engleski

Cytosolic distribution of Cd, Co, Cu, Fe, Pb, V and Zn in liver, gills and intestine of Vardar chub (Squalius vardarensis) from mining impacted rivers in Macedonia

Several metals, such as Co, Cu, Fe, and Zn, are essential micronutrients required for wide selection of physiological processes in living organisms. However, both essential metals and nonessential metals, such as Cd, Pb, and V, can be toxic, depending on their concentrations. As a response to increased metal exposure, binding of metals to various cytosolic biomolecules as ligands can be expected. Therefore, the assessment of potential metal toxicity requires the knowledge on metal speciation and distribution within cells. In this study, cytosolic distributions of seven metals associated with biomolecules of different molecular masses were analysed in the liver, gills and intestine of Vardar chub (Squalius vardarensis) caught in three differently contaminated rivers in north-eastern Macedonia in spring (May/June) of 2012. Comparison was made between metal distribution profiles obtained for two mining impacted rivers (Zletovska and Kriva), and one less contaminated river (Bregalnica), which was considered as a reference site. Cytosolic fractions were isolated from liver, gills and intestine of three chub specimens, one from each river, by homogenisation and subsequent centrifugation at 50000xg. Separation of cytosols in the fractions, which contained proteins of different molecular masses, was carried out by size exclusion high performance liquid chromatography, using Superdex™ 200 10/300 GL column (molecular mass range 10-600 kDa). Metal concentrations in obtained fractions were determined by high resolution inductively coupled plasma mass spectrometry. The distribution profiles of essential metals differed more between the organs than between the rivers, due to comparable cytosolic metal concentrations in organs of chub from all three rivers. Distribution profile of Co was characterized by several peaks. The first one occurred only in hepatic cytosol, within high molecular mass protein category (HMM ; >100 kDa). The other peaks occurred within medium (MMM ; 30-100 kDa), low (LMM ; 10-30 kDa) and very low molecular mass protein category (VLMM ; <10 kDa). The association of Co with VLMM protein fraction (maximum at 1.4 kDa) in all organs could be presumably explained as binding to known Co-containing compound, cobalamin (1.3 kDa). The elution times of Co peaks were almost identical in all tissues, but peak heights differed in accordance to cytosolic Co concentrations. Therefore, peaks in all hepatic cytosols and intestinal cytosol from the Bregalnica River were higher, narrower and sharper compared to gills and the other two intestinal samples. Observed increase of Co concentrations in hepatic and intestinal cytosols resulted with its increase predominantly within HMM and MMMregions. Copper distribution profile was characterized by a peak with maximum at elution time of 30 minutes (12.2 kDa), which coincided with elution time of metallothioneins (MTs). That peak was the highest in the liver, followed by intestine and then gills, which corresponded well with Cu cytosolic concentrations in those organs. Iron distribution profile in hepatic and intestinal tissues was characterized by two clear peaks. The maximum of the first peak corresponded to proteins of molecular mass around 400 kDa, such as ferritin (450 kDa), which serves as Fe storage protein, indicating probable participation of liver and intestine in Fe storage. The second Fe-peak appeared in all three tissues and covered the range of molecular masses from 10-60 kDa, which could possibly involve binding to known Fe-containing proteins, such as catalase (60 kDa) or transport protein myoglobin (17 kDa). Unlike other essential metals, the binding of Fe to ferritin differed between rivers, and was especially pronounced in liver in mining impacted rivers (Zletovska and Kriva), whereas in the gills the highest MMM peak was observed in the Kriva River, indicating higher Fe transport through gills, but not its storage. Interesting finding for Zn were different distribution profiles in three organs, not just regarding the peak heights, but also their elution times. In liver, three peaks of the following ranges of molecular masses were evident: 1) >300kDa, 2) 237-9.3 kDa, 3) 20.9-7.11 kDa. The last one, with elution time at 30 minutes, was the highest and coincided with MT elution time. In gills, the most of Zn was eluted in HMM and MMM regions, whereas MT peak was not observed. Contrary, in the intestine the highest Zn peak was found at elution time of 38 minutes (1.8 kDa) indicating Zn binding to small compounds. Distribution profiles of nonessential metals varied both between the organs and between the rivers, due to different exposure levels in the river water and their consequent accumulation in chub organs. In all organs, the most prominent Cd peak was found in the LMM protein region, with maximum at elution time of MTs (30 minutes, 12.2 kDa), same as previously described for Cu and hepatic Zn. The height of this peak corresponded well with cytosolic Cd concentrations, and was the highest in the liver in the Zletovska and Kriva River, followed by intestine and gills in the Zletovska River. In the gill cytosol, a small portion of cytosolic Cd was also found associated with HMM fractions, whereas in the intestinal cytosols, LMM peak did not only increased in height with increasing cytosolic Cd concentrations, but also expanded its width in direction of lower molecular masses. Distribution profiles of Pb from the Kriva River had the best resolved peaks, due to the highest cytosolic Pb concentrations in all three organs of chub from that river. The highest Pb concentrations and the highest peaks were found in the intestine, than liver, and they were the lowest in the gills. In liver, two sharp peaks were observed within HMM and MMM regions, with maxima at elution time of 23 minutes (105.7kDa), and 30 minutes, respectively. The second peak coincided with elution time of MTs. In gills, several peaks in HMM and MMM regions were found, but were rather small. In the intestine, Pb was mainly eluted in MMM protein region with clear, sharp peak with maximum at 27 minutes (35.9 kDa). Distribution profiles of V could be defined only for liver and intestine of chub from the Bregalnica River, because in all the other cases V concentrations were too low. In liver, sharp, narrow peak was obtained in HMM region (~500 kDa), whereas wide and much lower peak was observed in MMM region (181.3-12.2 kDa). A similar pattern was found in the intestine, but the peaks were much lower, due to lower cytosolic V concentrations. Presented cytosolic metal distribution profiles for three organs of Vardar chub, living under different conditions of metal exposure, were the first step towards recognition of specific metal-binding proteins, which could be further on used as biomarkers of metal pollution.

chub ; gills ; liver ; intestine ; metals ; distribution profiles ; proteins ; HPLC

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