydroxyflavone, curcumin, dihydrorobinetin, palmatine chloride, biochanin A, 2'-hydroxychalcone, and juglone) were identified as promising candidates for further development into useful anti-infective agents against infections caused by multi-drug-resistant bacterial pathogens which include collagenase A in their enzymatic set.

Procaine-The Controversial Geroprotector Candidate: New Insights Regarding Its Molecular and Cellular Effects.

Since its discovery in 1905 and its employment in everyday medical practice as a local anesthetic, to its highly controversial endorsement as an "anti-aging" molecule in the sixties and seventies, procaine is part of the history of medicine and gerontoprophylaxis. Procaine can be considered a "veteran" drug due to its long-time use in clinical practice, but is also a molecule which continues to incite interest, revealing new biological and pharmacological effects within novel experimental approaches. Therefore, this review is aimed at exploring and systematizing recent data on the biochemical, cellular, and molecular mechanisms involved in the antioxidant and potential geroprotective effects of procaine, focusing on the following aspects: (1) the research state-of-the-art, through an objective examination of scientific literature within the last 30 years, describing the positive, as well as the negative reports; (2) the experimental data supporting the beneficial effects of procaine in preventing or alleviating age-related pathology; and (3) the multifactorial pathways procaine impacts oxidative stress, inflammation, atherogenesis, cerebral age-related pathology, DNA damage, and methylation. According to reviewed data, procaine displayed antioxidant and cytoprotective actions in experimental models of myocardial ischemia/reperfusion injury, lipoprotein oxidation, endothelial-dependent vasorelaxation, inflammation, sepsis, intoxication, ionizing irradiation, cancer, and neurodegeneration. This analysis painted a complex pharmacological profile of procaine: a molecule that has not yet fully expressed its therapeutic potential in the treatment and prevention of aging-associated diseases. The numerous recent reports found demonstrate the rising interest in researching the multiple actions of procaine regulating key processes involved in cellular senescence. Its beneficial effects on cell/tissue functions and metabolism could designate procaine as a valuable candidate for the well-established Geroprotectors database.

Candidates for Repurposing as Anti-Virulence Agents Based on the Structural Profile Analysis of Microbial Collagenase Inhibitors.

The pharmacological inhibition of the bacterial collagenases (BC) enzymes is considered a promising strategy to block the virulence of the bacteria without targeting the selection mechanism leading to drug resistance. The chemical structures of the Clostridium perfringens collagenase A (ColA) inhibitors were analyzed using Bemis-Murcko skeletons, Murcko frameworks, the type of plain rings, and docking studies. The inhibitors were classified based on their structural architecture and various scoring methods were implemented to predict the probability of new compounds to inhibit ColA and other BC. The analyses indicated that all compounds contain at least one aromatic ring, which is often a nitrobenzene fragment. 2-Nitrobenzene based compounds are, on average, more potent BC inhibitors compared to those derived from 4-nitrobenzene. The molecular descriptors MDEO-11, AATS0s, ASP-0, and MAXDN were determined as filters to identify new BC inhibitors and highlighted the necessity for a compound to contain at least three primary oxygen atoms. The DrugBank database was virtually screened using the developed methods. A total of 100 compounds were identified as potential BC inhibitors, of which, 10 are human approved drugs. Benzthiazide, entacapone, and lodoxamide were chosen as the best candidates for in vitro testing based on their pharmaco-toxicological profile.

Zinc status, insulin resistance and glycoxidative stress in elderly subjects with type 2 diabetes mellitus.

Zinc deficiencies have been reported in numerous pathologies, such as diabetes mellitus, but also in the physiological process of ageing. Similarly, the end products of glycoxidation processes, advanced glycation end products (AGEs), are damaging compounds, a myriad of reports linking them to the development and progression of several age-associated chronic diseases. The aim of the present study was to analyze the relationships between zinc status, glycoxidative stress and insulin resistance (IR) in elderly subjects with type 2 diabetes mellitus (T2DM). A group of 52 non-smoking subjects (9 men and 43 women, aged 65-83 years) were enrolled in this cross-sectional study: 27 patients with T2DM, and 25 apparently healthy control subjects. Serum zinc (Zn) levels were assessed using a commercial kit based on an end-point colorimetric method, and serum AGEs were evaluated with a fluorimetric analytic procedure. The calculated glucose-to-zinc ratio (Gly/Zn), insulin-to-zinc ratio (Ins/Zn) and insulin-zinc resistance index (HOMA-IR/Zn) were further used to study the associations between serum Zn levels, secretory function of ß-pancreatic cells and AGEs. T2DM patients presented significantly higher serum insulin and Zn levels, as compared to the controls. We found a significant inverse correlation between Zn and AGEs, and a strong positive correlation between AGEs and the Gly/Zn ratio, suggesting that both Zn and AGEs are biomarkers that could reflect the persistence of hyperglycemia. We identified new surrogate biomarkers useful for the assessment of glycemic control with great potential for the development of preventive and therapeutic strategies for elderly diabetics, based on the evaluation of serum Zn levels.

The Radioprotective Effect of Procaine and Procaine-Derived Product Gerovital H3 in Lymphocytes from Young and Aged Individuals.

Ionizing radiation induces genomic instability in living organisms, and several studies reported an ageing-dependent radiosensitivity. Chemical compounds, such as scavengers, radioprotectors, and modifiers, contribute to reducing the radiation-associated toxicity. These compounds are often antioxidants, and therefore, in order to be effective, they must be present before or during exposure to radiation. However, not all antioxidants provide radioprotection. In this study, we investigated the effects of procaine and of a procaine-based product Gerovital H3 (GH3) on the formation of endogenous and X-ray-induced DNA strand breaks in peripheral blood mononuclear cells (PBMCs) isolated from young and elderly individuals. Interestingly, GH3 showed the strongest radioprotective effects in PBMCs from young subjects, while procaine reduced the endogenous amount of DNA strand breaks more pronounced in aged individuals. Both procaine and GH3 inhibited lipid peroxidation, but procaine was more effective in inhibiting mitochondria free radicals' generation, while GH3 showed a higher antioxidant action on macrophage-induced low-density lipoprotein oxidation. Our findings provide new insights into the mechanisms underlying the distinct effects of procaine and GH3 on DNA damage.

Drug metabolizing enzyme expression in rat choroid plexus: effects of in vivo xenobiotics treatment.

The presence of drug metabolizing enzymes in extrahepatic tissues such as the choroid plexus (CP) suggests that the CP, like the blood-brain barrier, affords a metabolic protection to the brain against xenobiotics. The CP, which is the principal site of formation of the cerebrospinal fluid (CSF), controls the exchange of many endogenous compounds and exogenous molecules between brain tissue and CSF. We present the changes in mRNA expression and enzymatic activities of UDP-glucuronosyltransferase, UGT1A6 isoform and NADPH-cytochrome P450 reductase, after in vitro treatment with xenobiotic molecules known to act in the liver as inducers or inhibitors of these drug metabolizing enzymes. Five study groups of male Sprague-Dawley rats were treated separately with 3-methylcholantrene (3-MC), phenobarbital (PB), dexamethasone (DEX), cyclosporine (CsA) or paraquat (PQ). Choroidal 1-naphthol glucuronidation activities were significantly induced by 3-MC and PQ administration (354 +/- 85 and 257 +/- 49 vs. 115 +/- 24 nmol/h per mg protein, in control group), whereas the other molecules were without effect. Accordingly, UGT1A6 mRNA expression, measured by RT-PCR, was 2.3-fold higher after 3-MC treatment and 2.1-fold higher after PQ administration. By contrast, reductase activities and mRNA expression remained unchanged in the isolated choroids plexus in these experimental conditions. We present for the first time evidences that the choroids plexus express transcripts for both UGT1A6 and NADPH-cytochrome P450 reductase, and their mRNA expression can be differently regulated by exogenous factors. These results emphasize that xenobiotics could modulate the biotransformation of exogenous and/or endogenous compounds in the choroids plexus, and underline the role of UGTs in the maintenance of brain homeostasis.

Spectrophotometric versus spectrofluorometric assessment in the study of the relationships between lipid peroxidation and metabolic dysregulation.

Reactive oxygen species are crucial to normal cell function, but are also part of the pathogenesis of multiple modern maladies. As such, sensitive, fast, and reliable methods of appreciating redox status are needed. We aimed to optimize the Amplex Red (AR) and ferric-xylenol orange (FOX) methods using human serum samples, rat tissue homogenates, and mitochondrial preparations. For AR, we intended to reduce probe concentration, maintaining method sensitivity, as well as extending its use from isolated lipoproteins samples, and readjust it for a high-throughput application. Also, we evaluated the usefulness of a modified xylenol orange-based spectrophotometric protocol, comparing and contrasting these methods in terms of clinical relevance and suitability for their further use in assessing redox status of various biological samples in different pathological conditions. Our results show that these optimized protocols are suitable for complex in vivo studies, as they require low quantities of sample and reagents, and are sensitive, rapid, and economical, with the option of adapting them for high-throughput analysis. For a better assessment of oxidative status of serum-derived samples, the two methods can be used concurrently, while for tissue-derived ones, either can be employed for the measurement of a global redox status.

Gender- and age-dependencies of oxidative stress, as detected based on the steady state concentrations of different biomarkers in the MARK-AGE study.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA. Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS.