Matthaios Kavvalakis

The atlas of dialkylphosphates; assessment of cumulative human organophosphorus pesticides' exposure §

Organophosphorus pesticides (OPs) are a group of chemicals with significant health interest, due to their wide spectrum of action and their excessive use both indoors (household) and outdoors (occupationally). The non-specific metabolites of OPs, dialkylphosphates (DAPs), are the most commonly used indicators for the assessment of cumulative OP exposure in humans. This review presents studies on human biomonitoring of OPs in the general population and in occupationally exposed humans. Furthermore, cases of OP intoxication determined by the measurement of DAP metabolites in various biological samples are included. In many studies, urine samples from both the general population and exposed populations have been analyzed mainly in Europe and America, while other matrices such as amniotic fluid, meconium, hair and blood have been less studied. A variety of analytical techniques were used for the determination of DAPs in these matrices. In studies measuring DAPs in urine samples, the detected concentrations ranged from 18 to 830ppb for the general population, while the corresponding values for exposed populations ranged from 29 to 1370ppb. Studies on amniotic fluid indicated DAP levels of 0.3-2.8ppb. Studies on meconium samples showed a concentration range of 0.5-16,000ppb. DAP levels in hair samples ranged from 40 to 165ppb for the general population and from 181.7 to 812.9ppb for the exposed population. Each matrix provides specific information on OP exposure, namely acute, long-term, chronic or prenatal. Meconium and hair can indicate cumulative exposure, while amniotic fluid is an indicator of fetal exposure to xenobiotics. Thus, various biological samples provide a more comprehensive view of OP exposure. In general, dimethylphosphate (DMP) and diethylphosphate (DEP) levels were higher in mainly urine samples, than other methyl and ethyl phosphates. In addition, results in the existing literature are sufficient to demonstrate the difference in levels of DAPs in general and occupationally exposed populations, mainly in urine and hair samples. However, more studies are needed to measure DAP levels in matrices such as amniotic fluid, meconium and hair to add to the literature and confirm existing data.

Multicomponent Analysis of Replacement Liquids of Electronic Cigarettes Using Chromatographic Techniques

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.

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

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.

Dialkyl phosphates in amniotic fluid as a biomarker of fetal exposure to organophosphates in Crete, Greece; association with fetal growth.

The aim of this study was to evaluate fetal exposure to organophosphate pesticides (OPs) by measuring their non-specific dialkyl-phosphate metabolites (DAPs) in amniotic fluid (AF), and to examine the potential association between prenatal exposure and fetal growth. AF samples were collected from 415 women during the second gestational trimester. The determined OPs metabolites were DMP, DMTP, DEP, DETP, and DEDTP. DAPs were extracted by liquid-solid extraction, derivatized and analyzed by gas chromatography-mass spectrometry. 97.8% of AF samples were positive for at least one DAP. DAPs levels did not differ between urban and rural areas. Macrosomic neonates have significantly higher sum levels of DMPs (p=0.043), which exerted a linear positive association with birth-weight centile (b=4.43, p=0.016). Conclusively, as DAPs are detectable in AF they may be used as a potential biomarker of fetal exposure to OPs. Sum levels of DMPs appear to be associated with birth weight independently of other covariates.

Cardiotoxicity in rabbits after long-term nandrolone decanoate administration

Abuse of anabolic androgenic steroids is linked to a variety of cardiovascular complications. The aim of our study was to investigate the possible cardiovascular effects of nandrolone decanoate on young rabbits using echocardiography, histology and monitoring of telomerase activity, oxidative stress and biochemical markers. Fourteen rabbits were divided into three administration groups and the control group. Doses of 4mg/kg and 10mg/kg of nandrolone decanoate, given intramuscularly and subcutaneously, two days per week for six months were applied. A 4-months wash-out period followed. Focal fibrosis and inflammatory infiltrations of cardiac tissue were observed in the high dose groups. Thiobarbituric acid-reactive species (TBARS) levels were significantly increased in the high dose groups, while catalase activity decreased. Myocardial Performance Index (MPI) is the main echocardiographic index primarily affected by nandrolone administration in rabbits. Despite the preserved systolic performance, histological lesions observed associated with distorted MPI values, point to diastolic impairment of the thickened myocardium due to nandrolone treatment. Oxidative stress accumulates and telomerase activity in cardiac tissue rises. Subcutaneous administration seems to be more deleterious to the cardiovascular system, as oxidative stress, telomerase activity and biochemical markers do not appear to return into normal values in the wash-out period.

Rapid method for the simultaneous determination of DDTs and PCBs in hair of children by headspace solid phase microextraction and gas chromatography-mass spectrometry (HSSPME/GC-MS).

The purpose of this study was to develop a rapid and cost efficient hair extraction method, using the headspace solid phase microextraction (HSSPME) technique for the simultaneous determination and biomonitoring of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) (DDT) and its isomers/metabolites and polychlorinated biphenyls (PCBs) in hair samples. A total of 72 head hair samples were collected from children living in urban and rural regions of the island of Crete. Two hundred milligrams of hair were digested under alkaline conditions and thermostated for 30 min at 90°C while a 65 µm PDMS/DVB fibre was exposed into the headspace of the vial. Analytical parameters of the method (time of incubation, agitation speed, recovery, precision, accuracy, carry over, matrix effect, linearity, and selectivity) were examined. Recoveries of the DDTs in the spiked hair samples were calculated from 42.3% for opDDD to 87.1% for opDDE, while recoveries for PCB congeners were from 52.6% for PCB138 to 96.6 % for PCB28. The method was applied for the analysis of authentic hair samples. Significant differences (p=0.001) of the burden to total DDTs (sumDDTs) as well as of the frequencies of detection of positive samples (p=0.020) were observed between the examined regions. Moreover, significant differences in the detected concentrations of PCB congeners were observed for PCB52 (p<0.001) and PCB28 (p=0.017) as well for their prevalence between urban and rural regions. Application of HSSPME for the biomonitoring of DDTs and PCBs biomarkers in hair was tested and successfully applied to the analysis of spiked and authentic hair samples. HSSPME was found to be substantially simpler and faster procedure than previous reported sample treatment procedures.

Buprenorphine and nor-buprenorphine levels in head hair samples from former heroin users under Suboxone® treatment.

In the current study, buprenorphine (BUP) and its major metabolite, nor-buprenorphine (NBUP), were determined in hair samples from former heroin users following Suboxone® treatment. Hair samples from 36 subjects were analyzed. The drugs of interest were isolated from hair by solid-liquid extraction with methanol and were determined by liquid chromatography-mass spectrometry, using an electrospray ionization interface. The analytical parameters of the method (such as linearity, limits of quantification, recovery, accuracy, and precision) were determined. The inter-quartile range of BUP levels was from 11.4 to 37.4 pg/mg (mean value 56.6 pg/mg) for the proximal hair segment, from 5.8 to 43.3 pg/mg for the middle hair segment (mean value 25.3 pg/mg), while a range from 4.3 to 33.9 pg/mg (mean value 105.2 pg/mg) for the distant to the root hair segment was determined. For NBUP the corresponding inter-quartile range was from 27.0 to 147.6 for the proximal segment (mean value 95.4 pg/mg), from 21.5 to 164.7 pg/mg for the middle segment (mean value 102.0 pg/mg) and from 20.4 to 103.6 pg/mg for the distant segment (mean value 156.8 pg/mg). The mean BUP/NBUP concentration ratio was 0.5. The daily dose of Suboxone® correlated significantly with BUP and NBUP levels in hair (p = 0.001 and p = 0.023) as well as with the BUP/NBUP ratio (p = 0.010). No significant correlation was found between the levels of BUP and NBUP and the duration of Suboxone® administration. The developed and validated method was successfully used for the determination of BUP and NBUP in hair samples collected from former heroin users under Suboxone® treatment.

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

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.

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

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. Rabbits 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.

Bisphenol A in soft drinks and canned foods and data evaluation

ABSTRACT Bisphenol A (BPA) is one of the most common industrial chemicals and known to exert endocrine disruption activity. The aim of this study was the quantification of BPA in food stuffs on the Greek market. The applied liquid chromatography-mass spectrometry method was validated for linearity, limit of quantification, accuracy, precision and recovery. About 41.7% of the canned solid phase samples, 25.0% of the canned liquid phase samples and 43.8% of the soft drinks were positive. Mean BPA concentrations (range) were 33.4 ± 4.4 ng/g (4.90 ± 0.64–66.0 ± 8.6 ng/g) in canned solid phase, 2.70 ± 0.08 ng/ml (1.90 ± 0.06–3.50 ± 0.11 ng/ml) in canned liquid phase and 2.30 ± 0.18 ng/ml (0.40 ± 0.03–10.2 ± 0.8 ng/ml) in soft drinks. The results of this study are comparable with those reported in the literature according to which higher concentrations of BPA were detected in the solid fraction of canned food compared to their liquid fraction.