Publications by authors named "Athanasios K Alegakis"

28 Publications

  • Page 1 of 1

Phthalate metabolites concentrations in amniotic fluid and maternal urine: Cumulative exposure and risk assessment.

Toxicol Rep 2020 19;7:529-538. Epub 2020 Apr 19.

Laboratory of Toxicology Science and Research, Medical School, University of Crete, 71003, Heraklion, Crete, Greece.

Phthalates are used in industry as plasticizers or additives in everyday products and they have been considered as endocrine disrupting chemicals. Maternal exposure during pregnancy has been associated with neonatal exposure, preterm birth and impacts in the reproductive and respiratory systems. The aim of this study is to determine six phthalate metabolites (mono isobutyl phthalate, miBP, mono n-butyl phthalate, mnBP, mono benzyl phthalate, mBzP, mono ethylhexyl phthalate, mEHP, mono 2-ethyl-5-hydroxyhexyl phthalate, mEHHP, mono 2-ethyl-5-oxohexyl-phthalate, mEOHP) in amniotic fluid and urine from 100 pregnant women. Participants answered questionnaires for the use of plastics and cosmetics, dietary habits, health effects, pregnancy problems, health and infant development. Positive amniotic fluid samples ranged from 1% to 21% and urine from 27% to 54%. The median levels for amniotic fluid were 2.3 μg/L - 10.7 μg/L and for urine 4.9 μg/L - 46.7 μg/L. The major results include significant correlations between urinary phthalates indicating their common sources of exposure, the frequent use of deodorant was significantly associated with higher urinary miBP (p = 0.050) and mnBP (p = 0.028) and a weak inverse association was found for the use of make-up products with mBzP (p = 0.053). The frequent use of plastic food containers was significantly associated with urinary mEHP (p = 0.026), and a positive trend was noticed for mEHP in amniotic fluid (p = 0.093). An association although weak was found between urinary mEHP and lower birth length (rs = 0.396, p = 0.062). No other associations were found for infant health problems or development. The daily intake of the total phthalates was calculated 5.4 μg/kg body weight/day which corresponds to hazard index 0.10 and exposure follows the declining trend that has been observed the last decades.
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http://dx.doi.org/10.1016/j.toxrep.2020.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186561PMC
April 2020

Biomonitoring of bisphenols A and S and phthalate metabolites in hair from pregnant women in Crete.

Sci Total Environ 2020 Apr 20;712:135651. Epub 2019 Nov 20.

Laboratory of Toxicology Science and Research, Medical School, University of Crete, 71003 Heraklion, Crete, Greece. Electronic address:

Phthalates, bisphenols A and S (BPA, BPS) are used as plasticizers and many of them are documented or suspected of being endocrine disruptors. Several studies indicate that exposure during pregnancy may affect the newborn's health and development. The aim of this cross-sectional study is the biomonitoring of seven phthalate metabolites, BPA and BPS in hair from 100 pregnant women in Crete. The most frequently detected compounds were monoethylhexyl phthalate (mEHP) (68%), mono isobutyl phthalate (miBP) (40%), BPA (37%), BPS (34%) and mono-n-butyl phthalate (mnBP) (28%). Phthalate metabolites were detected at medians from 19.5 to 44.4 pg/mg, BPA at 69.9 pg/mg and BPS at 3.5 pg/mg. Significant positive correlations between phthalate metabolites were found which indicated their common sources of exposure. The frequent use of plastics for food storage was strongly associated with mEHP (p = .013) and a weaker association was found for miBP (p = .063). The frequent use of cosmetics during or before pregnancy was associated with levels of phthalate metabolites in hair. More specifically, the use of hair spray before pregnancy was significantly correlated with monobenzyl phthalate (mBzP) (p = .041) and a trend was found for miBP (p = .066). The use of makeup products during pregnancy was strongly associated with miBP (p = .015) and the use of deodorant during pregnancy was inversely associated with mEHP (p = .021). Strong associations came up between mEHP and lower birth weight (Spearman correlation coefficient, r = -0.302, p = .021) and exposure to BPS was associated with increased body mass index of the participants (p = .036). Although data in literature on biomonitoring of the compounds in hair are limited, the findings of this study are promising and in agreement with existing data in hair or urine.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135651DOI Listing
April 2020

Discovery of potent telomerase activators: Unfolding new therapeutic and anti-aging perspectives.

Mol Med Rep 2019 Oct 23;20(4):3701-3708. Epub 2019 Aug 23.

Department of Clinical Pharmacy, University of Medicine and Pharmacy, Faculty of Pharmacy, 200349 Craiova, Romania.

Telomere length, a marker of cellular aging, decreases with age and it has been associated with aging‑related diseases. Environmental factors, including diet and lifestyle factors, affect the rate of telomere shortening which can be reversed by telomerase. Telomerase activation by natural molecules has been suggested to be an anti‑aging modulator that can play a role in the treatment of aging‑related diseases. This study aimed to investigate the effect of natural compounds on telomerase activity in human peripheral blood mononuclear cells (PBMCs). The tested compounds included Centella asiatica extract formulation (08AGTLF), Astragalus extract formulation (Nutrient 4), TA‑65 (containing Astragalus membranaceus extract), oleanolic acid (OA), maslinic acid (MA), and 3 multi‑nutrient formulas (Nutrients 1, 2 and 3) at various concentrations. The mean absorbance values of telomerase activity measured following treatment with some of the above‑mentioned formulations were statistically significantly higher compared to those of the untreated cells. In particular, in order of importance with respect to telomerase activation from highest to lowest, 08AGTLF, OA, Nutrient 4, TA‑65, MA, Nutrient 3 and Nutrient 2, triggered statistically significant increase in telomerase activity compared to the untreated cells. 08AGTLF reached the highest levels of telomerase activity reported to date, at least to our knowledge, increasing telomerase activity by 8.8 folds compared to untreated cells, while Nutrient 4 and OA were also potent activators (4.3‑fold and 5.9‑fold increase, respectively). On the whole, this study indicates that the synergistic effect of nutrients and natural compounds can activate telomerase and produce more potent formulations. Human clinical studies using these formulations are required to evaluate their mode of action. This would reveal the health benefits of telomerase activation through natural molecules and would shed new light onto the treatment of aging‑related diseases.
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http://dx.doi.org/10.3892/mmr.2019.10614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755196PMC
October 2019

Association of nutraceutical supplements with longer telomere length.

Int J Mol Med 2019 Jul 10;44(1):218-226. Epub 2019 May 10.

Department of Clinical Pharmacy, University of Medicine and Pharmacy, Faculty of Pharmacy, Craiova 200349, Romania.

Telomeres are nucleotide tandem repeats located at the tip of eukaryotic chromosomes that maintain genomic integrity. The gradual shortening of telomeres leads to cellular senescence and apoptosis, a key mechanism of aging and age‑related chronic diseases. Epigenetic factors, such as nutrition, exercise and tobacco can affect the rate at which telomeres shorten and can modify the risk of developing chronic diseases. In this study, we evaluated the effects of a combination of nutraceutical supplements (NS) on telomere length (TL) in healthy volunteers with no medical history of any disease. Participants (n=47) were selected from healthy outpatients visiting a private clinic and were divided into the experimental group (n=16), that received the NS and the control group (n=31). We estimated the length of single telomeres in metaphase spread leukocytes, isolated from peripheral blood, using quantitative‑fluorescent in situ hybridization (Q‑FISH) analysis. The length of the whole telomere genome was significantly increased (P<0.05) for the mean, 1st quartile and median measurements in the experimental group. Similar findings were observed for short TL (20th percentile) (P<0.05) for the median and 3rd quartile measurements in the NS group, compared to the control group. The beneficial effects of the supplements on the length of short telomeres remained significant (P<0.05) following adjustment for age and sex. Telomeres were moderately longer in female patients compared to the male patients. On the whole, the findings of this study suggest that the administration of NS may be linked to sustaining the TL.
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http://dx.doi.org/10.3892/ijmm.2019.4191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559326PMC
July 2019

Developing BIOTEL: A Semi-Automated Spreadsheet for Estimating Telomere Length and Biological Age.

Front Genet 2019 19;10:84. Epub 2019 Feb 19.

Laboratory of Toxicology, Medical School, University of Crete, Heraklion, Greece.

Telomere length (TL) is causally related to aging and several age-related diseases. Specifically, the abundance of short telomeres and the rate of telomere shortening are strong determinants of cell homeostasis. Thus, tools for analyzing and manipulating TL data can vastly improve research focused on aging. Aim: In this study, we developed a semi-automated worksheet, BIOTEL, to generate individual and group TL statistics and provide a crude estimation of biological age. Data from the Telomere Length Database Project (TLDP) were implemented to the spreadsheet to produce TL statistics. 150 participants were included, and their age was from 21 to 82 years, and the sex distribution ratio was 52.3%: 47.7% (male: female). Initially, we analyzed the fluorescence intensities of telomeres that were measured on metaphase spread leukocytes using three-dimensional (3D) quantitative-fluorescent hybridization (Q-FISH) procedures (3D DNA FISH) with a (C3TA2)3 peptide nucleic acid (PNA) probe. Raw data of fluorescence intensities, demographic data and medical records from the participants were imported into the worksheet. Basic statistical analyses of TL data were provided through BIOTEL, including TL percentiles, specialized charts for TL distribution including the percentage of critically short telomeres (< 3,000 kilobases), individual telomere profiles, and graphs of biological age vs. chronological age. BIOTEL ver. 2.4 is a functional semi-automated worksheet that calculates a wide range of TL statistics, thus a useful tool with applications in research of telomeres and biological age estimation.
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http://dx.doi.org/10.3389/fgene.2019.00084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389611PMC
February 2019

Application of metabolomics part II: Focus on fatty acids and their metabolites in healthy adults.

Int J Mol Med 2019 Jan 14;43(1):233-242. Epub 2018 Nov 14.

Laboratory of Toxicology and Forensic Sciences, Medical School, University of Crete, 71003 Heraklion, Greece.

Fatty acids (FAs) play critical roles in health and disease. The detection of FA imbalances through metabolomics can provide an overview of an individual's health status, particularly as regards chronic inflammatory disorders. In this study, we aimed to establish sensitive reference value ranges for targeted plasma FAs in a well‑defined population of healthy adults. Plasma samples were collected from 159 participants admitted as outpatients. A total of 24 FAs were analyzed using gas chromatography‑mass spectrometry, and physiological values and 95% reference intervals were calculated using an approximate method of analysis. The differences among the age groups for the relative levels of stearic acid (P=0.005), the omega‑6/omega‑3 ratio (P=0.027), the arachidonic acid/eicosapentaenoic acid ratio (P<0.001) and the linoleic acid‑produced dihomo‑gamma‑linolenic acid (P=0.046) were statistically significant. The majority of relative FA levels were higher in males than in females. The levels of myristic acid (P=0.0170) and docosahexaenoic acid (P=0.033) were significantly different between the sexes. The reference values for the FAs examined in this study represent a baseline for further studies examining the reproducibility of this methodology and sensitivities for nutrient deficiency detection and investigating the biochemical background of pathological conditions. The application of these values to clinical practice will allow for the discrimination between health and disease and contribute to early prevention and treatment.
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http://dx.doi.org/10.3892/ijmm.2018.3989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6257830PMC
January 2019

The metabolism of imidacloprid by aldehyde oxidase contributes to its clastogenic effect in New Zealand rabbits.

Mutat Res Genet Toxicol Environ Mutagen 2018 May - Jun;829-830:26-32. Epub 2018 Mar 19.

Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece. Electronic address:

Imidacloprid (IMI) is a systemic, chloro-nicotinyl insecticide classified in Regulation N° 1272/2008 of the European Commision as "harmful if swallowed and very toxic to aquatic life, with long-lasting effects". IMI is metabolized in vitro both by aldehyde oxidase (AOX) (reduction) and by cytochrome P450s enzymes (CYPs). In the present study, the AOX inhibitor sodium tungstate dihydrate (ST) was used to elucidate the relative contribution of CYP 450 and AOX metabolic pathways on IMI metabolism, in male rabbits exposed to IMI for two months. To evaluate the inhibition effectiveness, various metabolite concentrations in the IMI and IMI + ST exposed groups were monitored. DNA damage was also evaluated in micronucleus (MN) and single cell electrophoresis (SCGC) assays in both groups, along with oxidative stress (OS) with the inflammatory status of the exposed animals, in order to clarify which metabolic pathway is more detrimental in this experimental setting. A significant increase in the frequency of binucleated cells with MN (BNMN, 105%) and micronuclei (MN, 142%) was observed after exposure to IMI (p < 0.001). The increase in the ST co-exposed animals was less pronounced (BNMN 75%, MN 95%). The Cytokinesis Block Proliferation Index (CBPI) showed no significant difference between controls and exposed animals at any time of exposure (p > 0.05), which indicates no cytotoxic effect. Similarly, comet results show that the IMI group exhibited the highest achieved tail intensity, which reached 70.7% over the control groups, whereas in the IMI + ST groups the increase remained at 48.5%. No differences were observed between all groups for oxidative-stress biomarkers. The results indicate that the AOX metabolic pathway plays a more important role in the systemic toxicity of IMI.
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http://dx.doi.org/10.1016/j.mrgentox.2018.03.002DOI Listing
June 2019

Serum IGF-1, IGFBP-3 levels and circulating tumor cells (CTCs) in early breast cancer patients.

Growth Horm IGF Res 2017 04 16;33:28-34. Epub 2017 Feb 16.

Department of Surgical Oncology, University Hospital of Heraklion, Crete, Greece.

Objective: Insulin-like growth factor (IGF)-axis is involved in human oncogenesis and metastasis development for various solid tumors including breast cancer. Aim of this study was to assess the association between IGF-1, IGF-binding protein-3 (IGFBP-3) serum levels and the presence of circulating tumor cells (CTCs) in the peripheral blood of women diagnosed with early breast cancer (EBC), before and after adjuvant chemotherapy.

Design: 171 patients with early-stage breast adenocarcinomas were retrospectively evaluated. Immunoradiometric (IRMA) assays were employed for the in-vitro determination of IGF-1 and IGFBP-3 serum levels in blood samples collected after surgical treatment and before initiation of adjuvant chemotherapy. CTCs' presence was assessed through detection of cytokeratin-19 (CK-19) mRNA transcripts using quantitative real time reverse transcription polymerase chain reaction (RT-PCR). IGF-1, IGFBP-3 serum levels were correlated with CTCs' presence before and after adjuvant chemotherapy as well as with tumor characteristics including tumor size, axillary lymph node status, oestrogen (ER)/progestorene (PR) and human epidermural growth factor receptor 2 (HER2) receptor status. Log-rank test was applied to investigate possible association between IGF-1, IGFBP-3 serum levels and disease-free interval (DFI) and overall survival (OS).

Results: Before initiation of adjuvant therapy IGF-1, IGFBP-3 serum levels were moderately associated (Spearman's rho=0.361, p<0.001) with each other, while presenting significant differences across age groups (all p values<0.05). IGF-1 serum levels did not correlate with the presence of CTCs before initiation (p=0.558) or after completion (p=0.474) of adjuvant chemotherapy. Similarly, IGFBP-3 serum levels did not show significant association with detectable CTCs either before (p=0.487) or after (p=0.134) completion of adjuvant chemotherapy. There was no statistically significant association between the clinical outcome of patients in terms of DFI, OS and IGF-1(DFI: p=0.499; OS: p=0.220) or IGFBP-3 (DFI: p=0.900; OS: p=0.406) serum levels.

Conclusions: IGF-1 and IGFBP-3 serum levels before initiation of adjuvant chemotherapy are not indicative of CTCs' presence in the blood and do not correlate with clinical outcome of women with early-stage breast cancer.
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http://dx.doi.org/10.1016/j.ghir.2017.02.001DOI Listing
April 2017

Comparative Evaluation of Drug Deposition in Hair Samples Collected from Different Anatomical Body Sites.

J Anal Toxicol 2017 Apr;41(3):214-223

Laboratory of Toxicology, Medical School, University of Crete, Heraklion, 71003 Crete, Greece.

In this study, we focused on the validation of a method for the simultaneous detection and quantification of cannabinoids, cocaine and opiates in hair as well as on the distribution of the drugs deposition in hair collected from different anatomical body sites. The proposed analytical procedure was validated for various parameters such as selectivity, linearity, limit of quantification, precision, accuracy, matrix effect and recovery. Four hundred and eighty-one samples were collected during 2010-2015 from 231 drug abusers. A 6-h ultrasonic-assisted methanolic extraction was applied for the isolation of the drugs. The analysis was performed in an liquid chromatography-mass spectrometry system for the opiates and cocaine and in a gas chromatography-mass spectrometry system for the cannabinoids. Cocaine was the most frequent detected drug (68.8-80.5%) followed by cannabinoids (47.6-63.3%) and opiates (34.7-46.7%) depending on the body site that the samples were collected. The mean concentrations of Δ9-tetrahydrocannabinol (THC) were 0.63 ± 2.11 for head, 0.54 ± 1.03 for pubic, 0.34 ± 0.51 for axillary and 0.18 ± 0.18 ng/mg for chest hair samples. The values of cocaine were 6.52 ± 15.98, 4.64 ± 10.77, 6.96 ± 38.21 and 3.94 ± 6.35 ng/mg, while the values of 6-monoacetylmorphine (MAM) were 3.33 ± 5.89, 3.06 ± 9.33, 1.37 ± 1.37 and 16.4 ± 1.77 ng/mg for head, pubic, axillary and chest samples, respectively. Differences between the detected concentrations of cocaine and opiates between the hair samples of different anatomical sites, as well as the ratio of drug metabolites to the parent compounds were observed in some cases. Statistically significant differences in the mean detected levels were noticed for morphine and heroin between head and pubic hair and also for cocaine and benzoylecgonine, between head and axillary hair samples. Moreover, the ratio of MAM to morphine and THC to cannabinol seems to correlate statistically with the total opiate or cannabinoid detected concentrations. The above differences could be attributed to several parameters associated with the structure, morphology, growth rate and other characteristics of the collected hair.
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http://dx.doi.org/10.1093/jat/bkw127DOI Listing
April 2017

Long-term exposure to cypermethrin and piperonyl butoxide cause liver and kidney inflammation and induce genotoxicity in New Zealand white male rabbits.

Food Chem Toxicol 2016 Aug 16;94:250-9. Epub 2016 Jun 16.

Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece. Electronic address:

Cypermethrin (CY) is a frequently used class II pyrethroid pesticide, while piperonyl butoxide (PBO) plays a major role in the pesticide formulation of synthetic pyrethroids. Synthetic pyrethroids are metabolized in mammals via oxidation and ester hydrolysis. PBO can prevent the metabolism of CY and enhances its pesticide effect. While this potentiation effect reduces the amount of pesticide required to eliminate insects, it is not clear how this mixture affects mammals. In our in vivo experiment, New Zealand white male rabbits were exposed to low and high doses of CY, PBO, and their combinations, for 4 months. Genotoxicity and cytotoxicity were monitored by measuring binucleated cells with micronuclei (BNMN), micronuclei (MN) and the cytokinesis block proliferation index (CBPI) in lymphocytes. After two months of exposure, a statistically significant increase in the frequency of BNMN was observed for all exposed animals (p < 0.001) in a dose-dependent way. MN were significantly elevated compared to controls (p < 0.001), with high dose groups reaching a 442% increase when co-exposed. BNMN and MN continued to increase after four months. Histopathological examination of lesions showed damage involving inflammation, attaining lymphoplasmatocytic infiltration in the high dose groups. Both CY and PBO cause liver and kidney inflammation and induce genotoxicity.
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http://dx.doi.org/10.1016/j.fct.2016.06.016DOI Listing
August 2016

Long-term exposure of rabbits to imidaclorpid as quantified in blood induces genotoxic effect.

Chemosphere 2016 Apr 5;149:108-13. Epub 2016 Feb 5.

Center of Toxicology Science & Research, Division of Morphology, Medical School, University of Crete, Voutes Campus, Heraklion, 71003, Crete, Greece. Electronic address:

The present in-vivo study focuses on the genotoxic effect of the neonicotinoid pesticide imidacloprid (IMI) in rabbits. The purpose of the study was to establish a possible relationship between exposure to the pesticide (dose and duration) and genotoxicity. Furthermore, an analytical method for the simultaneous determination of IMI and its major metabolite 6-chloronicotinic acid (6-ClNA) in blood was developed and validated. The isolation of the two analytes from blood was performed by liquid-liquid extraction with dichloromethane. Analysis was performed by Liquid Chromatography - Atmospheric Pressure Chemical Ionization - Mass Spectrometry (LC-APCI-MS). The method was applied on the determination of IMI and 6-ClNA in serum samples obtained from rabbits fed with the insecticide at two low doses. Furthermore, parameters of genotoxicity and cytotoxicity were evaluated by measuring binucleated cells with micronuclei (BNMN), micronuclei (MN) and the Cytokinesis Block Proliferation Index (CBPI), in lymphocytes of exposed rabbits. The results revealed a genotoxic effect of IMI for both exposed groups. There were statistically significant differences in the frequencies of BNMN and MN between control and exposed groups but there was no dose-dependence, neither time-dependence of the genotoxic effect for the administered doses. This is the first time that long term exposure to IMI in rabbits was studied for the determination of its genotoxic effect. The genotoxic effect of IMI as it is depicted by the current study is in accordance with previous studies.
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http://dx.doi.org/10.1016/j.chemosphere.2016.01.040DOI Listing
April 2016

Downgrading the systemic condition of rabbits after long term exposure to cypermethrin and piperonyl butoxide.

Life Sci 2016 Jan 12;145:114-20. Epub 2015 Dec 12.

Laboratory of Toxicology, Medical School, University of Crete, Voutes, 71409 Heraklion, Crete, Greece. Electronic address:

Aim: The aimof this study is to clarify the effect of cypermethrin (CY) on the oxidative stress (OS) and inflammation status of animals exposed to it and the synergistic role of piperonyl butoxide (PB0).

Main Methods: Markers of oxidative stress, such as total antioxidant activity (TAC), protein carbonyls, hemoglobin (Hb), reduced glutathione (GSH), thiobarbituric-acid reactive substances (TBARS), along with the telomerase activity in PBMCs (peripheral blood mononuclear cells) were analyzed.

Key Findings: Oxidative stress markers showed statistically significant differences between groups in TAC (p b 0.001), GSH (p = 0.018) and CAT activity (p = 0.029), which depended on dose and combined effect of both compounds. Telomerase activity also showed a statistically significant difference between all groups (F = 43.48, df=6, 14, p b 0.001)with cypermethrin, piperonyl butoxide and the co-exposed groups being significantly different fromthe control group (p b 0.05). Significance: The observed results for TBARS, GSH, Hb, TAC, Crbnls and CAT from our exposed groups showed altered levels compared to control groups that could be linked to doses and combined effects of each chemical substance (cypermethrin and piperonyl butoxide). Oxidative stress markers suggest that cypermethrin, piperonyl butoxide and the co-exposed groups, induce oxidative stress as well as induction of telomerase activity.
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http://dx.doi.org/10.1016/j.lfs.2015.12.026DOI Listing
January 2016

Multicomponent analysis of replacement liquids of electronic cigarettes using chromatographic techniques.

J Anal Toxicol 2015 May 13;39(4):262-9. Epub 2015 Feb 13.

Center of Toxicology Science & Research, Division of Morphology, Medical School, University of Crete, Voutes Campus, Heraklion 71003, Crete, Greece

The electronic cigarette (e-cig) is an invention of the past few years and its popularity is rapidly growing all over the world. A rapid multicomponent analytical protocol for the analysis of the replacement liquids (e-liquids) of e-cig was developed using gas (GC) and liquid chromatography (LC)-mass spectrometry (MS). GC-MS-based methods were developed for the determination of the main humectants and polycyclic aromatic hydrocarbons (PAHs). For the determination and quantification of nicotine (NIC) and nitrosamines, appropriate LC-MS-based methods were developed. The approbated methods were applied for the analysis of 263 e-liquid samples obtained from the Greek market. The instruments response was linear; the limits of quantification ranged from 0.003 μg/mL for three PAHs to 1.187 μg/mL for glycerol. The precision was <16% for all analytes, while the mean accuracy ranged from 99.1% for NIC to 106.6% for the flavor 2,5-dimethylpyrazine. The measured concentrations of NIC were correlated with the theoretical concentrations as reported by the manufacturers. An analog relation between the concentration of the glycerol and of propylene glycol was noticed. The frequency of detection of flavors ranged from 30.4% for the methyl cyclopentenolone to 5.3% for 3.4-dimethoxybenzaldehyde. Nitrosamines and PAHs were not detected in any sample. Because a similar analytical protocol was not available from the existing literature so far, our study offers the advantage of complete analytical methods for rapid and simultaneous multicomponent identification.
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http://dx.doi.org/10.1093/jat/bkv002DOI Listing
May 2015

Risk assessment for children exposed to DDT residues in various milk types from the Greek market.

Food Chem Toxicol 2015 Jan 20;75:156-65. Epub 2014 Nov 20.

Laboratory of Toxicology, Department of Medicine, University of Crete, Voutes 71003, Heraklion, Greece. Electronic address:

The occurrence of residues of DDT and its metabolites was monitored in 196 cow milk samples of various pasteurized commercial types collected from the Greek market. Residue levels were determined by GC-MS analysis. In 97.4% of the samples at least one DDT isomer or one of the DDT metabolites was detected, in levels not exceeding the maximum permitted residue level by the EU. Hazard Index for both carcinogenic and non-carcinogenic effects was estimated under two assumptions: a) using DDT concentrations from positive samples and b) imputing LOD/2 as an arbitrary concentration for negative samples. No statistically significant differences in detected or summed residue (p > 0.05) concentrations between different milk types were observed, with the exception of specific metabolites of DDT in some milk types. Exposure assessment scenarios were developed for children aged 1, 3, 5, 7 and 12 years old based on estimated body weights and daily milk consumption. Hazard Indices for non-carcinogenic effects were below 0.109 covering also carcinogenic effects according to WHO approach. The cancer risk values for carcinogenic effects according to the US EPA Cancer Benchmark Concentration approach, ranged from 0.4 to 18. For both effects the highest values were calculated for the 1- to 3-year-old age groups.
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http://dx.doi.org/10.1016/j.fct.2014.11.012DOI Listing
January 2015

Biomonitoring of dialkylphosphate metabolites (DAPs) in urine and hair samples of sprayers and rural residents of Crete, Greece.

Environ Res 2014 Oct 28;134:181-7. Epub 2014 Aug 28.

Center of Toxicology Science & Research, Medical School, University of Crete, Heraklion 71003, Greece. Electronic address:

Purpose: The aim of this study was to evaluate the exposure of rural residents (control group) and occupational exposed population group of sprayers to organophosphorus pesticides (OPs) by measuring their non-specific dialkylphosphate metabolites (DAPs) in hair and in urine samples. All subjects (n=120) were residents of the municipality of Ierapetra, an area of intensive cultivation in Crete, Greece.

Methods: The determined OPs metabolites were DMP, DEP, DETP and DEDTP. Two different approaches were used for the analysis of the collected samples; solid-liquid extraction with sonication for hair and liquid-liquid extraction for urine. Gas chromatography-mass spectrometry (GC-MS) analysis was performed after derivatization of the isolated analytes.

Results And Discussion: The detection rates of DMP, DEP and DETP for both control and sprayers groups were high in both matrices, ranging from 91% to 100%. DEDTP was detected only in 9% of sprayers hair samples, while its detection rates in urine samples ranged from 83% to 90% for both population groups. Data analysis revealed significantly higher sumDAPs levels in urine of sprayers than in the urine of control group (p<0.001) and this is justified since sampling occurred during spraying periods. SumDAPs levels in hair samples of the sprayers were also significantly higher than in the hair of control group (p<0.001), confirming the long-term exposure to OPs. SumDAPs found levels in urine and hair samples of subjects were significantly correlated (Spearman׳s rho=0.728, p<0.001). Our study confirmed the elevated levels of DAPs in hair and urine samples in occupationally exposed group of sprayers in comparison to control group, even detected levels were similar in logarithmic scale.
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http://dx.doi.org/10.1016/j.envres.2014.07.012DOI Listing
October 2014

Development and application of GC-MS method for monitoring of long-term exposure to the pesticide cypermethrin.

Drug Test Anal 2014 Jun;6 Suppl 1:9-16

Center of Toxicology Science & Research, Medical School, University of Crete, Heraklion, Crete, 71409, Greece.

Cypermethrin (CPMN) is a synthetic pyrethroid used as an insecticide in large-scale commercial agricultural applications as well as for domestic purposes. In the present study a gas chromatography-mass spectrometry (GC-MS) based method was developed and validated for the quantitation of CPMN metabolites, 3-phenoxybenzoic acid (3-PBA) and cis- and trans- 3-(2,2-dichlorovinyl)-2,2-dimethyl-1-cyclopropane (cis- and trans- Cl2 CA). The developed method was applied for the monitoring of CPMN metabolites in hair of laboratory animals (rabbits) intentionally exposed per os to CPMN at 40 (low dose) and 80 (high dose) mg/kg weight/day for 16 weeks. The analytical method comprises three main steps: isolation of analytes from hair, analytes derivatization, and subsequent instrumental analysis by GC-MS. The limits of detection ensured by the method are 4.0, 3.9 and 1.0 pg/mg hair for cis-Cl2 CA, trans-Cl2 CA and 3-PBA, respectively. The instrument responce is linear (r(2)  > 0.99) in the investigated concentrations range from 25 to 1000 pg/mg. With and between-run precision as well as accuracy were estimated and found satisfactory. Analytes were efficiently isolated by solid-liquid extraction from hair with recoveries greater than 84.8% for cis-Cl2 CA, 87.2% for trans-Cl2 CA and 96.4% for 3-PBA. Rabbit's hair showed increasing levels for all metabolites (metabolites accumulation in a time and dose dependent manner) over time and in a dose-dependent manner. The developed experimental procedure could be used for biomonitoring of population exposure to CPMN.
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http://dx.doi.org/10.1002/dta.1601DOI Listing
June 2014

Social capital and adherence to cervical and breast cancer screening guidelines: a cross-sectional study in rural Crete.

Health Soc Care Community 2014 Jul 23;22(4):395-404. Epub 2014 Jan 23.

Department of Medicine, Faculty of Social Medicine, University of Crete, Heraklion, Greece.

Breast and cervical cancers are among the leading causes of female mortality. The reasons that make women adhere, or not, to screening guidelines are not only related to individual and health characteristics but are also placed in a wider social and cultural context. Social capital might facilitate the dissemination of relevant knowledge of and the adherence to cancer screening guidelines. This cross-sectional study explored the associations of individual-level social capital with breast and cervical cancer screening and the knowledge for the existence of relevant screening tests (Pap test and mammography) in the municipality of Gorgolaini, a rural area in Crete, Greece. A random sample of 131 of the 592 women of the 2001 electoral register were invited to participate in the study and 125 completed the Social Capital Questionnaire and two questions on self-reported health knowledge and behaviour (participation rate 95.4%). Women were eligible to participate if they were aged 35-75, had lived in the area for the last 10 years and were of Greek origin. Multiple logistic regressions were performed to establish associations among each social capital factor (total, participation in the community, value of life, tolerance for diversity, feelings of safety, family/friends connections) and knowledge of and adherence to breast and cervical cancer screening guidelines after adjustment for confounders. Our results suggest that early detection of breast and cervical cancers may be facilitated when taking into account the social context of the population.
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http://dx.doi.org/10.1111/hsc.12096DOI Listing
July 2014

Hypospadias in offspring is associated with chronic exposure of parents to organophosphate and organochlorine pesticides.

Toxicol Lett 2014 Oct 25;230(2):139-45. Epub 2013 Oct 25.

Department of Toxicology, Medical School, University of Crete, Heraklion, Greece. Electronic address:

We have currently evaluated the possible association between hypospadias and exposure to organophosphorus (OP) and organochlorine (OC) pesticides. For this purpose, we measured the dialkyl phosphate metabolites of organophosphate pesticides (DAPs) in the hair and blood, as well as OC pesticides (DDTs, HCHs) in the hair collected from children with hypospadias and their parents. The concentration of HCHs in the hair samples obtained from mothers was higher than that previously reported for people working in open cultivations, while the concentration of DDTs in the hair samples obtained from mothers, fathers and their children with hypospadias was much higher than that previously reported for occupationally exposed individuals. The DMP concentration in hair samples obtained from mothers was much higher not only from that reported for the general population, but even higher than that reported for occupationally exposed individuals. Furthermore, SUMDEPs and SUMDAPs in the hair samples obtained both from the hypospadiac boys, as well as from their parents were higher than the corresponding values previously reported for the general population. Our study supports the hypothesis that organophosphate and organochlorine pesticide exposure may be a potential risk factor for hypospadias.
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http://dx.doi.org/10.1016/j.toxlet.2013.10.015DOI Listing
October 2014

Risk assessment scenarios of children's exposure to aflatoxin M1 residues in different milk types from the Greek market.

Food Chem Toxicol 2013 Jun 21;56:261-5. Epub 2013 Feb 21.

TEI of Western Macedonia, Florina Branch, Department of Agricultural Products Management and Quality Control, Terma Kontopoulou, 53100 Florina, Greece.

Occurrence of aflatoxin M1 (AFM1) was determined in 196 milk samples (conventional, organic and kids milk) from the Greek market during November 2009 to June 2010. AFM1 content was analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA) commercial kit. Aflatoxin M1 was detected in 46.5% of the samples. 46.5% of the samples were found positive for AFM1. The most frequent range of detection was between 5 and 10 ng/l. Based on the EU regulation only 2 milk samples presented AFM1 levels higher than the maximum residue limits. Two different scenarios were used for the determination of hazard index: (a) scenario 1 using only positive (detected AFM1) samples and (b) scenario 2 when missing values where imputed with Limit of Detection (LOD) divided by 2. Significant statistical differences between different milk categories were presented only when the results were imputed with LOD/2 values. Exposure assessment scenarios were developed for ages 1, 3, 5, 7 and 12 and their respective estimated weights and daily milk consumption. Under the worst-case scenario all milk types presented a Hazard Index (HI) less than one. The highest HI values appear in the ages of 1-3.
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http://dx.doi.org/10.1016/j.fct.2013.02.024DOI Listing
June 2013

Diethyl phosphates accumulation in rabbits' hair as an indicator of long term exposure to diazinon and chlorpyrifos.

Forensic Sci Int 2012 May 22;218(1-3):106-10. Epub 2011 Oct 22.

Centre of Toxicological Sciences and Research Medical School, University of Crete, Voutes, Heraklion, 71409 Crete, Greece.

Long term exposure to organophosphate pesticides can be evaluated by quantitative analysis of their non-specific metabolites in hair matrix. The aim of this study was to determine whether these metabolites can be internally incorporated into the hair of rabbits exposed to diazinon and chlorpyrifos. The influence of dose and dose duration of each pesticide dosage were investigated. Three groups of rabbits were exposed to different dosages of diazinon (3.0 and 6.0mg/kg/day) and chlorpyrifos (18.0mg/kg/day) via drinking water. Hair samples were collected every month and analyzed for diethyl phosphate (DEP) and diethyl thiophosphate (DETP) by gas chromatography-mass spectrometry (GC-MS). The mean concentrations of the low-dose treated group, ranged from 112 to 257pg/mg for DEP and from 295 to 515pg/mg for DETP in hair. The high-dose treated group demonstrated a range of mean concentrations from 142 to 585pg/mg for DEP and from 406 to 988pg/mg for DETP in hair. For the chlorpyrifos treated group, the concentrations ranged from 138 to 1070 for DEP and from 554 to 886pg/mg for DETP. Analysis revealed the incorporation of these metabolites into the rabbit hair in a dosage and dose duration-dependent manner. These data confirms the ability of using hair analysis for diethyl phosphates to assess long-term OP exposure.
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http://dx.doi.org/10.1016/j.forsciint.2011.10.017DOI Listing
May 2012

Assessment of PCBs exposure in human hair using double focusing high resolution mass spectrometry and single quadrupole mass spectrometry.

Toxicol Lett 2012 Apr 22;210(2):225-31. Epub 2011 Aug 22.

Center of Toxicology Science and Research, Division of Morphology, Medical School, University of Crete, 71003 Heraklion, PO Box 1393, Crete, Greece.

Polychlorinated biphenyls (PCBs) levels were assessed in human hair samples, originating from two main agricultural regions of Greece. The analysis was performed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-double focusing high resolution mass spectrometry (GC-DFHRMS). The main analytical procedure involved hair decontamination, solid-liquid extraction and cleanup steps. The recoveries of PCBs ranged from 71.2% to 101.6%, with accuracies greater than 87.5% and the between-run precisions (%RSD) lower than 25% for all analytes. Differences in the frequencies of detection and the median values of PCBs were detected between the examined regions and between the applied analytical techniques. All Peloponnesus' hair examined samples were found positive for each examined PCB, while the percentage of the total positive samples ranged from 86.1% (for PCB 138) to 94.4% (for PCB 28 and 153 congeners) using GC-DFHRMS. The Cretan hair samples were less contaminated (SUM PCBs=0.61 and 1.47pg/mg) unlike the Peloponnesus' samples (SUM PCBs=24.68 and 38.74pg/mg) measured by GC-DFHRMS and GC-MS, respectively. PCBs with high chlorination gave lower concentration values compared to low chlorination PCBs in both populations. No significant differences were observed between women and men. The GC-DFHRMS technique provided higher percentage of positive samples and low PCBs median values, due to higher sensitivity and interferences from isobaric ions in the GC-MS technique and is therefore considered as a powerful tool for such assessments in hair specimens.
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http://dx.doi.org/10.1016/j.toxlet.2011.07.031DOI Listing
April 2012

Studying the association between musculoskeletal disorders, quality of life and mental health. A primary care pilot study in rural Crete, Greece.

BMC Musculoskelet Disord 2009 Nov 20;10:143. Epub 2009 Nov 20.

Clinic of Social and Family Medicine, Department of Social Medicine, School of Medicine, University of Crete, 74100 Heraklion, Crete, Greece.

Background: The burden of musculoskeletal disorders (MSD) on the general health and well-being of the population has been documented in various studies. The objective of this study was to explore the association between MSD and the quality of life and mental health of patients and to discuss issues concerning care seeking patterns in rural Greece.

Methods: Patients registered at one rural Primary Care Centre (PCC) in Crete were invited to complete the Nordic Musculoskeletal Questionnaire (NMQ) for the analysis of musculoskeletal symptoms, together with validated instruments for measuring health related quality of life (SF-36) and mental distress (GHQ-28).

Results: The prevalence rate of MSD was found to be 71.2%, with low back and knee pain being the most common symptoms. Most conditions significantly impaired the quality of life, especially the physical dimensions of SF-36. Depression was strongly correlated to most MSD (p < 0.001). Multiple logistic analyses revealed that patients who consulted the PCC due to MSD were likely to have more mental distress or impaired physical functioning compared to those who did not.

Conclusion: Musculoskeletal disorders were common in patients attending the rural PCC of this study and were associated with a poor quality of life and mental distress that affected their consultation behaviour.
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http://dx.doi.org/10.1186/1471-2474-10-143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785760PMC
November 2009

Long-term complication rates and survival of peritoneal dialysis catheters: the role of percutaneous versus surgical placement.

Semin Dial 2009 Sep-Oct;22(5):569-75. Epub 2009 Sep 11.

Department of Nephrology, Heraklion University Hospital, Crete, Greece.

Considerable controversy currently exists in the literature concerning the mode of catheter placement and its impact on the technical success of peritoneal dialysis (PD). We decided to compare the impact of the surgical versus the percutaneous insertion technique on peritoneal dialysis catheter (PDCs) complications and survival. Our study population comprised 152 patients in whom 170 PDCs were inserted between January 1990 and December 2007 at the main PD unit on the island of Crete. Eighty four catheters were surgically placed (S group) and 86 were placed percutaneously by nephrologists (N group). The total experience accumulated was 4997 patient-months. The overall complications did not differ between the two groups. Only early leakage was more frequent in N group than S group (10.3 versus 1.9 episodes per 1000 patient-months; p < 0.001). However, it was easily treated and did not constitute a cause of early catheter removal. Catheter survival was 91.1%, 80.7%, and 73.2%, in the S group versus 89.5%, 83.7%, and 83.7% for the N group at 1, 2, and 3 years, respectively (p = 0.2). Catheter survival has significantly increased over the last decade. Factors positively affecting PDC survival appeared to be the use of mupirocin for exit site care and the utilization of the coiled type of catheter, practices implemented mainly after 1999. Peritonitis-free survival and patient survival were not associated with the mode of placement, while in Cox regression analysis, were longer in patients treated with automated PD. The placement mode did not affect PD outcomes. Percutaneous implantation proved a safe, simple, low cost, immediately available method for PDC placement and helped to expand our PD program.
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http://dx.doi.org/10.1111/j.1525-139X.2009.00621.xDOI Listing
March 2010

Risk factors for ischaemic heart disease in a Cretan rural population: a twelve year follow-up study.

BMC Public Health 2007 Dec 18;7:351. Epub 2007 Dec 18.

Clinic of Social and Family Medicine, School of Medicine, University of Crete, Greece.

Background: Crete has been of great epidemiological interest ever since the publication of the Seven Countries Study. In 1988 a well-defined area of rural Crete was studied, with only scarce signs of coronary heart disease (CHD) despite the unfavorable risk profile. The same population was re-examined twelve years later aiming to describe the trends of CHD risk factors over time and discuss some key points on the natural course of coronary heart disease in a rural population of Crete.

Methods And Results: We re-examined 200 subjects (80.7% of those still living in the area, 62.4 +/- 17.0 years old). The prevalence of risk factors for CHD was high with 65.9% of men and 65.1% of women being hypertensive, 14.3% of men and 16.5% of women being diabetic, 44% of men being active smokers and more than 40% of both sexes having hyperlipidaemia. Accordingly, 77.5% of the population had a calculated Framingham Risk Score (FRS) > or = 15%, significantly higher compared to baseline (p < 0.001). The overall occurrence rate for CHD events was calculated at 7.1 per 1000 person-years (95% confidence interval: 6.8-7.3).

Conclusion: The study confirms the unfavorable risk factor profile of a well defined rural population in Crete. Its actual effect on the observed incidence of coronary events in Cretans remains yet to be defined.
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http://dx.doi.org/10.1186/1471-2458-7-351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234417PMC
December 2007

Pupil miosis within 5 minutes in darkness is a valid and sensitive quantitative measure of alertness: application in daytime sleepiness associated with sleep apnea.

Sleep 2006 Nov;29(11):1482-8

Dept of Psychiatry & Behavioural Sciences, Medical School PO BOX 2208, University of Crete, Heraklion 71003, Crete, Greece.

Study Objectives: The regulation of arousal and pupillary functions may be intimately linked via activity in the nucleus locus coeruleus. In this preliminary study, we tested the validity of the gradual pupillary miosis during 5 minutes in darkness, as a quantitative physiologic index of the arousal state of the brain.

Design: Cross-sectional assessment of 2 groups with between-group comparison and correlational analyses within the patient group.

Participants: Eleven unmedicated male patients recently diagnosed with obstructive sleep apnea (OSA) with no comorbid conditions who had undergone polysomnography to assess OSA severity and sleep variables, and 11 sex- and age-matched healthy controls.

Interventions: Sampling of the resting pupil diameter (RPD) over 5 minutes in darkness in the morning and in the afternoon hours, using an infrared video pupillometer.

Measurements And Results: The RPD was smaller, indicating a lower level of arousal, in the patient group compared with controls in both the morning and the afternoon; the RPD showed a significant circadian reduction in the afternoon only in the patient group. Within the patient group, the RPD correlated negatively with Epworth Sleepiness Scale scores and Arousal Index and positively with the lowest oxygen saturation during the night. Controlling for the effect of body mass index, the relationship between RPD and subjective sleepiness was lost, whereas the relationship with most of the objective indexes of OSA severity was improved.

Conclusions: The 5-minute pupillary miosis in darkness holds promise as a simple, fast-to-administer, valid, and sensitive test for the objective assessment of excessive daytime sleepiness.
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http://dx.doi.org/10.1093/sleep/29.11.1482DOI Listing
November 2006

Differential expression of matrix metalloproteinases in viral and non-viral chronic liver diseases.

Clin Chim Acta 2004 Nov;349(1-2):203-11

Liver Research Laboratory University of Crete Medical School, 71003 Heraklion, Crete, Greece.

Background: Fibrosis is a common consequence of chronic liver diseases irrespective of aetiology. Metalloproteinases play an important role in the fibrotic process participating in the balance between collagen synthesis and degradation. We examined whether matrix gelatinases and stromelysins are similarly involved in the development of viral (HCV, HBV) and non-viral (NASH) liver diseases.

Methods: Hepatic mRNA levels of matrix metalloproteinase MMP-2, MMP-9, MMP-10 and MMP-11 isolated from liver biopsies were measured by semi-quantitative RT-PCR. Seventy-three patients were examined in this study: non-diseased controls (10), patients with chronic hepatitis B (14), chronic hepatitis C (33) and non-alcoholic steatohepatitis (16).

Results: A significant increase of MMP-9 and MMP-10 expression was found in patients with non-viral (non-alcoholic steatohepatitis) liver disease. Patients with chronic hepatitis B and hepatitis C showed an increase in MMP-2 mRNA expression compared to controls. Moreover, chronic hepatitis B and hepatitis C patients had significantly different mRNA expression patterns.

Conclusions: These findings indicate that matrix metalloproteinases are differentially involved in the fibrotic process of viral and non-viral chronic liver diseases. Differences exist between HBV and HCV chronic hepatitis. Differences between early and late fibrosis indicate that in future studies, careful staging of patients is mandatory for interpretation of results.
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http://dx.doi.org/10.1016/j.cccn.2004.06.028DOI Listing
November 2004

Conductive keratoplasty for low to moderate hyperopia: 1-year results.

J Refract Surg 2003 Sep-Oct;19(5):496-506

University of Crete, Medical School, Vardinoyannion Eye Institute of Crete, Heraklion, Greece.

Purpose: To assess the safety, efficacy, and stability of conductive keratoplasty in the treatment of low to moderate hyperopia and evaluate the impact of the procedure on the quality of vision.

Methods: Thirty-six eyes (25 patients; 13 female and 12 male) were treated for hyperopia up to +3.25 D with a Refractec ViewPoint CK system and followed for 1 year. Mean age was 50.2 +/- 8.7 years (range 31 to 71).

Results: Before surgery, mean spherical equivalent refraction was +1.90 +/- 0.60 D (range +1.00 to +3.25 D), decreasing at 1 year after conductive keratoplasty to -0.06 +/- 0.80 D, and was within +/- 0.50 D of emmetropia in 68% (24 eyes) and within +/- 2.00 D in all eyes. Twelve months after conductive keratoplasty, uncorrected visual acuity was 20/20 or better in 19 eyes (50%) and 20/40 or better in 32 eyes (89%). No eye lost > or = 2 Snellen lines or had induced cylinder > or = 2.00 D. The mean root mean square values for higher order optical aberrations were 0.060 +/- 0.039 before and 0.174 +/- 0.170 at 3 months after surgery. No statistically significant changes in contrast sensitivity were noted.

Conclusions: Conductive keratoplasty for low to moderate hyperopia provided safe, effective, and stable results both in refraction and quality of vision.
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January 2004
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