This study aimed to evaluate the interrelationship between telomere length, telomerase activity and oxidative DNA damage in patients undergoing in vitro fertilization (IVF). This single-center, ...observational clinical study comprised 102 unselected, consecutive patients with various infertility diagnoses. Granulosa cells (GCs) and follicular fluid (FF) were analyzed simultaneously for telomere functions and for the marker of oxidative DNA damage, 8-hydroxy-2-deoxyguanosine (8-OHdG). An Absolute Human Telomere Lengths Quantification qPCR Assay kit and Telomerase Activity Quantification qPCR Assay kit (Nucleotestbio, Budapest, Hungary), as well as an 8-OHdG ELISA kit (Abbexa Ltd., Cambridge, United Kingdom) were used for analyses. Similar telomere lengths were found in GCs and FF, however telomerase activity was markedly depressed, while 8-OHdG levels were markedly elevated in FF compared with those in GCs (
< 0.01). Telomere lengths were independent of telomerase activity both in GCs and FF. However, GC 8-OHdG was inversely related to telomerase activity in GCs and FF (
< 0.05). Importantly, 8-OHdG levels both in GCs and FF had significant negative impact on the number of the retrieved and MII oocytes (
< 0.01), whereas FF 8-OHdG was negatively related further to the number of fertilized oocytes and blastocysts (
< 0.01). In conclusion, we could not confirm the direct association of telomere function and reproductive potential. However, oxidative DNA damage, as mainly reflected by 8-OHdG, adversely affected early markers of IVF outcome and clinical pregnancies.
Urinary biomarkers might provide non-invasive tool for monitoring of systemic processes. We aimed to investigate the time-course of urinary orosomucoid (u-ORM) excretion after cardiac surgery ...hypothesizing that u-ORM is an early and sensitive marker of systemic inflammatory activation.
During a 5-day follow-up study we monitored u-ORM levels in cardiovascular patients who underwent on-pump cardiac surgery (n=38). The patients baseline data were compared to healthy control individuals (n=40). u-ORM was measured by a newly developed automated turbidimetric assay and values were referred to urinary creatinine and expressed as u-ORM/u-CREAT (mg/mmol).
The cardiovascular patients showed slightly increased baseline u-ORM excretion compared to healthy controls (0.29 vs 0.08mg/mmol, p<0.001). After cardiac surgery, a rapid 10-fold elevation in u-ORM/u-CREAT levels was found. The values remained high till the 3rd postoperative day, and they then decreased significantly (p<0.01) on the 5th day after surgery. u-ORM/u-CREAT mirrored well the perioperative tendency of hs-CRP levels, but it did not follow the non-decreasing kinetics of serum ORM concentrations during the follow-up. u-ORM/u-CREAT correlated significantly (p<0.001) with inflammatory parameters (hs-CRP, se-ORM, WBC).
We described u-ORM as an early and sensitive marker of inflammatory activation. The rapid elevation of u-ORM/u-CREAT after surgery and its postoperative kinetics could reflect the magnitude of inflammatory response better than serum ORM and similar to hs-CRP. u-ORM measurements might provide a novel non-invasive tool for real-time monitoring of systemic inflammation, however further investigations are required to confirm it.
•Urinary orosomucoid was measured by a novel turbidimetric assay.•Urinary orosomucoid is a more sensitive inflammatory marker than serum orosomucoid.•Urinary orosomucoid mirrored the kinetics of CRP after cardiac surgery.•Good correlation between urinary orosomucoid and inflammatory parameters•Urinary orosomucoid is a non-invasive novel marker of inflammation.
Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2'R-ochratoxin A (2'R-OTA), which appears in the blood of coffee drinkers. Human serum ...albumin (HSA) binds 2'R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2'R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2'R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2'R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2'R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2'R-OTA than OTA from HSA. Fluorescence and binding constants of 2'R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2'R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.
Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into ...different interactions of OTA at the molecular level. OTA is found worldwide in many foods and drinks. Moreover, it can also be detected in human and animal tissues and body fluids, as well. Therefore, the development of highly sensitive quantitative methods for the determination of OTA is of utmost importance. OTA most likely forms complexes with divalent cations, both in cells and body fluids. In the present study, the OTA-zinc interaction was investigated and compared to OTA-magnesium complex formation using fluorescence spectroscopy and molecular modeling. Our results show that zinc(II) ion forms a two-fold higher stable complex with OTA than magnesium(II) ion. In addition, based on the enhanced fluorescence emission of OTA in its magnesium-bound form, a novel RP-HPLC-fluorescence detector (FLD) method was also established. Our results highlight that the application of magnesium chloride in alkaline eluents results in an approximately two-fold increase in sensitivity using the HPLC-FLD technique.
Citrinin (CIT) is a nephrotoxic mycotoxin produced by
Penicillium
,
Monascus
, and
Aspergillus
species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. ...During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (
K
~10
5
L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.
Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to ...investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with β-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of β-cyclodextrins on resazurin-based cell viability assay was also tested. Both resazurin and resorufin form stable complexes with the examined β-cyclodextrins (2.0-3.1 × 10³ and 1.3-1.8 × 10³ L/mol were determined as binding constants, respectively). Cells were incubated for 30 and 120 min and treated with resazurin and/or β-cyclodextrins. Our results suggest that cyclodextrins are able to interfere with the resazurin-based cell viability assay that presumably results from the following mechanisms: (1) inhibition of the cellular uptake of resazurin and (2) enhancement of the fluorescence signal of the formed resorufin.
Ochratoxin-A (OTA) is a mycotoxin with possibly carcinogenic and nephrotoxic effects in humans and animals. OTA is often found as a contaminant in agricultural commodities. The aim of the present ...work was to evaluate OTA-degrading and detoxifying potential of Cupriavidus basilensis OdblacR16 strain. In vivo administration of OTA in CD1 male mice (1 or 10 mg/kg body weight for 72 hours or 0.5 mg/kg body weight for 21 days) resulted in significant elevation of OTA levels in the blood, histopathological alterations- and transcriptional changes in OTA-dependent genes (annexinA2, clusterin, sulphotransferase and gadd45 and gadd153) in the renal cortex. These OTA-induced changes were not seen in animals that have been treated with culture supernatants in which OTA was incubated with Cupriavidus basilensis OdblacR16 strain for 5 days. HPLC and ELISA methods identified ochratoxin alpha as the major metabolite of OTA in Cupriavidus basilensis OdblacR16 cultures, which is not toxic in vivo. This study has demonstrated that Cupriavidus basilensis OdblacR16 efficiently degrade OTA without producing toxic adventitious metabolites.
The objective of this study was to find the correlation between the severity of perfusion abnormality detected by scintigraphy and the FFR value, as well as the localization of a particular coronary ...lesion. On the basis of FFR values and the corresponding left ventricular segments, we proposed a combined index to aim for better correlation with myocardial ischemia than the FFR parameter alone.
Twenty-eight patients (male: 22, female: 6, age 62±7.62) having FFR measurements and myocardial perfusion SPECT studies were enrolled in our retrospective analysis. FFR measurements on 36 vessels (20 LAD, 6 LCx, 10 RCA) with intermediate stenosis (40%-60%) were compared to the Tc-99m SestaMIBI myocardial perfusion SPECT studies. SPECT studies were performed before the invasive procedure in all cases. We introduced a new ischemic index, the left ventricular ischemic index (LVIi), by combining FFR values with the number of corresponding myocardial segments (N) LVIi=N x (1-FFR). This index correlated with the regional myocardial perfusion defects identified on the scintigrams. A perfusion reversibility score of 2 or above was considered indicative of active ischemia (regional difference score: rDSc). For the statistical analysis, we used linear regression analysis and receiver operating characteristic (ROC) curve analysis to compare the different parameters.
A close linear relationship was found between the LVIi and rDSc values (p<0.001) with linear regression analysis. When analyzing all FFR values independently of the localization of the lesions, they also correlated significantly to the rDSc, but this relation was not as close. LVIi predicted active ischemia (≥2 rDSc) on myocardial scintigraphy with 78% sensitivity and 94% specificity when the cutoff value was set to 0.96. FFR alone predicted ischemia on scintigraphy with 72% sensitivity and 94% specificity at the best 0.8 cut-off value. The area under the ROC curve was significantly higher for LVIi than FFR (0.94 vs. 0.87; p<0.05).
The scintigraphic data indicate that an LVIi >0.96 implies a clinically relevant stenotic lesion. In our opinion, FFR values, weighted with the corresponding left ventricular segments, should be taken into consideration for the best clinical decision-making.
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