Embrioni riba kao alternativni model u toksikologiji: revijalni pregled

Povećan broj ksenobiotika ugrožava životnu sredinu. Ova bioaktivna jedinjenja dospevaju u vodenu sredinu na različite načine i imaju značajan uticaj na akvatični živi svet. Za procenu opasnosti i rizika industrijskih hemikalija, proizvoda zaštite bilja, biocida, dodataka u hrani i lekova koriste se testovi na životinjama. Akutni test toksičnosti za ribe je obavezni korak u procesu ispitivanja toksičnosti hemikalija. Međutim njihova ekotoksikološka relevantnost je pod znakom pitanja. Povrh toga ribe se u ovim testovima podvrgavaju značajnom bolu i patnji. U skladu sa filozofijom 3r (redukcija/smanjenje, refinement/prečišćavanje, replacement/zamena) embrioni riba se smatraju metodom zamene ili prečišćavanja budući da ovi razvojni stadijumi nisu zakonski zaštićeni (Direktiva 2010/63/EU) i izgleda da osećaju manju bol i patnju. Cilj ovog pregleda bio je da se sumiraju dostupni podaci o testovima toksičnosti na embrionima riba i ukaže na moguće perspektive korišćenja ovog modela u savremenoj toksikologiji. Princip testa toksičnosti sa embrionima riba zasniva se na izloženosti vijabilnih embriona ksenobioticima, od oplođenja do kraja embriogeneze. Test toksičnosti sa embrionima riba validiran je u OECD studiji i prihvaćen kao OECD TG 236 96-h test za procenu toksičnosti za embrione riba. Određuje se letalnost koja je označena koagulacijom embriona, nedostatkom somita, ispravljanja repa ili otkucaja srca. Povrh smrtnosti, složeniji pokazatelji kao što je razvoj očiju, prisustvo krvotoka ili razvojne malformacije mogu se uočiti na stadijumima izvaljenih eleutero embriona. Mogućnost praćenja različitih pokazatelja čini embrione riba odličnim modelom za ispitivanje i razumevanje mehanizama toksičnosti i indikatorom mogućih nepovoljnih i dugotrajnih efekata. Na osnovu izvedenih istraživanja, test toksičnosti sa embrionima riba je razumna alternativa akutnom testu toksičnosti na ribama. Raspon mogućih aplikacija je značajan i ima mnogo perspektiva u budućnosti: kao što je dalja identifikacija molekularnih markera – indikatora načina delovanja ili uspostavljanje veze među efektima u kratkotrajnim testovima sa embrionima i dugotrajnih efekata na individue. Test toksičnosti sa embrionima riba takođe može da doprinese smanjenju broja eksperimenata na životinjama što je jedan od osnovnih principa EU legislative u oblasti dobrobiti

Efekti subhroničnog izlaganja zebrice, danio rerio sintetičkom mošusu

Policiklična jedinjenja sintetičkog mošusa su veoma rasprostranjena i koriste se u velikim količinama kao miris u proizvodima široke potrošnje. Zbog njihove česte upotrebe i otpuštanja mirisa, ova jedinjenja su postala prisutna svuda u životnoj sredini. Toksikološka istraživanja su potvrdila da jedinjenje mošusa predstavlja opasnost za vodene ekosisteme. Cilj ovog istraživanja bio je da se ocene efekti subhroničnog izlaganja sintetičkom mošusu tonalidu na rast riba i odgovore na oksidativni stress kod zebrice (Danio rerio). Test rasta kod mlađi zebrice izvršen je prema OECD metodu broj 215. U toku 28 dana, riba stara 30 dana je bila izložena koncentraciji tonalida (50; 500; 5.000 and 50.000 ng/l). Na kraju eksperimenta, sve ribe su žrtvovane, izmerene, određena je njihova specifična prosečna stopa rasta po tanku, a selektivni oksidativni stress markeri su analizirani u homogenatu celog tela (glutation S-transferaze, glutation reduktaze, glutation peroksidaze, katalaze i lipidne peroksidaze). U našem ekperimentu, nismo pronašli značajne razlike između kontrolne i eksperimentalne grupe u specifičnom prirastu, telesnoj težini i dužini. Međutim, primetili smo značajne promene kod većine oksidativnih stres markera naročito kod eksperimentalne grupe koja je bila izložena najvećoj koncentraciji mošusa (tonalida). U poređenju sa kontrolnom grupom, značajna povećanje je konstatovano u aktivnostima glutation S-transferaze (za koncentracije – 5.000 i 50.000 ng/l) i kataze (za koncentracije – 500, 5.000 i 50.000 ng/l). Sa druge strane, primećeno je značajno smanjenje aktivnosti glutation peroksidaze (za koncentraciju – 500 ng/l) u poređenju sa kontrolnom grupom. Nisu konstatovane promene u aktivnosti glutation reduktaze i nivou lipidne peroksidaze u poređenju sa kontrolnom grupom. Naši rezultati pokazuju da izlaganje zebrice tonalidinu ima značajan uticaj na oksidativne stres markere i enzime za detoksifikaciju. Promene u aktivnostima antioksidantnih enzima se mogu tumačiti kao adaptivni odgovor koji bi zaštitio organizam ribe od toksičnosti prouzrokovane tonalidinom

Leeches as Sensor-bioindicators of River Contamination by PCBs

The aim of the study was to evaluate the use of leeches of the genus Erpobdella as a means of assessing polychlorinated biphenyl contamination of watercourses. The River Skalice, heavily contaminated with PCBs, was selected as a model. The source of contamination was a road gravel processing factory in Rožmitál pod Třemšínem from which an estimated 1 metric ton of PCBs leaked in 1986. Levels of PCB were measured in leeches collected between 1992 to 2003 from 11 sites covering about 50 km of the river (the first sampling site upstream to the source of contamination and 10 sites downstream). The PCB indicator congeners IUPA no. 28, 52, 101, 118, 138, 153, and 180 were measured. Levels were highest at the four sampling sites nearest the source of pollution. The highest values of PCB congeners were found in 1992. PCB content decreased from 1992 to 2003 and with distance from the source. The study indicated that leeches of the genus Erpobdella are a suitable bioindicator of contamination in the surface layer of river sediments

1-Hydroxypyrene–A Biochemical Marker for PAH Pollution Assessment of Aquatic Ecosystem

The aim of the present study was to assess aquatic contamination by polycyclic aromatic hydrocarbons (PAH), using the 1-hydroxypyrene (1-OHP) content in fish bile as a biochemical marker. A total of 71 chub (Leuciscus cephalus L.) were collected from seven locations on the Svitava and Svratka rivers in and around the industrial city of Brno, Czech Republic. The levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection after deconjugation. Normalising the molar concentration of the biliary 1-OHP to the biliary protein content reduced sample variation. The content of 1-OHP was correlated with the PAH level in bottom sediment and semi-permeable membrane devices (SPMD), which was analyzed by a combination of HPLC/FLD and GC/MS methods. The highest mean values of 1-OHP were found in fish caught at the Svratka River at locations Modřice (169.2 ± 99.7 ng·mg−1 protein) and Rajhradice (152.2 ± 79.7 ng·mg−1 protein), which are located downstream from Brno. These values were significantly (P < 0.05) higher than those obtained from localities Kníničky (98.4 ± 66.1 ng·mg−1 protein) and Bílovice nad Svitavou (64.1 ± 31.4 ng·mg−1 protein). The lowest contents of PAH in sediment and SPMD were found at location Kníničky (1.5 mg·kg−1 dry mass and 19.4 ng·L−1, respectively). The highest contents of PAH in sediment and SPMD were found in Rajhradice (26.0 mg·kg−1 dry mass) and Svitava before junction (65.4 ng·L−1), respectively. A Spearman correlation test was applied to determine the relationship between biliary 1-OHP and the sum of PAH in sediment and SPMD. A positive, but no statistically significant correlation was found. The main impact sources of elevated level of PAHs in sites located downstream from Brno are most probably intensive industrial and agricultural activities and domestic waste

Effect of Cadmium Chloride on Metallothionein Levels in Carp

Due to anthropogenic activities, heavy metals still represent a threat for various trophic levels. If aquatic animals are exposed to heavy metals, we can obviously observe considerable toxicity. It is well known that organisms treated with heavy metals synthesize low molecular mass compounds rich in cysteine. In this work the effects of cadmium chloride (2.5, 5, 7.5, 10 and 12.5 mg/L) on common carp (Cyprinus carpio) was investigated. We determined cadmium content in tissue of muscle, liver and kidney by atomic absorption spectrometry with electrothermal atomization and content of metallothionein (MT) in the same tissues by the Brdicka reaction. Electrochemical methods can be considered as suitable and sensitive tools for MT determination in carp tissues. Results of our study showed a gradually enhancing of cadmium content in muscle with time and dose of cadmium chloride in water. MT levels in liver reached both high levels (above 130 ng/g) in fish exposed to 2.5, 5 and 7.5 mg/L and low level (to 50 ng/g) in fish exposed to 10 and 12.5 mg/L of cadmium chloride. This finding confirms that the synthesis of metallothioneins and binding capacity of these proteins is restricted

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5′-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP

Data from: Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards

Weight, development, and survival data for Phylloneta impressa juvenile spiders, Chrysoperla carnea lacewing larvae, and Harmonia axyridis ladybeetle larvae fed with pollen, aphids Rhopalosiphum padi, or spider mites Tetranychus urticae from SmartStax Bt maize or genetically closest non-Bt hybrid. ELISA Cry protein measurements of the previously described predators as well as of food items and maize leaves