Vladimir Mićović

GENOTOXICITY OF MARINE SEDIMENTS IN THE FISH HEPATOMA CELL LINE PLHC-1 AS ASSESSED BY THE COMET ASSAY

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

Metallothioneins and heat shock proteins 70 in marine mussels as sensors of environmental pollution in Northern Adriatic Sea

In an attempt to assess the intensity of environmental pollution in industrial zones of Kvarnerian Bay in Northern Adriatic Sea and the reactivity of Mytilus galloprovincialis to these changes, in this study we estimated the concentration of heavy metals at four locations in both sea-sediment and in the mussels. Further we tried to correlate these changes with seasonal variations in environmental temperature, pH and salinity, as well as with the expression of metallothioneins (MTs) and heat shock proteins (HSPs) in the digestive tract of the mussels. Sampling in vivo was performed monthly, during the year 2008, while under the laboratory conditions the reactivity of acclimated mussels were tested to increasing concentrations of CdCl(2) and to thermal stress. The data have shown that the induction of MTs and HSP isoforms of the 70-kDa size class were highly affected by model agents treatment including contamination of sea-sediment by Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution.

CYP1A induction potential and the concentration of priority pollutants in marine sediment samples – In vitro evaluation using the PLHC-1 fish hepatoma cell line

The use of in vitro biotests in combination with chemical determination of priority pollutants is considered a promising approach in environmental risk assessment. The main goal of this study was to evaluate the relationship between the CYP1A induction potential and the concentration of priority pollutants (PAHs, PCBs and heavy metals) in contaminated marine sediments. Six sediment samples characterized by different types of pollution were collected from the Bay of Kvarner, Croatia. CYP1A induction potency was determined in vitro by the measurement of ethoxyresorufin-O-deethylase (EROD) activity in PLHC-1 fish hepatoma cells. The results were compared to the potency of the model CYP1A inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expressed in 2,3,7,8-TCDD equivalents. All of the tested sediment samples were able to induce CYP1A activity in a dose-dependent manner. On a general scale, there was a good correlation between CYP1A induction and the concentration of priority pollutants in the tested samples. However, some samples, which had relatively low levels of priority pollutants, exhibited a strong CYP1A induction response. Therefore, apart from the confirmed usability and sensitivity of the EROD determination in the PLHC-1 cells as a suitable in vitro model in ecotoxicology, the results of this study indicate that the list of priority pollutants usually determined in the attempt to evaluate the risk of adverse effects to marine wildlife should be reconsidered.

Pregnancy Loss and Maternal Methemoglobin Levels: An Indirect Explanation of the Association of Environmental Toxics and Their Adverse Effects on the Mother and the Fetus

The aim of this epidemiologic study was to point out a relationship between the exposure to products of coal combustion, and complications in pregnancy where one third of causes of stillbirth are still unknown. In the town of Labin (Croatia) a coal-powered thermoelectric power plant is the single major air polluter. We compared the records of miscarriages, premature births and stillbirths in two periods: the control and the exposure period. Data on reproductive loss was based on the records of pregnant women visiting for regular monthly pregnancy checkups. At the time of the epidemiological prospective study, 260 women (n = 138 in the clean period and n = 122 in the dirty period) were considered representative. The data were processed using Chi square and correlation tests. The frequencies of miscarriages and stillbirths were significantly lower in the control than in the exposure period (p < 0.05). Methemoglobinemia and stillbirths recorded over the “exposure” period are significantly higher than in the “control” period (p = 0.0205). The level of methemoglobin in the bloodstream is an worthy biomarker, predictor and precursor of environmental toxics’ adverse effects on the mother and fetus, and can indirectly explain the unrecognized level of fetal methemoglobin. Methemoglobin and heme, having prooxidant properties, also cause the early and late endothelial dysfunction of vital organs. Despite our retrospective epidemiological study findings, we emphasize that the rate of reproductive loss represents a hypothetical risk, which needs to be confirmed with further fetal clinical and anatomopatholgical researches about the effects of methemoglobin catabolism products on the fetal CNS.

The value of an operating microscope in peripheral nerve repair. An experimental study using a rat model of tibial nerve grafting.

Summary. The aim of this study was to test the hypothesis that the use of an operating microscope improves the results of peripheral nerve repair. Tibial nerve grafting was carried out on 48 Fischer rats divided into 2 groups: in one, a loupe was used, and in the other a surgical microscope. At 5 months after grafting, recovery was evaluated by functional, electromyographic, and morphometric tests. The mean motor nerve conduction velocity was 26.77±9.37 m/sec in the group where the loupe was used compared with 44.19±11.36 m/s when the microscope group was used. The soleus muscle weight and the diameter of myelinated fibres also confirmed better regeneration in the microscope group. These results clearly indicate that it is essential to use the microscope for peripheral nerve repair.Résumé. Le but de l’étude est de faire tester l’hypothèse que l’aide du microscope améliore les résultats de la chirurgie du nerf périphérique. La greffe du nerf tibial a été pratiquée sur 48 rats Fischer, repartis en deux groupes, l’un avec l’aide de la loupe et l’autre avec l’aide du microscope. Cinq mois après la greffe les résultats ont été soumis à une évaluation fonctionnelle, électromyographique et morphométrique. Les moyennes de la vitesse de la transmission motrice du nerf dans le groupe assisté par la loupe et celui assisté par le microscope étaient 26.77±9.37 m/s et 44.19±11.36 m/s respectivement. L’équivalent du test clinique, le poids du muscle soleus ainsi que les mésures du diamètre des fibres myelinisées ont aussi démontré une meilleure régénération, dans le groupe assisté par microscope. Ces resultats montrent clairement que l’aide du microscope est essentielle pour la réfection du nerf périphérique.

The role of interfascicular nerve grafting after gunshot wounds

Summary. In this study the authors present the results of 44 patients with 49 interfascicular nerve grafting procedures necessitated by gunshot wounds. The aim was to establish the effectiveness of microsurgical interfascicular nerve grafting for war injuries. The 49 interfascicular nerve grafting procedures were performed between July 1991 and January 1993. All but one were performed within 6 months of injury. Nerve recovery was assessed independently by a neurologist. The M/S scale was used for median, ulnar and radial nerves, and the grading scale recommended by Millesi was used for the sciatic, tibial and peroneal nerves. Follow-up was for between 2.5 and 3.5 years. Patients scoring at least M3/S3+, or Grade 2 on the Millesi scale, were considered to have useful functional recovery which occurred in high median (0 out of 1), low median (4 out of 9), high ulnar (0 out of 2), low ulnar (5 out of 11), high radial (4 out of 6), low radial (2 out of 2), sciatic (5 out of 9), peroneal (4 out of 7), and tibial (2 out of 2). The extent of recovery correlated inversely with the patient’s age and the length of the graft, but was independent of the time between injury and surgery and of presumed projectile energy. Our results suggest that microsurgical interfascicular nerve grafting is of value in the management of wartime nerve injuries.Résumé. Dans cette étude les auteurs présentent les résultats obtenus sur 44 patients avec 49 greffes interfasciculaires pratiquées sur des lésions de nerfs consécutives aux blessures par balle ou éclat d’obus pendant la guerre. Le but de l’étude était d’établir l’efficacité des greffes interfasciculaires effectuées par techniques microchirurgicales. Les 49 greffes ont été pratiquées entre juillet 1991 et janvier 1993, toutes, excepté une, dans les 6 mois qui suivaient la blessure. La guérison des patients a étéévaluée indépendamment par le neurologue. L’échelle M/S a été utilisée pour le nerf médian, ulnaire et radial, tandis que l’échelle de Millesi a été utilisée pour le nerf sciatique, sciatique poplité interne et sciatique poplité externe. Le suivi a été entre 2 ans et demi et 3 ans et demi. Les résultats des patients qui ont obtenu au moins M3/S3+ ou le grade 2 d’après l’échelle de Millesi ont été considérés comme une bonne récupération fonctionnelle. Ces cas de bonne récupération ont été les suivants. Pour le nerf médiant haut 0 sur 1, pour le médiant bas 4 sur 9, pour le nerf ulnaire haut 0 sur 2, pour l’ulnaire bas 5 sur 11, pour le radial haut 4 sur 6, pour le radial bas 2 sur 2, pour le sciatique 5 sur 9, pour le sciatique poplité externe 4 sur 7 et pour le sciatique poplité interne 2 sur 2. L’importance de la récupération a été inversement proportionnelle à l’âge du patient et à la longueur de la greffe, mais elle ne dépendait pas de la durée écoulée entre la blessure et l’intervention chirurgicale, ni de la force du projectile. Nos résultats suggèrent que les techniques microchirurgicales pour les greffes de nerfs interfasciculaires sont efficaces sur les lésions nerveuses dans les blessures de guerre.

The responses of the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity in sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a polluted site: implications for their use in environmental risk assessment

The present study investigates the response of three hepatic biomarkers in adult sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a wastewater outlet of an oil refinery with fish caged at a pristine site used as controls. The biomarkers that were investigated were the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity. In addition, we have measured the levels of polycyclic aromatic hydrocarbons (PAHs) and selected heavy metals (lead, cadmium, mercury, copper and zinc) in sediment samples at the polluted site. Although the polluted site had high environmental levels of PAHs and heavy metals, there was no difference in hepatic EROD activity and HSI between fish caged at the polluted site and controls. On the other hand, GST activity was significantly lower in fish caged at the polluted site compared to controls. Our results point out that the studied biomarkers have limited use in environmental risk assessment studies, at least when caged adult sea bass is used as the sentinel species and complex toxicant mixtures are involved.

Evaluation of equivalence between different methods for enumeration of fecal indicator bacteria before and after adoption of the new Bathing Water Directive and risk assessment of pollution

The quality of bathing water is of considerable public importance due to the possibility of fecal contamination. In 2009, Croatia implemented the new European Bathing Water Directive (BWD, 2006/7/EC) establishing stricter microbiological standards for new parameters with new reference methods. This study aims to evaluate the equivalence of different methods according to the old and revised BWD and to provide the possibility of data comparison. Furthermore, the directive requires the establishment of the bathing water profile (BWP) for pollution risk assessment. The estimation of consistency of pollution risk assessment with obtained microbiological results was also performed. Six marine beaches of the Municipality of Rijeka (Croatia) were examined during the 2009 season. Statistical analysis showed equivalence between determination methods for fecal contamination indicators. Based on the current water classification results, the need for correction of estimated pollution risks and recommendations for inclusion of historical microbiological data during BWP enactment was noticed.

Associations between Adherence to the Mediterranean Diet and Lifestyle Assessed with the MEDLIFE Index among the Working Population

The adherence to the Mediterranean diet is beneficial for cardiovascular diseases prevention. The study aim is to use Mediterranean lifestyle (MEDLIFE) questionnaire for estimation of Mediterranean lifestyle habits among the working population and to establish MEDLIFE score correlation with the risk factors for cardiovascular diseases. In the study has participated 366 workers from Croatia, which fulfilled MEDLIFE and validated food frequency questionnaire (FFQ) questionnaire. The multivariate logistic regression was performed to evaluate the association between MEDLIFE index, workers’ obesity and cardiovascular diseases risk. The lowest adherence to Mediterranean lifestyle was associated to younger, low education, body fat above acceptable ranges and unfavorable lipid profile. Significant association to Mediterranean lifestyle was more among women (p = 0.002), middle aged (p = 0.02), highly physically active (p = 0.009) and those who play collective sports >2 h/w (p = 0.001), having body fat within acceptable range (p = 0.003), total cholesterol less (p = 0.03) and high-density lipoproteins (HDL-C) (p = 0.04) more than recommended. Inverse significant association was for high educational level (p = 0.02). The Mediterranean lifestyle adherence is associated to lower risk factors for cardiovascular diseases among studied working population. MEDLIFE index revealed that physical activity and conviviality are better ingrained among younger population but not the Mediterranean diet.

Methemoglobinemia as biomarker and precursor of brain capillary oxidative damage link to ferric iron accumulation and originator of neurodegenerative disease

Our main aim is to make a contribution to the establishment of sources of oxidants as the key factors in understanding the role that oxidants and oxid