Copyright © 2004, Springer Netherlands.The digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island (Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantify
the relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, through
light microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cu and Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetric
density of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein is suggested as a biomarker of Cu exposure in terrestrial mollusks.Centro de Investigação em
Recursos Naturais (CIRN), University of the Azores

Amaral, André

Anselmo, Henrique

Cunha, Regina Tristão da

Rodrigues, Armindo

BioMetals

Repositório da Universidade dos Açores

BioMetals 17: 625–629, 2004.© 2004 Kluwer Academic Publishers. Printed in the Netherlands. 625The connective tissue index of Helix aspersa as a metal biomarkerAndre´ Filipe Santos Amaral∗, Henrique Anselmo, Regina Maria Pires Toste Tristão da Cunha& Armindo Santos RodriguesUniversidade dos Açores, Departamento de Biologia, R. Mãe de Deus, 9500 Ponta Delgada, São Miguel, Açores,Portugal ∗Author for correspondence (E-mail: aamaral@notes.uac.pt)Received 12 September 2003; Accepted 8 March 2004; Published online October 2004Key words: Helix aspersa, digestive gland, connective tissue, copper, ironAbstractThe digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island(Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantifythe relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, throughlight microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cuand Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetricdensity of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein issuggested as a biomarker of Cu exposure in terrestrial mollusks.IntroductionThe digestive gland is the most important organ parti-cipating in xenobiotic detoxification in mollusks. Thisorgan is responsible for the production of digestiveenzymes, nutrient absorption and digestion, endocyt-osis of food substances, food storage and excretion(Owen 1996). The digestive gland epithelium of ter-restrial gastropods is composed of three cell types(Dimitriadis 2001): digestive cells, calcium cells, andexcretory cells. The most frequent cell type to befound in this epithelium of Stylommatophora is the di-gestive cell, which is directly involved in the digestiveprocess. Excretory cells are less numerous and con-cerned with the excretion of wastes. The calcium cellsare characterized by the presence of calcium granules,occurring in large numbers in Helix aspersa (approx-imately 103 per cell), for the most part in the form ofphosphate (Fournié & Chétail 1982, Simkiss & Taylor1994). Besides epithelial cells, digestive gland alsocomprises connective tissue, which is composed byrhogocytes (pore cells), pigment cells, and fibroblasts,among others. Rhogocytes are implicated in hemocy-anin formation, collagen synthesis, and accumulationof calcium leading to development into calcium cells,and metal detoxification (Luchtel & Deyrup-Olsen2001).Among the multiple functions attributed to the di-gestive gland of mollusks, it is also known to be amajor target site for metal accumulation, involved inmetal sequestering and release (Ireland & Marigómez1992, Soto 1995). Such actions show histological andhistochemical changes that can be used as biomarkersof metal exposure.The aim of this study was to determine if anim-als from qualitatively different areas are significantlydifferent from each other in metal metabolism, and ifdifferences in that metabolism can be detected in thedigestive gland epithelium and connective tissue.Material and methodsThe Azores archipelago is located in the North At-lantic Ocean at the triple junction of Eurasian, Africanand North American plates, characterized by a com-plex tectonic settlement, where the seismic and vol-canic phenomena are common (Nunes et al., 1993).The Azores archipelago is remote from heavy in-dustrial activities despite of the expanding tourism,626meaning that its volcanic activity must be consideredwhen studying the availability of trace metals. Thefour sampling sites of the present work are locatedin the island of São Miguel: (1) Fuel deposits, withscarce vegetation, is next to a commercial seaport, andstands next to a road with a car traffic frequency ofabout 500 cars per hour; (2) Rotunda of Belém, thoughhaving more vegetation than Fuel deposits, is the mainpoint of entry and exit from Ponta Delgada, which isthe main city, and for this it has one of the highest cartraffic frequency (about 2000 cars per hour); (3) Back-yard, inside the historic zone of Ponta Delgada (lessthan 100 cars per hour); and (4) Lombadas (controlstation), with dense vegetation, partially composed byendemic species, stands 20 m from a rarely used roadand 4 km from the nearest village.Sixty adult snails of H. aspersa, fifteen per site,were collected. In the laboratory, the digestive glandsof snails were excised under a Wild M3 stereomicro-scope.For chemical analysis, digestive glands were dried(130 ◦C), digested in nitric acid, and finally dissolvedin 0.1 N HNO3. Metal concentrations (Al, Cd, Cu, Fe,Pb) were measured by an atomic absorption spectro-photometer (GBC 906AA).One tissue sample from each digestive gland wasfixed in neutral buffered formaldehyde (Hopwood1996), dehydrated in alcohol, cleared in methylben-zoate (overnight), rinsed in benzene, embedded inparaffin and sectioned at 7 µm thickness.A set of sections was submitted to an autometal-lographic procedure (Soto et al. 1998) in order todemonstrate metals while another was stained withhaemalum and eosin (Martoja & Martoja-Pierson1970) to estimate the relative volumetric density ofcalcium cells and connective tissue cells. Paraffinsections were dewaxed in xylene, hydrated in ethanol-water mixtures and left in an oven at 37 ◦C untilcompletely dried. Tissues sections were covered witha photographic emulsion (Ilford Nuclear Emulsion L4)under safety light conditions. After drying for 30 minin total darkness, sections were rinsed in a developerbath (1:5, b/w Ilford PQ Universal) for 15 min, rinsedin a stop bath (1% acetic acid) for 1 min, and finallyrinsed in a fixative bath (1:10, b/w Ilford Hypam) for10 min.The relative volumetric density of black silver de-posits (VvBSD) was estimated using the M168 WeibelMultipurpose Test System (Weibel 1979). The samemethodology was applied to the haemalum and eosinsections to estimate the relative volumetric densityTable 1. Metal concentrations in the digestive glandof H. aspersa (µg metal/g dry tissue).Sampling stations Al Cu FeFuel Deposits 223,15 209,48 670,01Rotunda of Bele´m 171,74 130,72 486,78Backyard 40,47 73,26 237,21Lombadas 583,33 49,02 171,88of calcium cells (VvCC) and the relative volumetricdensity of connective tissue cells (VvCTC). Beforeproceeding with statistical analysis, VvBSD, VvCCand VvCTC values were normalized using the arcsinetransformation (Sokal & Rohlf 1995).A one-way analysis of variance was performed(Sokal & Rohlf 1995) in order to determine the ef-fect of locality (L) on the VvBSD, VvCC and VvCTClevels in the digestive gland of H. aspersa. This wascomplemented with a Duncan test (p ≤ 0.05), usingthe SPSS software (SPSS Inc., Microsoft Co.). Correl-ations between VvBSD, VvCC, VvCTC and metals werealso determined.ResultsMetal accumulation has been detected in the digest-ive gland of H. aspersa from all four sampling sites(Table 1). Values of Cd and Pb were very low in allsnails.The calcium cell composition of the digestivegland epithelium sections, from the four samplingsites, did not differ (Duncan test, p > 0.05). However,when observing the stroma of the gland, Rotunda ofBelém and Fuel deposits evidenced more and largerconnective tissue cells than Lombadas and Backyard(Figures 1A–B and 2). The VvCTC of Rotunda ofBelém presented the highest value, being significantlydifferent from the Backyard and Lombadas (Duncantest, p ≤ 0.05) but not from the Fuel deposits (Duncantest, p > 0.05) (Figure 2).Metals occurred essentially inside the connectivetissue cells, mainly rhogocytes, and few in the digest-ive cells and the basal lamina beneath it, while calciumcells were devoid of BSD (Figure 1C–D). Metal accu-mulation was significantly higher in Rotunda of Belémand Fuel deposits when compared with Lombadas andBackyard (Duncan test, p ≤ 0.05). There were no627Fig 1. (A) Section of the digestive gland of a specimen from Lombadas, stained with haemalum and eosin, showing normal connective tissuecells (ctc), calcium cells (cc) and excretory cells (ec). (B) Section of the digestive gland of a specimen from Rotunda of Bele´m, stained withhaemalum and eosin. Note the acini with normal digestive cells (dc), calcium cells (cc) and excretory cells surrounded by large connectivetissue cells (ctc). (C) Autometallographic demonstration of metal deposits (arrow head) in the digestive gland of a specimen from Lombadas.Note the scarcity of metal deposits. D- Autometallographic demonstration of metal deposits in the digestive gland of a specimen from Rotundaof Bele´m. Note the abundance of metal deposits (arrow head) present in the connective tissue cells (ctc) and their absence in calcium cells (cc).Scale bar = 10 µm.Fig 2. (Semi)quantification, by means of volume density, of metals (VvBSD), calcium cells (VvCC) and connective tissue cells (VvCTC) [×¯±se] in the digestive gland of H. aspersa. a. u.= arbitrary units.628Table 2. Pearson’s correlation between variables. ∗, p ≤ 0.5; ∗∗, p ≤ 0.01.Variable VvBSD VvCTC VvCC Al Cu FeVvBSD 0,565∗∗ 0,146. . . −0,382∗∗ 0,751∗∗ 0,779∗∗VvCTC 0,311*.. −0,322*.. 0,540** 0,566**VvCC −0,110. . . 0,179. . . 0,169. . .significant differences between Rotunda of Belém andFuel deposits (Duncan test, p > 0.05) (Figure 2).According to the one-way analysis of variance,locality (L) affected significantly VvBSD (F = 33.398;p ≤ 0.01) and VvCTC (F = 10.389; p ≤ 0.01) but notVvCC (F = 0.754; p > 0.05). A significant Pearsoncorrelation (p ≤ 0.01) was detected between VvBSDand VvCTC (Table 2).DiscussionAutometallography has been earlier reported to be auseful and sensitive technique for the localization andquantification of metal deposits in cellular compart-ments of marine and terrestrial mollusks (Marigómezet al. 1996, Soto et al. 1996, Marigómez et al.1998, Soto et al. 1998). In the present work, metalswere visualized under the light microscope as BSDin the connective tissue but not in the calcium cellsof the digestive gland epithelium. These observationsare in agreement with previous results of Zaldibaret al. (1998) for freshwater snails. The BSD extent(VvBSD) in the digestive gland presented significantdifferences between sites with different metal bioavail-ability, being Cu and Fe the most correlated to VvBSDlevels.Changes in relative cell-type proportion (RCP) ofthe digestive gland have been proposed as a metal bio-marker that reflects high levels of bioavailable metalsin the environment (Marigómez et al. 1998). Previouslaboratory experiments recorded high BSD extent ac-companied by changes in RCP of the digestive glandepithelium (Marigómez et al. 1996, 1998). In thepresent study, quantification of calcium cells (VvCC)revealed that snails from all studied sites exhibitedvery similar epithelial composition, but in contrast,snails from the two sites with highest metal bioavail-ability demonstrated more and larger connective tissuecells. This increase in the proliferation of connectivetissue cells was also related to Cu and Fe, and has beenearlier reported in winkles after their exposure to Cuand Zn (Soto et al. 1999). A possible explanation tothis event is the existence of Fe and Cu granules insiderhogocytes (Haszprunar 1996) and the role of thesecells in the formation of hemocyanin and metal de-toxification (Luchtel & Deyrup-Olsen 2001), meaningthat rhogocytes may react promptly and earlier thanepithelial calcium cells when facing high levels of Feand Cu exposure in the environment. One fact support-ing this is the exclusive occurrence of Cu-MT mRNAin rhogocytes (Chabikovsky 2003). Thus, changes inconnective tissue rhogocytes may be used as an earlywarning in Fe and, especially, Cu contamination.AcknowledgementsThe authors thank Professor Patricia Garcia for helpfuldiscussions and comments, and Dr. Jorge Medeirosfor imaging assistance. The present work has beenfinancially supported by Centro de Investigação emRecursos Naturais (CIRN), University of the Azores.ReferencesChabicovsky M, Niederstätter H, Thaler R, Hödl E, Parson W, Ross-manith V, Dallinger R. 2003. Localization and quantification ofCd- and Cu-specific metallothionein isoform mRNA in cells andorgans of the terrestrial gastropod Helix pomatia. Toxicol ApplPharm 190, 25–36.Dimitriadis VK. 2001 Structure and function of the digestive sys-tem in Stylommatophora. In: Barker GM, ed. The Biology ofTerrestrial Molluscs. Wallingford: CABI Publishing: 237–257.Fournié J, Chétail M. 1982 Accumulation calcique au niveau cel-lulaire chez les mollusques. 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The connective tissue index of Helix aspersa as a metal biomarker

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