Određivanje biogenih amina i endotoksina u lignji, muzgavcu, škampima i dagnji pohranjenima na sobnoj temperaturi

Little research has been published on the indicators of spoilage in Mediterranean molluscan shellfi sh and crustaceans. Thus is why we studied changes in the concentrations of endotoxin and four biogenic amines (histamine, putrescine, tyramine and cadaverine) in European common squid (Loligo subulata, Lamarck, 1798), musky octopus (Eledone moschata, Lamarck, 1798), Norway lobster (Nephrops norvegicus, Linnaeus, 1758), and mussel (Mytilus galloprovincialis, Lamarck, 1819) from the Adriatic Sea stored at room temperature for 24 h. Endotoxin load in fresh squid, Norway lobster, and mussel (<1 EU mg-1) indicated good microbiological quality of raw samples. Biogenic amine index (as the sum of histamine, putrescine, tyramine, and cadaverine) correlated well with endotoxin load in squid (r=0.978, p<0.001) and musky octopus (r=0.874, p<0.01). A good correlation was also found between endotoxin and putrescine in Norway lobster (r=0.777, p<0.05). The highest endotoxin load was found in decomposed mussels and was associated with histamine alone. In conclusion, increase in biogenic amine levels is species-specific. Endotoxin analysis could be used for rapid assessment of microbiological quality of cephalopods and shellfish.Istraživanja o pokazateljima kontaminacije mekušaca i člankonožaca iz mediteranskih zemalja su rijetka. Cilj ovoga rada bio je ispitivanje koncentracije endotoksina i četiriju biogenih amina (histamin, putrescin, tiramin i kadaverin) u lignji (Loligo subulata, Lamarck, 1798), muzgavcu (Eledone moschata, Lamarck, 1798), škampima (Nephrops norvegicus, Linnaeus, 1758) i dagnji (Mytilus galloprovincialis, Lamarck, 1819) iz Jadranskog mora koji su bili pohranjeni na sobnoj temperaturi tijekom 24 h. Količina endotoksina u svježoj lignji, škampima i dagnji (<1 EU mg-1) upućuje na njihovu dobru mikrobiološku kvalitetu. Visoka korelacija ustanovljena je između indeksa biogenih amina (suma histamina, putrescina, tiramina i kadaverina) i endotoksina u lignji (r=0,978, p<0,001) i muzgavcu (r=0,874, p<0,01). Također je ustanovljena visoka korelacija između endotoksina i putrescina u škampima (r = 0,777, p<0,05). Najveća razina endotoksina (povezana s porastom histamina) ustanovljena je kod dagnje. U zaključku, porast biogenih amina je species-specifi čan. Analiza endotoksina mogla bi se primijeniti kao brza metoda za određivanje mikrobiološke ispravnosti mekušaca i člankonožaca

Određivanje elemenata u kosi metodom ICP-MS: odabir odgovarajuće procedure pranja uzorka

Human hair is a biological sample that is, unlike blood, noninvasively collected and can be used in the assessment of element intake. Before analysis, hair samples need to be washed to eliminate external contamination, for which no standard procedure exists. The present study evaluated the efficiency of different processes for washing hair samples (by non-ionic detergent, acids, solvents, and their mixtures), including ultrasonication before ICP-MS element (As, Ca, Cd, Cu, Fe, Hg, Mg, Mn, Mo, Pb, Se, and Zn) analysis. All tested washing procedures using detergent or solvents were satisfactory, while nitric and hydrochloric acid solutions yielded to “released” and lost elements due to damaged hair (visible discolouration and hair impairment). The application of ultrasonication improved washing efficiency up to 10 %, depending on the tested element and washing procedure. This work is licensed under a Creative Commons Attribution 4.0 International License.Ljudska kosa je biološki uzorak koji se, za razliku od krvi, skuplja neinvazivno i može rabiti u procjeni unosa elemenata. Prije analize uzorke kose potrebno je oprati kako bi se odstranila vanjska onečišćenja za što ne postoje standardni postupci. Istraživanjem je procijenjena učinkovitost različitih postupaka pranja uzoraka kose (neionskim detergentom, kiselinama, otapalima i njihovim mješavinama) uključujući primjenu ultrazvuka prije analize elemenata (As, Ca, Cd, Cu, Fe, Hg, Mg, Mn, Mo, Pb, Se, and Zn) metodom ICP-MS. Ispitivani postupci pranja bili su zadovoljavajući, izuzev primjenom dušične i klorovodične kiseline, pri čemu se elementi “otpuštaju” i gube zbog oštećivanja kose (vidljive su promjena boje i pucanje vlasi). Primjena ultrazvuka pospješila je učinkovitost pranja uzoraka do 10 % ovisno o ispitivanom elementu i postupku pranja. Ovo djelo je dano na korištenje pod licencom Creative Commons Imenovanje 4.0 međunarodna

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

Introduction: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. Materials and methods: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. Results: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (CT) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). Conclusion: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification

Izloženost endotoksinu u drvoprerađivačkoj industriji: odnos između razina izmjerenih u zraku i na tlu

Wood processing is usually performed in environments with large amounts of endotoxin-rich bioaerosols that are associated with a variety of health effects. The aim of this preliminary study was to assess the relation between endotoxin levels in settled and airborne dust in wood-processing industry. Ten pairs of airborne and settled dust samples were collected in a sawmill and parquet manufacture of two wood-processing plants in Croatia. Endotoxin was assayed with a chromogenic end-point LAL (Limulus amebocyte lysate) method. The results showed that endotoxin levels in airborne respirable dust were above the proposed occupational exposure limit of 125 EU m-3 and could be considered hazardous for the respiratory system. In settled dust they ranged between 229.7 EU mg-1 and 604.3 EU mg-1 and in airborne dust between 166.8 EU mg-1 and 671.6 EU m-3, but there was no signifi cant correlation between them (Spearman’s rho=0.358, P=0.310). This study points to sawmill settled dust as endotoxin reservoir and suggests that it may add to already high exposure to airborne endotoxins associated with wood processing. Investigations of the relation between settled and airborne endotoxin levels should be continued to better understand the sources and sites of endotoxin contamination in wood-processing industry.Drvoprerađivačka industrija radna je sredina s izloženošću velikim količinama bioaerosola bogatog endotoksinom, što je povezano s učincima na zdravlje izložene radne populacije. Cilj ove studije bio je procijeniti odnos između razina endotoksina u sedimentiranoj prašini i zraku (aerosoliziranoj prašini) drvoprerađivačkih radnih prostora. Deset parova uzoraka zraka i sedimentirane prašine skupljeno je u dvije tvornice za preradu drva (pilanama i pogonima za proizvodnju parketa) u Hrvatskoj. Endotoksin je izmjeren kromogenom “end-point” LAL (Limulus amebocitni lizat) metodom. Rezultati su pokazali da razine endotoksina u aerosoliziranoj respirabilnoj prašini drvoprerađivačkih pogona prelaze preporučene granice izloženosti za radne prostore od 125 EU m-3, pa se mogu smatrati štetnima za dišni sustav izloženih radnika. Razine endotoksina izmjerene u sedimentiranoj i aerosoliziranoj prašini kretale su se u sličnu rasponu [(229.7 do 604.3) EU mg-1 i (166.8 do 671.6) EU m-3], ali nije utvrđena značajna korelacija razina endotoksina između parova uzoraka sedimentirane i aerosolizirane prašine (Spearman rho=0.358, P=0.310). Ovi rezultati upućuju na to da uz velike količine proizvedene i aerosolizirane drvne prašine u sklopu radnih operacija u pilanama, razini endotoksina u aerosoliziranoj respirabilnoj prašini može pridonijeti i uskovitlana sedimentirana prašina koja je dodatni rezervoar endotoksina. Istraživanje odnosa između razina endotoksina u sedimentiranoj i aerosoliziranoj prašini treba nastaviti kako bi se bolje defi nirali izvori i mjesta kontaminirana endotoksinom u pilanama

Mercury Disposition in Suckling Rats: Comparative Assessment Following Parenteral Exposure to Thiomersal and Mercuric Chloride

Due to the facts that thiomersal-containing vaccine is still in use in many developing countries, and all forms of mercury have recognised neurotoxic, nephrotoxic, and other toxic effects, studies on disposition of ethylmercury and other mercury forms are still justified, especially at young age. Our investigation aimed at comparing mercury distribution and rate of excretion in the early period of life following exposure to either thiomersal (TM) or mercuric chloride (HgCl2) in suckling rats. Three experimental groups were studied: control, TM, and HgCl2, with 12 to18 pups in each. Both forms of mercury were administered subcutaneously in equimolar quantities (0.81 μmol/kg b.w.) three times during the suckling period (on the days of birth 7, 9, and 11) to mimic the vaccination regimen in infants. After the last administration of TM or HgCl2, total mercury retention and excretion was assessed during following six days. In TM-exposed group mercury retention was higher in the brain, enteral excretion was similar, and urinary excretion was much lower compared to HgCl2-exposed sucklings. More research is still needed to elucidate all aspects of toxicokinetics and most harmful neurotoxic potential of various forms of mercury, especially in the earliest period of life

Kadmij i olovo u uzorcima vučje jetre: optimizacija metode razgradnje uzoraka uz pomoć mikrovalova

A microwave-assisted digestion method for the determination of cadmium (Cd) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS) was optimised on certified reference material (CRM) (bovine liver, BCR-185R) and wolf liver samples. Different factors influencing digestion efficiency (temperature, time, composition of the digestion mixture, sample mass) were tested. Validation included linearity (up to 200 μg L-1 for Cd and Pb), detection (0.003 μg L-1 for Cd and 0.035 μg L-1 for Pb), and quantification (0.008 μg L-1 for Cd and 0.081 μg L-1 for Pb) limits. Good agreement between measured and certified values was achieved in all conditions, with recoveries ranging from 94 % to 111 % for Cd and from 95 % to 105 % for Pb. The precision of the method, expressed as relative standard deviation, was up to 3 % for Cd and 8 % for Pb. The best digestion parameters (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0.1 g of CRM) based on accuracy and precision were applied on two wolf liver samples to evaluate the need for the predigestion step (freeze-drying) and appropriate mass of the sample. Freeze-drying improved precision and minimising the tissue mass to 0.1 g reduced the matrix effect. Using these optimised digestion conditions, we determined Cd and Pb in 40 wolf livers collected in Croatia, and their medians (0.055 μg g-1 and 0.107 μg g-1, respectively) were in the range of previously reported data for the grey wolf.Certifi cirani referenti materijal (CRM) govedskih jetara (BCR-185R) i uzorci vučjih jetara koristili su se za optimizaciju metode razgradnje uzoraka prije mjerenja kadmija (Cd) i olova (Pb) masenom spektrometrijom induktivno spregnute plazme (ICP-MS). Ispitivani su različiti uvjeti koji utječu na učinkovitost razgradnje (temperatura, vrijeme, sastav otopine za razgradnju, masa uzorka). Validacijom je obuhvaćeno ispitivanje linearnosti (do 200 μg L-1 Cd/Pb), granice detekcije (0,003 μg L-1 za Cd, 0.035 μg L-1 za Pb) i kvantifikacije (0,008 μg L-1 za Cd, 0,081 μg L-1 za Pb). Postignuto je dobro slaganje izmjerenih i certificiranih vrijednosti u svim ispitivanim uvjetima uz izračunati raspon iskorištenja 94 % do 111 % za Cd i 95 % do 105 % za Pb. Najveća relativna standardna devijacija, kao mjera preciznosti, iznosila je 3 % za Cd i 8 % za Pb. Najbolji uvjeti za razgradnju (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0,2 g CRM), izabrani prema točnosti i preciznosti, primijenjeni su na uzorke vučjih jetara pri procjeni odgovarajuće mase uzorka i potrebe za uvođenjem dodatnog koraka prije razgradnje (liofilizacije). Liofilizacija poboljšava preciznost pa je preporučujemo pri pripremi uzoraka tkiva. Također, najmanja masa uzorka izabrana je ne samo zbog bolje preciznosti nego i zbog najmanjeg utjecaja matrice te potrebne količine tkiva. Medijani Cd (0,055 μg g-1) i Pb (0,107 μg g-1 mokre mase) dobiveni analizom 40 uzoraka vučjih jetara skupljenih u Hrvatskoj bili su u rasponu vrijednosti objavljenih u literaturi za sivog vuka

Utjecaj godišnjeg doba na koncentraciju prašine i endotoksina u zraku nastambe za kokoši nesilice

In the present study, air quality in a cage-housed laying hen house was investigated throughout the seasons by assessing dust and endotoxin concentrations. Measurements were done twice a month during the 1-year production cycle. The mean levels of dust and endotoxins ranged from 0.60 mg/m3 in May to 2.83 mg/m3 in November, and from 203.15 EU/m3 in August to 745.53 EU/m3 in April. Significantly higher concentrations of dust and endotoxins in the poultry house were determined in the autumn and winter seasons, and endotoxins in the spring season as well, compared to the summer (P<0.05 all). The results suggested that the cooler periods of the year pose a greater risk for the welfare and performance of laying hens, but also for the health of humans working in these settings in terms of the levels of airborne dust and endotoxins.U ovom radu istraživana je kvaliteta zraka u nastambi kavezno držanih kokoši nesilica u svim godišnjim dobima utvrđivanjem koncentracije prašine i endotoksina. Mjerenja su provođena dva puta na mjesec tijekom jednogodišnjeg proizvodnog ciklusa. Srednje vrijednosti koncentracije prašine i endotoksina kretale su se od 0,60 mg/m3 (svibanj) do 2,83 mg/m3 (studeni), odnosno od 203,15 EU/m3 (kolovoz) do 745,53 EU/m3 (travanj). Značajno veće koncentracije prašine i endotoksina u peradarniku ustanovljene su ujesen i zimi, endotoksini također i u proljeće, u usporedbi s ljetom (P<0,05 sve). Iz rezultata istraživanja može se zaključiti da hladnija razdoblja godine predstavljaju veći rizik za dobrobit i proizvodnost kokoši nesilica, ali i za zdravlje zaposlenika u ovim nastambama s obzirom na koncentraciju prašine i endotoksina u zraku

Metal(loid) exposure assessment and biomarker responses in captive and free-ranging European brown bear (Ursus arctos)

We investigated the level of five non-essential metal(loid)s (As, Cd, Hg, Tl, Pb) and nine essential metals (Mg, Ca, Mn, Fe, Co, Cu, Zn, Se, Mo) in hair and blood components of captive and free-ranging European brown bear populations in Croatia and Poland. Metal(loid) associations with biomarkers of oxidative stress (superoxide dismutase, SOD ; glutathione- peroxidase, GSH-Px ; malondialdehyde, MDA) and metal exposure (metallothionein, MT) were estimated in this top predatory mammal. Lead was the most abundant non-essential metal(loid) in both blood and hair, with 4 of 35 individuals having blood levels over 100 µg/L. A positive association was found between Pb level and SOD activity in blood. Free-ranging bears had higher blood SOD activity, Mn, Zn and Cd levels, hair Co, Cd, Tl and Pb compared to captive individuals, while the opposite was true for Mg and hair Ca thereby reflecting habitat and diet differences. With increasing age, animals showed lower levels of SOD activity and certain essential metals. Females had higher SOD activity and blood levels of some essential metals than males. Hair showed a higher Fe and Co level when sampled during the growth phase and was not predictive of non- essential metal(loid) blood levels. The established metal(loid) baseline values will enable future risk assessment in both captive and wild European brown bear populations

Potentially Toxic Elements in Water, Sediments and Fish from the Karstic River (Raša River, Croatia) Located in the Former Coal-Mining Area

The assessment of the environmental quality of a sensitive karst aquatic system under the centuries-long anthropogenic influence of the coal mining industry is important for both improving the quality of water resources and protecting aquatic wildlife and human health. In this study, we investigated the anthropogenic impact on the aquatic environment of the upper and middle course of the Raša River through the analysis of a suite of metal(loid)s in three aquatic compartments (water, sediment, fish) using inductively coupled plasma mass spectrometry (ICP-MS). Concentrations of inorganic constituents in water were low, while the chemical composition of stream sediments mainly reflected the geological background of the area, indicating the origin of metal(loid)s from predominantly natural sources. Although comparison with PEC-Q values indicated that existing sediment quality conditions could pose a threat to benthic organisms with regard to Cr and Ni, the constant vertical profiles of these elements suggested their natural origin from the weathering of flysch. Element levels in the muscle of targeted fish species were in accordance with the values typical for low-contaminated freshwater systems, while levels of Cd, Pb and Hg were mostly below the European regulatory limits for toxic elements in foods, indicating that the low concentrations of most contaminants in muscles of fish from the Raša River do not present a risk to humans or other consumers. The obtained data indicated a generally low contamination status of the western part of the Raša River basin with regard to the analyzed inorganic elements