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Nickel toxicity in the hepatopancreas of an isopodPorcellio scaber (Oniscidea)

M. Tarnawska a,*, P. Migula a, W. Przybyłowicz b,c, J. Mesjasz-Przybyłowicz b,M. Augustyniak a

a Department of Animal Physiology and Ecotoxicology, University of Silesia Katowice, Bankowa 9, 40-007 Katowice, Polandb Materials Research Group, iThemba LABS, Somerset West, South Africa

c Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Krakow, Poland

Available online 24 February 2007

Abstract

This study is focused on recognizing how the functional role of hepatopancreas, the main metal storage organ in woodlice, is affectedby the excess of nickel, a toxic element to soil invertebrates. Chronic Ni toxicity (24 weeks) was studied on four groups of woodlice kepton dry shredded maple leaves contaminated with Ni at average concentrations of 0.1 lg g�1 (control), 8.0 lg g�1 (Ni1), 75 lg g�1 (Ni2)and 270 lg g�1(Ni3) dry weight. Micro-PIXE mapping of elemental distribution in the hepatopancreas of the Porcellio scaber woodlicewas used to study relations between nickel and other elements in individuals exposed to different metal concentrations in the diet. Datawere processed using GeoPIXE II software. Transmission electron microscopy (TEM) was used to check the relations between ultra-structural changes in hepatopancreatic cells and nickel tissue burden.

Elemental mapping showed a dose-related nickel bioaccumulation in the hepatopancreas at concentrations from 3 lg g�1 (uncontam-inated control animals) to nearly 840 lg g�1 (Ni3). Generally, nickel was distributed uniformly in small aggregations. A combined eval-uation of elemental maps and electronograms showed that aggregations of nickel in arbitrarily selected micro-areas in PIXE maps couldbe the granular structures observed in TEM electronograms. The mechanism of Ni sequestration in the hepatopancreas could be similarto this used for cadmium or lead. The sequences of ultrastructural changes, which follow the increased burdens of Ni in the hepatopan-creatic cells, were: the increase of intracellular electron-dense granules, increase in the number of myelin-like structures, intensified mito-chondrial swelling and appearance of concentrically arranged, rough endoplasmic reticulum.� 2007 Elsevier B.V. All rights reserved.

PACS: 89.60.�k; 82.80.Yc; 82.80.�d; 87.23.�n

Keywords: Micro-PIXE; Transmission electron microscopy; Hepatopancreas; Porcellio scaber; Nickel; X-ray microanalysis; Nuclear microprobe

1. Introduction

In metal-polluted soil ecosystems isopods are likely to beexposed to the highest concentrations of metals, which arepresent mostly in the leaf-litter layer [1].

Excess of metals in the diet is a stressful factor whichmight cause a series of protective responses or changes,eventually leading to cellular death. Hepatopancreas, the

main internal organ of isopods, plays important role inhandling both essential metals involved in normal physio-logical processes [2] and nonessential ones [3,4]. The struc-ture of hepatopancreatic cells of woodlice can be affectedby both internal and external factors such as moulting,daily cycle of secretion, starvation, and presence of metalsin the food [5–8]. Alterations of cellular ultrastructure werepreviously often used for assessing the effects of metalpollution by several authors [3,9–11], but studies relatedto nickel influence on hepatopancreatic cells are limited.The aim of this study was to identify a distribution patternof nickel and some other elements in this organ, as well as

0168-583X/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.nimb.2007.02.082

* Corresponding author. Tel.: +48 32 359 1203; fax: +48 32 258 77 37.E-mail address: dybowska@us.edu.pl (M. Tarnawska).

www.elsevier.com/locate/nimb

Nuclear Instruments and Methods in Physics Research B 260 (2007) 213–217

NIMBBeam Interactions

with Materials & Atoms

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to identify Ni-induced cellular alterations in woodlicechronically exposed to the metal.

2. Material and methods

2.1. Animal culture

Individuals of woodlice (Porcellio scaber, Latreille 1804)were collected from a control site near Myszkow, southernpart of Poland. Only specimens of at least 10–15 mm inlength and mean body mass of 50 mg were used in the test.They were fed on dry, senescent maple leaves (Acer platano-

ides). The experiment was carried out on both sexes ofwoodlice. They were acclimatized for 2 weeks in groups of10 animals in plastic boxes (16 · 10 · 5 cm), with a 5 mmgypsum base in a 16:8 h light:dark photoperiod at 18 �C(±2 �C) [4]. After this period four groups were set up. Thetest animals were fed with the same food, pre-soaked for24 h in solutions of NiCl2 of 10, 100 and 1000 mg of Ni l�1),representing low (Ni1), medium (Ni2) and high (Ni3) nickellevels which could possibly be found in their natural diet.Control woodlice were given uncontaminated leaves.

2.2. Nuclear microprobe measurements

After 24 weeks of Ni exposure the animals were anaes-thetised, hepatopancreas was dissected and immediatelypreserved by impact cryofixation against a cold metal blockusing a Leica EM CFC Cryoworkstation, next freeze–driedin a Leica EM CFD Cryosorption Freeze Dryer andmounted between two layers of the Formvar film. TheFormvar film was coated with a thin carbon layer fromthe side of the incoming proton beam to prevent chargebuild-up during measurements.

Microanalyses were performed using a nuclear micro-probe at the Materials Research Group, iThemba LABS,South Africa [12]. Particle-induced X-ray emission (PIXE)and proton backscattering (BS) were used simultaneously.In this study, a proton beam of 3.0 MeV energy and cur-rent of 200–300 pA was focused to a 3 lm · 3 lm spotand scanned over selected areas of square or rectangular

shape with variable number of pixels (up to 128 · 128).Normalization of results was done by using the integratedbeam charge, collected simultaneously from a Faraday cuplocated behind the specimen and from the insulated speci-men holder. Data were collected in list-mode using XSYSacquisition system and next processed using GeoPIXE II[13,14]. Elemental maps were generated using Dynamic

Analysis method refined after the experiment by usingmatrix composition matching selected regions of interestwithin scanned areas rather than using average matrixcomposition from the whole scanned area. This procedurewas based on PIXE and BS spectra extracted from theseregions. Mean concentrations from regions were also calcu-lated using full deconvolution of PIXE spectra. The matrixcomposition and areal density were obtained from theanalysis of corresponding BS spectra using a RUMP simu-lation package [15] and experimental, non-Rutherfordcross-sections for C and O at a laboratory angle of 170�[16]. Other experimental details were described earlier [17].

2.3. Transmission electron microscopy

Animals were sacrificed at 9:00–10:00 a.m. taking intoaccount possible structural changes in hepatopancreaticcells resulting from their circadian secretory cycles [6].After decapitation hepatopancreas was isolated, trans-ferred to 2.5% glutaraldehyde in 0.1 M phosphate bufferpH 7.4 at 4 �C for 24 h and postfixed with 1% OsO4 (2 h)and embedded in an embedding mixture Epon, 812 [18].Ultrathin sections were cut with Leica ultracut UCT25ultramicrotome, next stained with uranyl acetate and leadcitrate [19] and examined with Hitachi H500 transmissionelectron microscope.

3. Results

3.1. Elemental distributions in the hepatopancreas ofP. scaber

Elemental mapping showed dose-related nickel bioaccu-mulation in the hepatopancreas at concentrations from

Table 1Concentration of selected elements in the hepatopancreas of Porcellio scaber based on PIXE spectra extracted from selected regions of the hepatopancreasmarked in Fig. 1(a)–(d)

Elements Experimental groups

Control Ni1 Ni2 Ni3

Cl 5312 ± 54 (22) 4957 ± 33 (29) 2943 ± 61 (16) 10361 ± 160 (51)K 5589 ± 53 (4) 9740 ± 101 (7) 4153 ± 43 (3.1) 3963 ± 108 (16)Ca 1897 ± 15 (2.3) 3577 ± 35 (4.5) 4730 ± 70 (1.9) 44388 ± 591 (13)Mn 7.1 ± 0.7 (0.68) 18 ± 1 (1.6) 16.4 ± 0.5 (0.59) 7 ± 2 (2.4)Fe 151 ± 2 (0.64) 112 ± 2 (1.5) 492 ± 4 (0.56) 164 ± 4 (2.1)Ni 3 ± 2 (1.5) 13 ± 5 (3.9) 203 ± 8 (1.4) 838 ± 43 (7.1)Cu 3873 ± 46 (2) 11844 ± 147 (5.5) 3204 ± 45 (2) 4452 ± 67 (9.4)Zn 1094 ± 15 (2.3) 4193 ± 54 (6.1) 3909 ± 43 (2.2) 2148 ± 39 (8.9)Br 9 ± 1 (1.6) 29 ± 3 (3.8) 23 ± 1 (1.4) 36 ± 6 (5.2)Sr 10 ± 1 (1.5) 47 ± 4 (3.2) 68 ± 2 (1.7) 125 ± 7 (4.8)

Mean values ± uncertainties and minimum detection limits at 99% confidence level (in brackets) are reported in lg g�1.

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3 lg g�1 in the control group to nearly 840 lg g�1in the(Ni3) group (Table 1). Due to low concentrations, Ni maps

for the control and (Ni1) groups show only backgroundfluctuations. In maps for the animals from the (Ni2) and

Fig. 1. Quantitative maps of elemental distributions in selected areas of the hepatopancreas of woodlice Porcellio scaber. (a) – control group (control); (b)– (Ni1) group; (c) – (Ni2) group; (d) – (Ni3) group. MAX – maximum concentration value; respective concentrations (lg g�1) are shown below every map.

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(Ni3) groups Ni showed a uniform distribution pattern ofsmall aggregations (Fig. 1(c)–(d)). The (Ni3) group wasalso characterized by the highest amounts of Cl and Ca.The distribution of Cl, Ca and K was also even and partlyoverlapping each other (Fig. 1(a)–(d)). Cu and Zn differ indistribution arrangement. They were located mainly on theborder between tubular units. This is well demonstrated inthe map of (Ni2) woodlice (Fig. 1(c)). Fe showed the samepattern and was significantly enriched in the (Ni2) group.The concentration of Mn was too low to allow observationof distribution pattern in any group.

3.2. Cellular effects of Ni on the hepatopancreas of P. scaber

A single layer of epithelial hepatopancretic cells of con-trol woodlice comprise two types of cells: B and S. The Bcells had numerous lipid droplets (Fig. 2). Both types ofcells had microvilli in their apical part. The cytoplasmwas characterised by granular endoplasmatic reticulum(rER), frequently present as concentric structures.

In comparison with control animals the hepatopancre-atic cells of Ni-stressed isopods displayed numerous struc-tural alterations. Electronograms of hepatopancreaticepithelium of (Ni1) animals showed small vesicles of gran-ular rER (Fig. 3). Myelin-like structures (mls) and intracel-lular granules (g) with an electron-dense material werepresent in the (Ni1) and (Ni2) woodlice (Figs. 3, 4). (Ni2)woodlice were characterized by localization of all cell struc-tures in the basal cellular part and presence of cytoplas-matic granules with electron-dense deposits at differentstages of packing. Electron-dense granules and swelledmitochondria were observed in the cells of (Ni3) animals(Fig. 5). Moreover, these cells had concentrically arrangedrER cisternae and lysosomes.

4. Discussion and conclusions

Nickel distribution in the analyzed areas of hepatopan-creas of P. scaber was even, although in the map from

Fig. 2. Control group, TEM, portion of the hepatopancreas of Porcellio

scaber, inside two cells (transverse section). B-cell; li – lipid droplets; m –mitochondria; n – nucleus; S – S-cell.

Fig. 3. (Ni1) group, TEM, portion of the hepatopancreas of Porcellio

scaber, basal part of the cell (transverse section). bla – basal lamina; m –mitochondria; rER – rough endoplasmatic reticulum.

Fig. 4. (Ni2) group, TEM, portion of the hepatopancreas of Porcellio

scaber, basal part of the cell (transverse section). g – electron-densegranules; li – lipid droplets; m – mitochondria; mls – myelin-likestructures.

Fig. 5. (Ni3) group, TEM, portion of the hepatopancreas of Porcellio

scaber, inside the cell (transverse section). g – electron-dense granules; m –mitochondria; rER – rough endoplasmatic reticulum.

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(Ni3) woodlice the points with the highest metal contentwere present in larger conglomerations (Fig. 1(d)). Assu-redly, these places refer to granular structures (Fig. 5),which were also earlier identified in the hepatopancreaticcells of isopods exposed to Cd and Pb [10,11]. Dose-relatedNi accumulation in the hepatopancreas confirms that thisorgan is important in its sequestration, as it was previouslyproved for other metals (Zn, Cd, Pb and Cu) [3]. From ourstudies it is now clear how Ni is distributed (micro-PIXEmaps) when present in the diet in excess, and the resultscan be compared to those obtained for other elementswhich were given to animals at physiological ranges of con-centrations. Hepatopancreas and the hindgut are bothimportant in metal sequestration and protection againsttheir toxicity. A comparison between these Ni-stressedorgans showed that Ni contents in hepatopancreasincreased proportionally to a dietary metal, while in thehindgut it was more flexible, probably due to differencesbetween digestibility rates of isopods from experimentalgroups. The highest values were in the less Ni-stressed(Ni2) group woodlice [20]. Hepatopancreas is its eventualplace of accumulation. This explains more uniform distri-bution of Ni in this organ as seen from the maps, whilein the hindgut more aggregated areas are easily recognised.The resultant toxic effects observed through documentedultrastructural alterations were different for these twoorgans.

Exposure of cells to chemical stress can evoke protectivecellular adaptations, or/and pathological changes, whichoften lead to cellular death [1]. According to Korslootet al. [21] nickel caused mostly secondary effects on thehindgut epithelium, which might reflect alterations of thecross-membrane transport of ions and water. Moreover,it caused mitochondrial dysfunctions suggesting the respi-ratory metabolism [20].

For the hepatopancreas, more protective effects wereidentified in the endoplasmic reticulum with granularforms, which can be identified as Ca-immobilized metalaggregations, showing a better preparation of this organto accumulate metals. On the other hand, lysosome-likestructures and rough endoplasmic reticulum give the evi-dence of more serious alterations caused by nickel, becausethey are one of the most important symptoms of apoptosis.This feature can also be explained as one of the pre-adap-tive trade-off mechanisms. Isopods accumulate metalsinstead of their elimination which is energetically costly.Energy gained is thus allocated into the body growth ofdeveloping larvae. Maturity is reached earlier and earlierthey can start to reproduce. Effects of Ni-stress observedin isopods could be used as biomarkers of chronic nickelexposure.

Acknowledgements

This study was financially supported by the Polish StateCommittee of Scientific Research (KBN) (Grant No. 2PO4G 094 26, 2004-2005). It forms part of the Joint SouthAfrican–Polish project ‘‘Ecophysiological studies on natu-ral feeders of South-African nickel-hyperaccumulatingplant Berkheya coddii for its use as a metal bioremediationtool’’, supported by the South African National ResearchFoundation and the Polish State Committee for ScientificResearch (KBN) in Poland. Opinions expressed and con-clusions arrived at, are those of the authors and are notnecessarily to be attributed to the NRF.

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