Leda Kovatsi

Histopathological lesions, oxidative stress and genotoxic effects in liver and kidneys following long term exposure of rabbits to diazinon and propoxur.

PURPOSE The aim of the present study was to investigate the effects of diazinon and propoxur on liver and kidneys, following long term exposure of rabbits. METHODS Ten New Zealand white female rabbits were used. The animals were divided into 5 groups, consisting of 2 animals each. Diazinon (groups 1 and 2) and propoxur (groups 3 and 4) were administered at 2 different doses, and group 5 served as the control group. Histopathological lesions in the liver and kidneys, oxidative stress and oxidative DNA damage were evaluated. RESULTS Both pesticides induced focal inflammation and fibrosis in the liver and kidneys. The low dose of propoxur induced a significant increase in total antioxidant capacity (TAC), with no difference in reduced glutathione (GSH), while the high dose of propoxur induced an increase in GSH with no change in TAC. For diazinon-exposed animals, the opposite findings were observed. Both diazinon and propoxur induced a statistically significant oxidative DNA damage in the liver and kidneys and a subsequent increase in telomerase activity in these tissues, possibly as a counteracting mechanism. Furthermore, systemic inflammation, as depicted by the dose-dependent increase in telomerase activity in peripheral blood mononuclear cells (PBMCs), was observed in propoxur treated animals. CONCLUSIONS Histopathological lesions, oxidative stress and genotoxic effects were induced in liver and kidneys following long term exposure of rabbits to diazinon and propoxur.

The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA

Recently, the field of predicting phenotypes of externally visible characteristics (EVCs) from DNA genotypes with the final aim of concentrating police investigations to find persons completely unknown to investigating authorities, also referred to as Forensic DNA Phenotyping (FDP), has started to become established in forensic biology. We previously developed and forensically validated the IrisPlex system for accurate prediction of blue and brown eye colour from DNA, and recently showed that all major hair colour categories are predictable from carefully selected DNA markers. Here, we introduce the newly developed HIrisPlex system, which is capable of simultaneously predicting both hair and eye colour from DNA. HIrisPlex consists of a single multiplex assay targeting 24 eye and hair colour predictive DNA variants including all 6 IrisPlex SNPs, as well as two prediction models, a newly developed model for hair colour categories and shade, and the previously developed IrisPlex model for eye colour. The HIrisPlex assay was designed to cope with low amounts of template DNA, as well as degraded DNA, and preliminary sensitivity testing revealed full DNA profiles down to 63pg input DNA. The power of the HIrisPlex system to predict hair colour was assessed in 1551 individuals from three different parts of Europe showing different hair colour frequencies. Using a 20% subset of individuals, while 80% were used for model building, the individual-based prediction accuracies employing a prediction-guided approach were 69.5% for blond, 78.5% for brown, 80% for red and 87.5% for black hair colour on average. Results from HIrisPlex analysis on worldwide DNA samples imply that HIrisPlex hair colour prediction is reliable independent of bio-geographic ancestry (similar to previous IrisPlex findings for eye colour). We furthermore demonstrate that it is possible to infer with a prediction accuracy of >86% if a brown-eyed, black-haired individual is of non-European (excluding regions nearby Europe) versus European (including nearby regions) bio-geographic origin solely from the strength of HIrisPlex eye and hair colour probabilities, which can provide extra intelligence for future forensic applications. The HIrisPlex system introduced here, including a single multiplex test assay, an interactive tool and prediction guide, and recommendations for reporting final outcomes, represents the first tool for simultaneously establishing categorical eye and hair colour of a person from DNA. The practical forensic application of the HIrisPlex system is expected to benefit cases where other avenues of investigation, including STR profiling, provide no leads on who the unknown crime scene sample donor or the unknown missing person might be.

Epigenetic mechanisms in metal toxicity.

The true understanding of epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current flurry of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses various molecular mechanisms of epigenetic regulation of gene expression, and expands the discussion with examples of heavy metal-induced alterations of gene expression and the associated epigenetic changes.

Mitochondrial control region sequences from northern Greece and Greek Cypriots

Entire mitochondrial control region data were generated for population samples of 319 unrelated individuals from northern Greece and 91 unrelated individuals from Cyprus. The samples from northern Greece have been previously typed for 15 nuclear short tandem repeat (STR; Kovatsi et al., Forensic Sci. Int. 159:61–63, 2006).

Population genetics of Y-chromosome STRs in a population of Northern Greeks☆

Seventeen Y STR loci were typed in a population sample of 191 unrelated male individuals from Northern Greece. Haplotypes are presented for the following loci: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448. The overall haplotype diversity was 0.9992. This database study provides significant additional information for the application of Y-chromosomal STRs to forensic identification efforts in Greece by nearly doubling both the number of individuals and the number of Y-loci typed from Greek populations. These samples have been previously typed for autosomal STRs [L. Kovatsi, T.J. Parsons, R.S. Just, J.A. Irwin, Genetic variation for 15 autosomal STR loci (PowerPlex 16) in a population sample from northern Greece, Forensic Sci. Int. 159 (2006) 61-63] and the mitochondrial DNA control region [J. Irwin, J. Saunier, K. Strouss, C. Paintner, T. Diegoli, K. Sturk, L. Kovatsi, A. Brandstatter, M.A. Cariolou, W. Parson, T.J. Parsons, Mitochondrial control region sequences from northern Greece and Greek Cypriots, Int. J. Legal Med. 122 (2008) 87-89].

p16 promoter methylation in Pb2+-exposed individuals

Background. One of the principle symptoms of lead poisoning is the development of neurological disorders. Neuronal response is closely related to DNA methylation changes. Aim. In this study, we estimated p16 methylation in nine individuals exposed to lead using methylation-specific polymerase chain reaction followed by analysis of the methylated cytosine content of the product by thermal denaturation. Results. We found that, based on lead blood concentration, lead-exposed individuals were divided into two groups. Among highly exposed individuals (blood Pb2+ concentration = 51–100 μg/dL), we observed complete CpG methylation, whereas for low Pb2+ concentrations (blood Pb2+ concentration = 6–11 μg/dL), we observed partial methylation. Conclusion. Our results show that among lead-overexposed individuals, p16 methylation is frequent and extensive, and suggest that DNA methylation could be involved in the mechanism by which lead induces neurotoxicity.

Association of reduced zinc status with angiographically severe coronary atherosclerosis: a pilot study.

In vitro studies attribute antiatherogenic and insulin-like properties to zinc (Zn). However, only a few conflicting clinical data exist concerning the relationship between Zn and coronary artery disease (CAD) as well as glycemic indices. We studied 72 patients without prior history of myocardial infarction or revascularization procedures, who underwent coronary angiography for evaluation of chest pain. Coronary artery disease severity was estimated using 3 angiographic scores. Zn in serum and 24-hour urine, as well as serum Zn/24-hour urine Zn ratio were determined. Serum Zn was not associated with CAD prevalence and severity. However, urinary Zn loss was significantly higher among patients with CAD and showed a positive association with CAD severity. Serum Zn/24-hour urine Zn ratio was inversely associated with CAD, as well as with diabetes mellitus prevalence, fasting glucose, and glycated hemoglobin levels. Low serum Zn/24-hour urine Zn ratio is associated with angiographically severe atherosclerosis and impaired glucose homeostasis.

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

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.

Novel strategies for sample preparation in forensic toxicology

This paper provides a review of novel strategies for sample preparation in forensic toxicology. The review initially outlines the principle of each technique, followed by sections addressing each class of abused drugs separately. The novel strategies currently reviewed focus on the preparation of various biological samples for the subsequent determination of opiates, benzodiazepines, amphetamines, cocaine, hallucinogens, tricyclic antidepressants, antipsychotics and cannabinoids. According to our experience, these analytes are the most frequently responsible for intoxications in Greece. The applications of techniques such as disposable pipette extraction, microextraction by packed sorbent, matrix solid-phase dispersion, solid-phase microextraction, polymer monolith microextraction, stir bar sorptive extraction and others, which are rapidly gaining acceptance in the field of toxicology, are currently reviewed.

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6‐monoacetylmorphine in vitreous humor

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 μL.