Environ. Sci. Technol. 2005, 39, 5620-5625
Evidence for Biogenic Pyromorphite Formation by the Nematode Caenorhabditis elegans B . P . J A C K S O N , * ,† P . L . W I L L I A M S , ‡ A. LANZIROTTI,§ AND P. M. BERTSCH† Advanced Analytical Center for Environmental Sciences, Savannah River Ecology Laboratory, University Of Georgia, Aiken, South Carolina 29803, Department of Environmental Health Science, University Of Georgia, Athens, Georgia 30602-2102, and Consortium for Advanced Radiation Sources, University of Chicago, Chicago, Illinois 60637
The determination of chemical speciation and spatial distribution is a prerequisite for a mechanistic understanding of contaminant bioavailability and toxicity to an organism. We have employed synchrotron X-ray techniques to study Cu and Pb speciation and spatial distribution in the soil nematode Caenorhabditis elegans. Nematodes were exposed to each metal ion singly or simultaneously in solution for 24 h and were then rinsed thoroughly and preserved in formalin for transportation to the National Synchrotron Light Source. Experiments were conducted at the microprobe beamline X26A employing a focused beam of approximately 10 µm in diameter. Nematodes were mounted in agar gel on Kapton tape. Two-dimensional elemental maps for Cu- and Pb-exposed nematodes were collected in fluorescence mode. Copper was homogeneously distributed throughout the body of the nematode, but Pb exhibited a high degree of localization in the nematode, exclusively in the anterior pharynx region. Detectable localized concentrations of Pb in C. elegans occurred at aqueous exposure concentrations of 2.4 µM. Micro X-ray diffraction of these Pb hotspots gave a diffraction pattern indicating a crystalline Pb solid that was consistent with the Pb phosphate, pyromorphite. Biogenic inorganic phosphate granule formation is relatively common in soil invertebrates; however, these phosphates are typically amorphous, and we believe that this is the first report of crystalline pyromorphite formed internally in an organism.
Introduction Effective risk assessment methodologies for metal-contaminated soils are essential for protecting human and ecosystem health. Most regulatory procedures set limits based on acceptable total metal concentrations in soils. However, it is well established that total metal concentrations are often poorly correlated with either toxicity or mobility of contaminants, and increasingly, the argument is made that risk assessments should incorporate measures of bioavailable contaminant concentration. Purely chemical measures for assessing trace element availability have been developed, * Corresponding author e-mail:
[email protected]; phone: 803 725 0854; fax: 803 725 3309. † Savannah River Ecology Laboratory, University of Georgia. ‡ Department of Environmental Health Science, University of Georgia. § University of Chicago. 5620
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but such measures do not consider uptake of a contaminant at the cell surface or the variety of mechanisms by which an organism can detoxify a trace element. An alternative approach is to explicitly consider bioavailability through use of a bioassay. In this case, the organism response to, or uptake of, a contaminant is used to assess bioavailability (1, 2). Standardized soil toxicity testing methods have been developed utilizing earthworms (Eisenia fetida) (3) and the free-living soil nematode Caenorhabditis elegans (4). C. elegans is a native soil invertebrate, it is relatively easy to culture, has a short life cycle, and can be used in aqueous or soil matrixes; as such it is well suited for soil toxicity testing. The molecular biology of C. elegans has been extensively studied; the complete cell lineage has been described and the genome has been sequenced (5, 6). Transgenic strains of C. elegans have been developed for use in environmental studies, including strains where a lacZ or gfp reporter genes have been fused to the heat shock protein, hsp16, gene or the metallothionein gene (7-9). A variety of other sublethal endpoints including behavior, growth, feeding, and reproduction have been developed with this nematode (10). The advantage is that sublethal responses are easily measured, and the assays are often much more sensitive than conventional lethality-based endpoints. Undoubtedly, the use of biological endpoints and the monitoring of protein expression in response to environmental stress are significant advances in methodology for predicting bioavailability and toxicity. However, a fuller understanding of these processes can only be gleaned from a molecular level approach. Organisms possess various cellular “defense” mechanisms for dealing with exposure to excess concentrations of trace elements (11). Metallothioneins, a class of low molecular weight, cysteine-rich proteins, are known to play an important role in binding excess trace metal cations in both vertebrates and invertebrates (12), including C. elegans (7, 13). Another class of cysteine-rich polypeptides, the phytochelatins, which previously have been identified in plants and some fungi, has recently been shown to play a role in metal detoxification in C. elegans (14). Invertebrates are also known to produce inorganic granules, usually amorphous Ca carbonates or phosphates, and it has been suggested that adsorption or coprecipitation of contaminants into these granules could be a detoxification mechanism (15, 16). Knowledge of the spatial distribution of a contaminant within an organism is important in determining the site of toxic action or in identifying processes whereby trace metals are selectively sequestered. The spatial distribution of hsp16 expression in response to a number of trace metal cations, As, and paraquat has been reported (9). For the metal cations studied (Pb, Hg, Zn, Cu, and Cd), it appeared that each elicited a spatially discrete hsp response. However, hsp16 expression, or, more precisely, detection of expression through staining, was only detectable in a relatively small fraction of the total population of worms ranging from a maximum of 16% of the exposed population for Pb to 43% for Hg. This study reports on the use of synchrotron X-ray spectroscopy to study uptake and spatial distribution in two dimensions of Pb and Cu in C. elegans. Synchrotron X-ray techniques are nondestructive, high resolution, and element specific and are increasingly being employed for mapping of trace element distributions and determining trace element speciation in bacterial, plant, and animal matrixes (17-21) and in microfocused elemental mapping in biomedical research (22, 23). Synchrotron X-ray fluorescence (SXRF) gives specific information about the elemental composition of a 10.1021/es050154k CCC: $30.25
2005 American Chemical Society Published on Web 06/18/2005
FIGURE 1. Representative SXRF maps for Cu (A-C) and Pb (D-F) exposed nematodes. Dark to light colors represent an increasing fluorescence intensity proportional to an increase in the elemental concentration. sample and in this respect it is similar to EDS measurements in electron microscopy. However, unlike the latter technique, SXRF analysis is performed at room temperature on samples in their natural state. This technique can also provide chemical speciation information through near-edge (XANES) or extended X-ray absorption fine structure (EXAFS) spectroscopy and crystalline phases can be identified by SXRD analyses (24).
Materials and Methods Nematode Exposures. Wild-type strain N2 C. elegans obtained from the Caenorhabditis Genetics Center (Minneapolis, MN) were used in this experiment. Three-day-old, age-synchronized cultures were derived from egg plates following the methods of Donkin and Williams (25). Two water-soluble, reagent-grade toxicants were tested: CuCl2‚ 2H2O (Aldrich, Milwaukee, WI) and Pb(NO3)2 (Fisher Scientific, Fairlawn, NJ). Nematodes were loaded into 1-mL wells
of a 12-well tissue culture plate. Each well contained K medium (0.032 M KCl, 0.051 M NaCl) (26) with or without the toxicant. Plates were placed in a 20 °C incubator for a 24-h exposure period. Following each experimental exposure, approximately 100 nematodes were preserved in formaldehyde and stored at 4° C prior to shipment to the National Synchrotron Light Source. Nematodes were mounted on Kapton tape in agar gel for exposure to the X-ray beam. SXRF Spectroscopy. X-ray fluorescence spectroscopy was conducted at the microprobe beamline X-26A, National Synchrotron Light Source, Brookhaven National Laboratory. The incident X-ray beam was tuned to 13 keV (for Pb LR and Cu KR) or 8.5 keV (for Cu KR) using a Si(111) channel-cut monochromotor. The incident beam was focused using Rhcoated Kirkpatrick-Baez focusing optics to an approximate size of 10 × 10 µm2. Energy-dispersive XRF data were collected using a Canberra SL30165 Si(Li) detector or a Canberra 9-element Ge Array detector. Two-dimensional elemental VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Optical (A) and XRF (B and C) images of a nematode exposed to both Cu (B) and Pb (C). maps of nematodes were performed using step sizes on the order of 10-30 µm in X and Y dimensions depending on the orientation of the nematode on the agar slide (the size of a nematode is approximately 100 µm in width and 1 mm in length). Dwell time per pixel was varied between 2 and 10 s, depending on an initial estimate of the elemental XRF signal and the area to be mapped. Micro XRD of elemental hotspots was conducted using a Brucker SMART 1500 charge-coupled device (CCD) system. This CCD system is optimized for collection of data out to higher 2θ angles and on very weakly diffracting samples. Since X26A is optimized for spatially collimated microbeams, it is possible to obtain high-resolution microdiffraction data on very small (10 µm) crystals. Data were processed using the Fit2D software package for integrating 2D Debye Scherrer rings to one-dimensional 2θ scans (27).
Results and Discussion Representative two-dimensional SXRF elemental maps for C. elegans individuals exposed to either Cu or Pb clearly 5622
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showed that the spatial distribution of Cu and Pb within the nematode differed (Figure 1). Lead was almost exclusively located in the pharynx; this region contains a high density of neurons and our observation of localization of Pb is consistent with the known neurotoxic effects of this element (28). Our results are in agreement with a study of Pb accumulation in a nematode (Panagrolaimus superbus), which used transmission electron microscopy (TEM)-EDS to show spatial localization of Pb in the oesophageal region (29). Jones and Candido (30) found that C. elegans respond to a variety of stressors, including heavy metals, by a cessation or reduction of pharangeal pumping. Hence, in this case, it is possible that the nematodes ceased pumping upon Pb exposure, essentially localizing exposure to the pharynx. Copper displayed no discernible localization but was relatively homogeneously distributed through the nematode. While the relatively low Cu concentrations at any one point in the nematode precluded further determination of Cu binding environment, the relatively homogeneous distribution of this element suggests that it impacts a number of cell
types. C. elegans produces phytochelatins and metallothioneins (14), and both of these thiol-rich peptides can bind Cu both in normal cell homeostasis and in response to excess concentrations. The spatial distribution of Cu and Pb shown in Figure 1 was consistent in mapping numerous nematodes exposed to Cu and Pb in different experiments over approximately a two-year period. Additionally, although nematodes were generally fed during the 24-h toxicity test, we also had treatments without feeding and these exhibited the same qualitative elemental distribution pattern for Cu and Pb. For soil invertebrates the importance of the external surface (cuticle) as opposed to ingestion as the primary pathway for uptake is not known. However, our results consistently show higher internal and lower cuticular concentrations for Pb which supports ingestion as the primary exposure pathway. It is not possible to discount diffusion across the cuticle as a route of exposure for Cu. While the elemental maps appear to show reduced fluorescence at the exterior surface of the nematodes, this may result from the reduced sample thickness at the surface of this tubular organism and the fact that at the surface some portion of the X-ray beam does not interact with the sample. To date, the principle method for imaging a response of nematode C. elegans to various environmental stressors has been the spatial detection of the heat shock protein, hsp16. Molecular fusions of β-galactosidase reporter LacZ E. coli genes have provided an elegant methodology for detection and spatial localization of hsp16 production within the nematode after exposure to the contaminant. One such study reports on a highly specific response after Pb(NO3)2 exposure in the muscle cells of the posterior bulb of the pharynx (9), which is consistent with the element specific response reported herein. However, this study also reports on a specific response to CuCl2 in muscles and neurons in the anterior end of the pharynx, which is not consistent with our element specific determinations for Cu that show fairly homogeneous distribution of Cu throughout the animal. Similarly, Dennis et al. (31) reported a localized pharynx staining upon exposure to Cu (and other divalent cations) alone, but when exposed to the metal ion in the presence of a surfactant a homogeneous staining response throughout the worm was reported. However, hsp16 expression in C. elegans is inherently nonspecific and occurs in response to a wide range of stressors including heat, metal cations, metalloid oxyanions, and organic compounds such as fungicides. While such a wide response has advantages in a real-world mixedcontaminant system it does not provide information on the actual bioavailable contaminant. More fundamentally, localized hsp16 expression upon exposure to an environmental stressor does not necessarily mean that this is also the site of toxic action of that stressor, i.e., it does not provide molecular or spatially resolved information regarding the actual contaminant. In most real systems organisms are exposed to multiple stressors. The response of C. elegans to multiple metal stressors in aqueous solution has been studied using both lethality and nonlethal hsp16-gfp expression (32-34). This latter study found that many binary and ternary metal cation mixtures acted synergistically such that response was greater than the sum of individual cation responses. In particular Cu appeared to have a strong synergistic effect with the other metal cations tested. Another stress response of C. elegans that has been reported is a decrease or cessation of pharyngeal pumping (feeding) in the presence of sublethal concentrations of toxicants (30). To investigate these observations using the element specific SXRF technique, nematodes were exposed to Cu or Pb at their respective LC50 while the third treatment was exposed simultaneously to both Cu and Pb at their respective LC50. Qualitatively, the spatially distribution
FIGURE 3. Energy-dispersive spectra for (A) Pb-exposed nematode and (B) Cu-exposed nematode. of Cu and Pb in the two metal exposure (Figure 2) was essentially identical to the spatial distribution observed in the single metal ion exposures (Figure 1). Quantitative statistical comparison between the treatments was conducted by comparing the per pixel fluorescence intensity on a selected area of three worms from each treatment by oneway analysis of variance. Statistical analysis revealed that Cu uptake was significantly reduced (p < 0.001) when nematodes were exposed to both Pb and Cu. This observation supports a reduction in pharangeal pumping in response to metal exposure. In general, 24-h lethality responses of C. elegans for various metal cations, as indicated by LC50 concentrations, are relatively high, e.g., 125 and 93 µM for Pb and Cu respectively (33). The highly localized nature of Pb accumulation in C. elegans suggested that SXRF could be an effective tool for identifying exposure to Pb at more environmentally relevant concentrations. To test this, nematodes were exposed to Pb at concentrations of 0.24, 2.4, 24, and 240 µM for 24 h. A minimum of 5 nematodes from each treatment were selected for SXRF mapping to qualitatively determine how consistently nematodes within a treatment had a detectable Pb uptake. No Pb hotspots were identified in the two-dimensional maps for the 0.24 µM exposure; however, localized Pb areas were detected in all nematodes examined in each of the three higher treatments. Comparison of full energy dispersive spectra of Cu- and Pb-exposed nematodes also illustrates the almost exclusive occurrence of the Pb cation at the Pb hotspot for the exposed nematode. For the Cu-exposed nematodes, while Cu was the primary cation it was only slightly elevated compared to other endogenous cations (Figure 3). The highly localized Pb accumulation revealed by SXRF mapping coupled with the almost exclusive presence of Pb at this hotspot, as indicated by the energy-dispersive spectra, suggested that this hotspot was a Pb solid phase within the nematode. A particularly intense Pb hotspot from the 240 µM treatment was examined using the µ-XRD capability of the VOL. 39, NO. 15, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Integrated 2θ vs intensity plot of the XRD pattern from Pb hotspot in comparison to pyromorphite. X26A microprobe beamline. Characteristic Debye-Scherrer diffraction rings were observed (Figure 4, inset) indicating that Pb was bound in a crystalline material. Comparison with diffraction data of crystalline minerals in the JCPDS database identified the Pb mineral as pyromorphite, Pb5(PO4)3Cl (Figure 4). A previous study has reported on purportedly noncrystalline nanoparticles with high concentrations of Pb, P, S, and Ca in thin sections of the oesophageal region of the free-living nematode P. superbus using TEMEDS (29). The production of membrane-bound inorganic granules occurs in cells of virtually every animal phylum (35). However, these granules have been consistently identified as amorphous Ca(Mg) phosphates or pyrophosphates that, if their function is trace metal detoxification, protect the organism through coprecipitation of contaminant cations. The extracellular production of nanoscale pyromorphite by the soil bacterium Burkholderia cepacia has been demonstrated in biofilms formed on Al2O3 (36). Our results now demonstrate the internal biogenic formation of pyromorphite within an organism. Pyromorphite minerals are isostructural with the common biogenic Ca phosphate mineral apatites, and natural Pb phosphates often occur as Pb-Ca solid solutions (37). However, pyromorphites are approximately 20 orders of magnitude more insoluble than apatites; hence it is probable that if Pb is localized in an area of high PO4 concentration or of granular Ca phosphate precipitation, then pyromorphite will precipitate. Other soil invertebrates, notably earthworms, also produce these amorphous phosphate granules; therefore we hypothesize that in Pbcontaminated soils these organisms may also produce pyromorphite granules. Lead phosphates are insoluble and geochemically stable over a wide range of pH, ionic strength, and temperature (37). Interestingly, despite its very low solubility, under certain situations pyromorphite may be 5624
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solublized by organic acid producing soil fungi, which utilize it as a phosphate source while precipitating Pb as the oxalate salt (19). Nonequilibrium conditions and soil humic acids have also been shown to increase Pb phosphate solubility over thermodynamic predictions (38). Nevertheless, phosphate addition to Pb-contaminated soils and subsequent pyromorphite precipitation has been shown to lower Pb bioavailability and is employed as an in situ stabilization and remediation technology (39-42). Our results suggest that nematodes, which are indigenous soil organisms, detoxify Pb through pyromorphite precipitation. Given the high density and turnover of roundworms in soil, biogenic pyromorphite formation may contribute to controlling Pb solubility and cycling in soils.
Acknowledgments This research was supported by the Environmental Remediation Sciences Division of the Office of Biological and Environmental Research, U.S. Department of Energy through the Financial Assistant Award No. DE-FC09-96SR18546 to the University of Georgia Research Foundation. Research was carried out in part at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, under Contract No. DE-AC02-98CH10886. Use of the Beamline X26A was supported by the Department of Energy, Basic Energy Science’s Geosciences Research Program under Grant No. DE-FG02-92ER14244. The nematode strain used in this study was provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.
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Received for review January 24, 2005. Revised manuscript received May 17, 2005. Accepted May 17, 2005. ES050154K
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