Lubomira Tothova

Salivary markers of oxidative stress in oral diseases

Saliva is an interesting alternative diagnostic body fluid with several specific advantages over blood. These include non-invasive and easy collection and related possibility to do repeated sampling. One of the obstacles that hinders the wider use of saliva for diagnosis and monitoring of systemic diseases is its composition, which is affected by local oral status. However, this issue makes saliva very interesting for clinical biochemistry of oral diseases. Periodontitis, caries, oral precancerosis, and other local oral pathologies are associated with oxidative stress. Several markers of lipid peroxidation, protein oxidation and DNA damage induced by reactive oxygen species can be measured in saliva. Clinical studies have shown an association with oral pathologies at least for some of the established salivary markers of oxidative stress. This association is currently limited to the population level and none of the widely used markers can be applied for individual diagnostics. Oxidative stress seems to be of local oral origin, but it is currently unclear whether it is caused by an overproduction of reactive oxygen species due to inflammation or by the lack of antioxidants. Interventional studies, both, in experimental animals as well as humans indicate that antioxidant treatment could prevent or slow-down the progress of periodontitis. This makes the potential clinical use of salivary markers of oxidative stress even more attractive. This review summarizes basic information on the most commonly used salivary markers of oxidative damage, antioxidant status, and carbonyl stress and the studies analyzing these markers in patients with caries or periodontitis.

Salivary markers of oxidative stress and antioxidant status: influence of external factors.

BACKGROUND: Salivary markers of oxidative stress and antioxidant status represent promising tool for the research of oral diseases. One of the criteria is the validation of these biomarkers from the perspective of the confounding and modifying factors. AIM: To examine the effect of circadian rhythm, tooth-brushing and ascorbic acid treatment on selected salivary markers of oxidative and carbonyl stress, and antioxidant status. SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from 19 healthy participants three times during a day, before and after tooth-brushing, and before and after the administration of vitamin C (250 mg). Advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGEs), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) were measured. RESULTS: Salivary AGEs levels varied significantly during the day (p < 0.05) with the highest concentrations in the morning. FRAP levels varied during the day (p < 0.01) with the highest concentrations in the afternoon. Tooth-brushing decreased AGEs (p < 0.05) and TBARS levels (p < 0.01) and increased FRAP levels (p < 0.05). Single intake of vitamin C significantly decreased AGEs (p < 0.001) and increased both FRAP (p < 0.01) and TAC (p < 0.01) concentrations. CONCLUSION: Significant daily variations were observed in salivary AGEs and FRAP levels. Tooth-brushing and treatment with vitamin C decreased carbonyl stress and increased the antioxidant status. These results are important from the perspective of using saliva for the research of oral diseases.

Effects of testosterone and estradiol on anxiety and depressive-like behavior via a non-genomic pathway

Besides their known slow genomic effects, testosterone and estradiol have rapid effects in the brain. However, their impact on mood-related behavior is not clear. The aim of this study was to investigate the non-genomic pathway of testosterone and estradiol in the amygdala in relation to anxiety and depressive-like behavior. Sham-operated and gonadectomized male rats (GDX) supplemented with testosterone propionate, estradiol, or olive oil were used. Five minutes after administration, anxiety and depression-like behavior were tested. Estradiol increased anxiolytic behavior in the open-field test compared to the GDX group, but administration of testosterone had no significant effect. Besides, c-Fos expression in the medial nucleus of the amygdala significantly increased after testosterone treatment compared to the GDX group, while no significant difference was observed in the central and the basolateral nuclei of the amygdala in the testosterone-treated group compared to the GDX group. In conclusion, estradiol had an anxiolytic effect via a rapid pathway, but no rapid effect of testosterone on anxiety was found. Further studies elucidating whether the rapid effect is mediated by a non-genomic pathway are needed.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d‐dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT‐PCR assay for expression of epithelial (CK19) or epithelial‐mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme‐linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Phage therapy of Cronobacter-induced urinary tract infection in mice

BACKGROUND Cronobacter spp. is an opportunistic pathogen causing rare but dangerous cases of meningitis, sepsis and urinary tract infection. Phage therapy overcomes antibiotic resistance and represents an alternative approach to standard antimicrobial treatment. There are no published studies on the use of phages against Cronobacter spp. in vivo. The aim of our study was to prove the effects of isolated Cronobacter-specific phages on renal colonization in a model of urinary tract infection in mice. MATERIAL/METHODS Urinary tract infection was induced by transurethral application of Cronobacter turicensis (1011 CFU/ml). Simultaneously, isolated Cronobacter-specific phages were administered intraperitoneally (1011 PFU/ml). After 24 hours, kidneys and bladder were collected and used for cultivation and analysis of gene expression and oxidative stress markers. RESULTS Phage therapy reduced the number of Cronobacter colonies in the kidney by 70%. Higher levels of malondialdehyde were reduced by phage therapy without affecting the antioxidant status. The expression of pro-inflammatory cytokines tumor necrosis factor-alpha and monocyte chemoattractant protein-1 increased by the infection and was attenuated by phage therapy. CONCLUSIONS Phage therapy proved effective in the prevention of ascending renal infection in a murine model of urinary tract infection. Long-term effects and safety of the treatment are currently unknown. Further studies should test phage therapy in other Cronobacter infection models.

Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes

Abstract Objective: To determine amniotic fluid total antioxidant capacity (TAC), ferric-reducing antioxidant power (FRAP) and thiobarbituric acid-reacting substances (TBARS), markers of oxidative stress, in pregnancies complicated by preterm prelabor rupture of membranes (pPROM) and their correlation to microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA). Methods: One-hundred thirty-eight women with singleton pregnancies complicated by pPROM were included in this study. Amniotic fluid was collected by transabdominal amniocentesis at the time of admission and amniotic fluid concentrations of TAC, FRAP and TBARS were measured. Result: The presence of MIAC and/or HCA did not show any significant differences in the amniotic fluid TAC, FRAP and TBARS concentrations. Positive correlations between gestational age at sampling and amniotic fluid TAC and FRAP concentrations were found (TAC: rho = 0.32; p = 0.0002; FRAP: rho = 0.36; p < 0.0001). A negative correlation between gestation age at sampling and amniotic fluid TBARS concentrations was identified (rho = –0.25; p = 0.004). Conclusions: Oxidative stress is associated with pPROM as indicated by the presence of markers tested in the amniotic fluid; however, oxidative stress markers tested are not influenced by the presence of MIAC or HCA.

Sex differences of oxidative stress markers in young healthy subjects are marker-specific in plasma but not in saliva

Background: Several studies showed there are sex differences in oxidative stress. An observational study analysing oxidative stress markers in young healthy men and women is lacking. Moreover, it is unclear whether the differences are related to sex hormones. Aim: The primary goal was to analyse differences in oxidative stress markers with regard to sex in plasma of young healthy subjects and whether differences are related to sex hormones. The secondary study compared oxidative stress markers in plasma with salivary samples. Methods: Plasma and saliva samples were analysed from 158 young healthy probands. Established spectro-photometric/fluorometric methods were used to quantify oxidative stress markers. Sex hormones were measured using ELISA kits. Results: In plasma, malondialdehyde and advanced glycation end products were significantly higher in women. Advanced oxidation protein products and the ferric reducing ability of plasma were higher in men. Sex hormones were not associated with oxidative stress markers. In saliva, analysed markers of antioxidant status were higher in men, but no sex differences were found in other markers. Conclusion: Observed parameters showed marker-specific sex differences in plasma, but these differences were not related to sex hormones. Plasma and saliva concentrations of biomarkers did not correlate, reflecting oral but not systemic conditions.

Effect of exogenous testosterone on oxidative status of the testes in adult male rats

The aim of this study was to investigate the testosterone‐induced changes in the oxidative status of testes in adult male rats treated either with testosterone or after blockade of androgen receptors with cyproterone acetate. A total of 40 intact rats were divided into four groups: a control group receiving sterile oil, the testosterone group receiving testosterone isobutyras, the cyproterone group receiving cyproterone acetate and the combination group receiving both testosterone isobutyras and cyproterone acetate. Treatments were carried out for 2 days by intramuscular application. Parameters of oxidative stress and the expression levels of the steroidogenic acute regulatory protein (StAR) gene were measured in testes. Significantly increased TBARS and advanced glycation end products (AGEs) levels were found in the testosterone group when compared to the control group. The °1 ferric‐reducing ability of the tissue and total antioxidative capacity were lower in the testosterone group in comparison with the control group. Gene expression analysis revealed significant downregulation of the StAR gene in the testes of rats in the testosterone and combination groups with respect to control animals. In conclusion, administration of exogenous testosterone influences the lipid peroxidation and carbonyl stress and decreases the antioxidant defence in the testes. These data might have implications for male fertility in humans.

Oxidative Stress and Antioxidants in the Diagnosis and Therapy of Periodontitis

Oxidative stress has been implicated in the pathogenesis of numerous diseases. However, large interventional studies with antioxidants failed to show benefits in the prevention or treatment of cardiovascular diseases, cancer, or diabetes mellitus. Numerous clinical studies have confirmed the association of oxidative stress markers and periodontitis. Technical and biological variability is high for most of the analyzed markers and none of them seems to be optimal for routine clinical use. In a research setting, analysis of a palette of oxidative stress markers is needed to cover lipid peroxidation, protein oxidation, and the antioxidant status. The source of reactive oxygen species and their role in the pathogenesis of periodontitis remains unclear. Interventional experiments indicate that oxidative stress might be more than just a simple consequence of the inflammation. Small studies have confirmed that some antioxidants could have therapeutic value at least as an addition to the standard non-surgical treatment of periodontitis. A clear evidence for the efficiency of antioxidant treatment in large patient cohorts is lacking. Potentially, because lowering of oxidative stress markers might be a secondary effect of anti-inflammatory or antibacterial agents. As the field of research of oxidative stress in periodontitis gains attraction and the number of relevant published papers is increasing a systematic overview of the conducted observational and interventional studies is needed. This review summarizes the currently available literature linking oxidative stress and periodontitis and points toward the potential of adjuvant antioxidant treatment, especially in cases where standard treatment fails to improve the periodontal status.

Umbilical cord blood markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes

Abstract Objective: To determine umbilical cord blood total antioxidant capacity (TAC), ferric reducing antioxidant power (FRAP), thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs) and markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) and their associations with microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA), funisitis and selected aspects of short-term neonatal morbidity. Materials and methods: One hundred and sixty-five women with singleton pregnancies complicated by PPROM were included in this study. Blood samples were obtained by venipuncture from the umbilical cord vein after the delivery of the newborn. The umbilical cord blood concentrations of TAC, FRAP, TBARS and AGEs were measured. Results: The presence of MIAC, HCA and funisitis did not show differences in the umbilical cord blood TAC, FRAP, TBARS and AGEs concentrations. Positive correlations were found between the gestational age at sampling and umbilical cord blood TAC and AGEs concentrations (TAC: rho = 0.26; p = 0.001; AGEs: rho = 0.35; p < 0.0001). There was no association between umbilical cord blood TAC, FRAP, TBARS and AGEs concentrations and selected aspects of short-term neonatal morbidity. Conclusions: Oxidative stress is associated with PPROM, as indicated by the presence of markers tested in the umbilical cord blood; however, the evaluated oxidative stress markers are not influenced by the presence of MIAC and/or HCA, and funisitis or subsequent development of selected aspects of short-term neonatal morbidity.