Articles pubs.acs.org/acschemicalbiology
Methylmercury Targets Photoreceptor Outer Segments Malgorzata Korbas,†,‡ Barry Lai,§ Stefan Vogt,§ Sophie-Charlotte Gleber,§ Chithra Karunakaran,† Ingrid J. Pickering,∥,⊥ Patrick H. Krone,‡,⊥ and Graham N. George∥,⊥,* †
Canadian Light Source Inc., 44 Innovation Boulevard, Saskatoon, SK S7N 2V3, Canada Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada § X-ray Science Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, United States ∥ Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada ⊥ Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada ‡
ABSTRACT: Human populations experience widespread low level exposure to organometallic methylmercury compounds through consumption of fish and other seafood. At higher levels, methylmercury compounds specifically target nervous systems, and among the many effects of their exposure are visual disturbances, including blindness, which previously were thought to be due to methylmercury-induced damage to the visual cortex. Here, we employ high-resolution X-ray fluorescence imaging using beam sizes of 500 × 500 and 250 × 250 nm2 to investigate the localization of mercury at unprecedented resolution in sections of zebrafish larvae (Danio rerio), a model developing vertebrate. We demonstrate that methylmercury specifically targets the outer segments of photoreceptor cells in both the retina and pineal gland. Methylmercury distribution in both tissues was correlated with that of sulfur, which, together with methylmercury’s affinity for thiolate donors, suggests involvement of protein cysteine residues in methylmercury binding. In contrast, in the lens, the mercury distribution was different from that of sulfur, with methylmercury specifically accumulating in the secondary fiber cells immediately underlying the lens epithelial cells rather than in the lens epithelial cells themselves. Since methylmercury targets two main eye tissues (lens and photoreceptors) that are directly involved in visual perception, it now seems likely that the visual disruption associated with methylmercury exposure in higher animals including humans may arise from direct damage to photoreceptors, in addition to injury of the visual cortex.
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assumed that observed significant damage to the visual cortex10 is either the sole or primary cause of methylmercury induced visual problems. In previous work, we used a larval zebrafish model with synchrotron-based X-ray fluorescence imaging (XFI) to determine mercury localization in developing vertebrates.2,14,15 We observed accumulation of methylmercury species in the eye, in both lens and retina. Remarkably, methylmercury levels in the lens increased even after removal of fish from treatment solutions, indicating that mercury redistributed from other tissues acted as a source for uptake by the lens.15 These results clearly show that methylmercury targets the eye and that longterm accumulation in lens and retina may present a significant and, to date, neglected problem. Here, we use high resolution XFI to pinpoint the cellular localization of mercury within retina following acute methylmercury exposure. We identify the outer segments of photoreceptors as the main subcellular target, not only in the retina but also in the pineal gland of the exposed zebrafish larvae. These results suggest that direct methylmercury action on sensory cells may be partly
he compounds of mercury are more toxic than those of any other nonradioactive heavy element, yet human exposure is widespread. In the environment, mercury is found in a range of different chemical forms, each with distinct bioaccumulation and toxicological profiles.1,2 Organometallic mercury forms, in particular methylmercury, are highly neurotoxic and have been responsible for large-scale catastrophic poisonings of human populations in Japan and Iraq.3 Developing fetuses and young children are particularly susceptible to methylmercury effects;4 this became evident in the 1950s during a large-scale poisoning in Minamata Japan, when in utero exposure to maternally ingested methylmercury resulted in severe child abnormalities at birth, despite minimal maternal symptoms. Fortunately, such large-scale acute exposures are rare, in part due to increased risk awareness. Nevertheless, chronic low-level exposure to methylmercury is widespread due to its natural occurrence in marine fish5,6 and other seafood.7 While available studies provide conflicting conclusions on the extent of the risks,8,9 it is clear that mercury exposure troubles many communities, and the World Health Organization has classified mercury among the top-ten chemicals of major public health concern. Visual disturbances including blindness are prominent among the many symptoms of acute methylmercury poisoning,10−12 and it is widely © 2013 American Chemical Society
Received: June 28, 2013 Accepted: August 5, 2013 Published: August 5, 2013 2256
dx.doi.org/10.1021/cb4004805 | ACS Chem. Biol. 2013, 8, 2256−2263
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Figure 1. Typical 5 × 5 μm2 resolution X-ray fluorescence map of a larval zebrafish head section, showing (A) a histological image stained with methylene blue and (B) the Hg XFI of the adjacent unstained serial section following methylmercury treatment (5 dpf zebrafish larvae exposed to 1 μM CH3HgCl for 36 h); eye lens (el), retina (re), brain (br), and optic nerve (on).
without mercury exposure. We estimate this dose to be close to 20% of the LC50, sufficiently low that the fish did not die but still had observable levels of mercury in their tissues. No major differences in localization and concentration of naturally occurring elements (P, S, Ca, Zn) could be detected between sections from control and exposed fish. Consistent with previous XFI studies of eukaryotic cells,17 the phosphorus signal correlated with the localization of retina and lens cell nuclei (Figure 2) coincident with high concentrations of nucleic acids. All three retinal nuclear layers were clearly distinguishable in the maps of phosphorus as well as zinc and calcium (Figure 2), with the highest zinc and calcium levels detected in the retinal pigmented epithelium (rpe). The melanosomes (pigmented cells) of rpe contain the pigment melanin, which is known to bind essential ions as well as toxic metals.18 Previous studies have detected various metals such as aluminum, zinc, copper, calcium, manganese, molybdenum, iron, lead, mercury, and cadmium in the human retinal pigmented epithelium.19,20 In the present work, we also consistently observe low levels iron, copper, and selenium (at up to 0.06, 0.02, or 0.07 μg/cm2, respectively) in zebrafish larvae rpe, with much lower levels in other parts of the retina or in the lens. Sulfur levels, of interest because of mercury’s high affinity for thiolate donors, were highest in the eye lens at up to 18.6 μg/cm2 in the vicinity of the lens core (Figure 2), probably originating primarily from crystallins (lens proteins) cysteine and methionine residues with some contribution from glutathione.21 Sulfur content in the retina was at least 10-fold lower and was almost uniformly distributed except in the outer plexiform layer and the outer segments of the photoreceptors, where it was slightly elevated (Figure 2). As expected, mercury was only detected in the methylmercury-treated animals (Figure 2); as we previously reported,14 mercury and sulfur distribution differed, with mercury concentrated in a thin (∼10 μm) layer closer to the edge of the lens (0.6−0.8 μg/cm2) while sulfur levels increased from lens periphery to core. In contrast, retinal mercury levels correlated with sulfur levels. Low mercury levels (up to 0.06 μg/cm2) were detected across the whole retina with the highest levels in the outer plexiform layer and the photoreceptor outer segments (up to 0.2 μg/cm2). The tricolor map (Figure 2B), in which phosphorus, zinc, and mercury distributions are overlaid, demonstrates that the photoreceptors outer segments (os) specifically take up mercury. This clearly shows the mercuryrich layer of photoreceptor cells separating the phosphorus-rich photoreceptor inner segments layer (is) from the high-zinc retinal pigmented epithelium (rpe). Figure 2B also shows slightly lower mercury concentrations relative to other retinal
responsible for visual disturbances associated with methylmercury exposure.
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RESULTS AND DISCUSSION
Our previous studies on methylmercury in developing zebrafish larvae point to the lens and the retina as the main structures of the fish visual system specifically targeted by methylmercury. Due to the limited spatial resolution of the X-ray beams used in these previous studies, we were not able either to identify the types of cells accumulating the highest methylmercury levels or to examine subcellular localization. Here, by probing the affected tissues with a submicrometer sized beam, we achieved an unprecedented resolution allowing us not only to detect mercury unequivocally in certain types of cells but also to correlate its accumulation with distributions of other biologically relevant elements such as phosphorus, sulfur, calcium, and zinc. Figure 1 shows an XFI map (5 × 5 μm2 pixel size) of mercury distribution in a typical head section of a 5 days post fertilization (dpf) zebrafish larva (36-h exposure) showing distinct accumulation in the exterior of the eye lens and in the retina. Figure 2 shows a high resolution XFI map (500 × 500 nm2 pixel size) of the eye of a different section, equivalent to the area highlighted in Figure 1. Like the human retina, the zebrafish retina is composed of three layers of nerve cell bodies (outer and inner nuclear layers and the ganglion cell layer) separated by two layers of synapses (outer and inner plexiform layers). The retina cellular architecture is clearly discernible in hematoxylin and eosin (H&E) stained sections from 5 dpf zebrafish (Figure 2; “horizontal” and “vertical” are referenced to the retinal layers). The outer nuclear layer (onl) contains photoreceptor cell bodies whereas the inner nuclear layer (inl) contains cell bodies of the bipolar, horizontal, and amacrine cells. As shown in Figure 2, photoreceptor cells are composed of (a) an outer segment (os), comprising stacks of membranes containing the visual pigment molecules such as rhodopsins; (b) an inner segment (is), the polarized body of the photoreceptor with the nucleus residing basally; and (c) the synaptic terminal where neurotransmission to retinal interneurons of the retina occurs.16 The outer plexiform layer (opl) contains synaptic connections between the photoreceptors and bipolar and horizontal cells. The area of synaptic connections where the bipolar and amacrine cells are linked to the ganglion cells is the inner plexiform layer (ipl). Figure 2 compares phosphorus, sulfur, calcium, zinc, and mercury distributions in unstained 3-μm thick eye sections from 5 dpf zebrafish larvae following 48-h exposure in water to 500 nM CH3HgCl with those from control fish of the same age 2257
dx.doi.org/10.1021/cb4004805 | ACS Chem. Biol. 2013, 8, 2256−2263
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zinc and phosphorus, single photoreceptor cells became discernible, clearly showing mercury specifically targeting the outer segments of photoreceptors. In earlier work2,14 using a beam diameter of 5−10 μm, we suggested that methylmercury accumulated predominantly in lens epithelial cells. Using higher resolutions (500 nm), we now can more accurately determine the precise cellular localization of mercury in the developing lens (Figure 4). Surprisingly, following a 48-h treatment with CH3HgCl, the highest mercury concentrations (up to 1 μg/cm2) were detected in the secondary fiber cells immediately underlying the lens epithelial cells rather than in the lens epithelial cells (marked by a concentrated calcium signal), which had significantly lower mercury levels (up to 0.1 μg/cm2). Mature fibers located more centrally to the lens did not accumulate these high mercury levels. Since the lens high mercury ring was offset by approximately 10 μm from the lens epithelial monolayer, this was not resolved in our earlier 5−10 μm resolution XFI.2,14 Low levels of mercury were also detected in the cornea (up to 0.06 μg/cm2) (Figure 4). We previously observed mercury accumulation in the pineal gland of methylmercury-treated zebrafish larvae.2 The pineal gland is a small endocrine gland in the vertebrate brain that produces melatonin. In lower vertebrates it contains cells that show a distinctive resemblance to eye photoreceptor cells. Figure 5A shows high (500 nm) resolution XFI of the pineal gland in 3-μm thick sections of 5 dpf larvae treated for 48 h with CH3HgCl and untreated control larva. Treated and control samples showed no major differences in localization and concentration of P, Zn, and S, with no mercury detected in the control. As expected, and like the eye, phosphorus was localized in the cell nuclei, but in contrast to retina, the maps of pineal zinc were less similar to those of phosphorus, showing more uniform zinc distribution, especially in the skin epithelium. Interestingly, sulfur and mercury distributions were very similar and almost uniform except for some small oval-shaped areas (3 μm diameter or smaller) with sharp spikes (>factor 3) in both mercury and sulfur concentrations. The colocalization of mercury with sulfur is evident in tricolor maps overlaying these elements with zinc (Figure 5A). Comparing micrographs of the pineal glands and their respective mercury and sulfur distributions shows that Hg/S-rich spots correspond to dark structures in the optical images (Figure 5B), the position and morphology of which is consistent with the outer segments of the pineal photoreceptors. According to previous studies on the larval zebrafish pineal gland, the outer segments of the pineal photoreceptors appear dark in light and electron microscopy and are centrally positioned in the gland,22 often displaying loose whorls of outer segment membrane encircling an empty center. This morphology can be seen in the tricolor plot of mercury, sulfur, and zinc distribution in the section of methylmercury-treated larvae (Figure 5A, second row). The Hg/S molar ratios of these mercury and sulfur colocalized deposits varied between 0.010 and 0.015. Although selenium often associates with mercury, its concentration in both treated and untreated sections was less than 0.015 μg/cm 2 , substantially lower than that of mercury. Since the sulfur-rich deposit also was observed in the untreated pineal gland section and due to the relatively low Hg/S molar ratio in these black structures, it appears these are not composed predominantly of mercuric sulfide or selenide. We made additional observations of these Hg/S-dense structures, using scanning transmission Xray microscopy (STXM) 23 on the same section imaged by XFI.
Figure 2. Quantitative elemental distributions in retina and lens from CH3HgCl-exposed and unexposed zebrafish larvae. (A) Comparison of P, S, Ca, Zn, and Hg distributions in transverse sections of fish eyes from 5 dpf larvae exposed for 48 h to 500 nM CH3HgCl (right) with those from control fish (left), measured using XFI. Each measured (unst) section is paired with the adjacent section’s histological image (H&E). Individual element quantities are plotted on a common scale for exposed and control sections; eye lens (le), ganglion cell layer (gcl), inner plexiform layer (ipl), inner nuclear layer (inl), outer plexiform layer (opl), retinal pigmented epithelium (rpe), outer nuclear layer (onl), and photoreceptors outer (os) and inner segments (is). (B) Overlay of P (red) with Zn (green) and Hg (blue) in the eye section from CH3HgCl-exposed larva.
layers in the part of the outer nuclear layer occupied by the horizontal and bipolar cells (more densely spaced cells), as compared with the other retinal layers. Figure 3A overlays a differential (Nomarski) interference contrast micrograph and mercury maps of a retina section of another CH3HgCl-treated larva, clearly showing preferential mercury accumulation in the photoreceptors (os) as well as in the outer plexiform layer. Figure 3B shows XFI of the retinal photoreceptor layer at still higher resolution (150 × 150 nm2 pixel size), exhibiting a similar overall pattern to that of Figure 2 with high zinc levels in the retinal pigmented epithelium and mercury and sulfur colocalization in the photoreceptors (os). When the high-resolution mercury signal was superimposed on 2258
dx.doi.org/10.1021/cb4004805 | ACS Chem. Biol. 2013, 8, 2256−2263
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Figure 3. Preferential localization of mercury in the photoreceptors outer segments of CH3HgCl-exposed zebrafish larvae. (A) Mercury map of the eye section (right) superimposed on the differential interference contrast micrograph of the same section and compared with the H&E histological image of the adjacent section (left), showing mercury localization in the outer segments of photoreceptors and in the outer plexiform layer; eye lens (le), ganglion cell layer (gcl), inner plexiform layer (ipl), inner nuclear layer (inl), outer plexiform layer (opl), and retinal pigmented epithelium (rpe). (B) Quantitative, high resolution (150 nm pixel size) elemental distributions of P, S, Zn, and Hg in the photoreceptors cells of CH3HgCl-exposed zebrafish larva. The overlay of P (red), Zn (green) and Hg (blue) shows the inner (is) and outer segments (os) of photoreceptors as resolved by high resolution XFI.
In summary, using high-resolution XFI, we have identified the outer segments of both the retinal and the pineal photoreceptors as preferential uptake sites for methylmercury. For retina, the second highest mercury uptake site was the outer plexiform layer with lower mercury levels in the other retinal layers. Methylmercury distribution in the retina and pineal gland was correlated with that of sulfur, which, together with methylmercury’s affinity for thiolate donors suggests involvement of protein cysteine residues in methylmercury binding. Very few studies of methylmercury and retina or pineal gland have been reported.25−27 Goto et al.25 examined methylmercury chloride effects upon electro-retinograms in rats and observed dose- and time-dependent changes, suggesting that photoreceptors are sensitive to methylmercury.25 Weber et al.26 showed long-term functional changes in the retinal bipolar cells of the adult zebrafish following developmental exposure to methylmercury chloride.26 Mela et al.27 reported autometallography of methylmercury treated adult zebrafish retina, observing apparent high mercury density in the outer and inner nuclear layers, although low sensitivity and low specificity are problems with autometallography, which relies on surface adhesion of silver ions to mercury rich deposits.28 Indeed, in a comparison between autometallography and XFI of mussel, XFI revealed tissue areas containing cadmium but not stained with silver.29 Compared with many other techniques, XFI not only has much higher sensitivity but is a direct probe that does not rely on chemical reactions. The mechanisms of methylmercury transport into the retina and then specifically into the photoreceptor outer segments are, at present, unknown. Nutrients delivered to retinal cells from the blood must first cross a monolayer of cells with complex tight junctions called the blood−retina barrier (BRB). The BRB comprises retinal capillary endothelial cells (the inner BRB) and retinal pigmented epithelium (RPE) cells (outer BRB).30 In humans, the outer BRB regulates the supply of essential nutrients from choroidal vessels lining RPE to photoreceptors, whereas the inner BRB controls the access of solute molecules from intraretinal capillaries to the other retinal cells such as ganglion cells, bipolar cells, horizontal cells, and amacrine cells.30 Zebrafish larvae have functional inner and outer BRB by 3 dpf13,31 and both the retinal endothelial cells, and the RPE
Figure 4. High resolution elemental distributions in CH3HgClexposed zebrafish lens. (Top panel) Maps of Ca and Hg distributions in a transverse lens section (area shown by red box) from 5 dpf larva exposed for 48 h to 500 nM CH3HgCl measured using XFI. The measured (unst) section is paired with the adjacent section’s histological image (H&E); lens epithelium (ep), cornea (cr). The cross marks the position of the lens epithelial cells. (Bottom panel) Comparison of Ca (green), S (red), and Hg (blue) distributions in the same lens.
Since our specimen thickness (3 μm) and embedment in carbonyl-containing methacrylate precluded C K-edge measurements, we employed N K-edge STXM using the characteristic protein nitrogen 1s→π*(amide) absorption at 401 eV to map protein levels.24 Figure 6 shows a transmission difference map calculated by subtracting transmitted X-ray intensity at (401 eV) and below (395 eV) this transition. The difference image shows contrast, especially in the areas darkly stained in the optical micrograph. This demonstrates that mercury concentrates in pineal gland spots rich in both protein and sulfur, the morphology of which is consistent with the outer segments of the pineal photoreceptors. 2259
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Figure 5. Quantitative elemental distributions in pineal glands from CH3HgCl-exposed and unexposed zebrafish larvae. (A) Comparison of P, S, Zn, and Hg distributions in transverse sections of pineal glands from 5 dpf larvae exposed for 48 h to 500 nM CH3HgCl (two top rows) with those from control fish (bottom row), measured using XFI. Each measured (unst) section is paired with the adjacent section’s histological image (H&E). Black boxes mark the pineal glands in the transverse head sections. Individual element quantities are plotted on a common scale for exposed and control sections. The overlay of S (red), Zn (green) and Hg (blue) confirms the colocalization of S and Hg. (B) The mercury (right) and sulfur (middle) maps of the pineal gland from an exposed larva superimposed on the differential interference contrast micrograph of the same section (left) to facilitate the localization of mercury and sulfur in the outer segments of the pineal photoreceptors (black spots) of the CH3HgCl-exposed zebrafish larva.
cells express a variety of transporters.30 One such transporter is the Na+-independent large neutral amino acid transporter (system L), which is present in both inner and outer BRB in humans and other mammals32,33 and which has been implicated in transport of methylmercury L-cysteinate.34 However, preferential uptake of methylmercury by the outer segments of photoreceptors may be due to other factors such as their high concentrations of cysteine-rich opsin proteins,35 the thiolate groups of which might directly bind methylmercury. For example, human and zebrafish rhodopsin (rod opsin) contain 10 and 14 cysteine residues, respectively;36 moreover, 6 of 10 thiolates in bovine rhodopsin are able to bind to methylmercury, disturbing the rhodopsin’s normal photolytic sequence.37 In mouse retina, it has been estimated that one rod cell has ∼108 rhodopsin molecules, which are densely assembled in the rod disc membranes (∼30 000−55 000 rhodopsin molecules/μm2),38,39 giving a considerable titer of thiolates for prospective methylmercury binding. Since the pineal gland lies outside the blood-brain barrier, pineal photoreceptors will have unrestricted access to methylmercury directly from the circulating blood. Previous studies demonstrated rhodopsin expression in the zebrafish
pineal gland.40 Thus, it is plausible that the spots with dense protein and colocalized mercury and sulfur detected in the pineal glands of methylmercury-exposed larvae comprise rhodopsin molecules with methylmercury bound to their −SH groups. While the pineal gland does not respond to light in adult mammals, it may do so at early life stages, as much of the photoreceptive organization is present in the pineal of neonatal mammals.41 It is thus possible that specific methylmercury uptake by the pineal photoreceptors may also take place in developing mammals. In conclusion, using high-resolution XFI, we have revealed that methylmercury specifically accumulates in secondary lens fibers as well as photoreceptor outer segments of both retina and pineal gland. Since methylmercury targets two main eye tissues (lens and photoreceptors) that are directly involved in visual perception, it now seems likely that the visual system consequences of methylmercury exposure may have their roots not only in the visual cortex of the brain but, surprisingly, in the eye tissues themselves. 2260
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of 3 μm thickness were cut on a microtome using glass knives. Of two adjacent sections, one was mounted on a glass slide and stained with H&E stain, while the other, intended for synchrotron XFI, was fixed on a Si3N4 membrane (area 2 × 2 mm2, 500 nm thickness, Silson Ltd., Northampton, England) without any further processing. X-ray Fluorescence Imaging (XFI). X-ray fluorescence images were collected at the Advanced Photon Source (Argonne, IL, U.S.A.) using beamlines 2-ID-D and 2-ID-E (Figures 2−6), or 20-ID-B (Figure 1) with the storage ring operating in continuous top-up mode at 102 mA and 7.0 GeV. The incident X-ray energy was set to 13.45 keV and the intensities of X-ray fluorescence and scatter were monitored using a silicon-drift Vortex detector (SII NanoTechnology U.S.A. Inc.). Incident and transmitted X-ray intensities were measured with nitrogen-filled ion chambers. Experiments used a Rh-coated silicon mirror for harmonic rejection and a Si(111) double crystal monochromator (2-ID-D, 20-ID-B) or a single bounce Si(220) crystal monochromator for beam delivery onto focusing optics. The 20-ID-B 5 μm diameter microfocused beam was generated by Kirkpatrick−Baez (K-B) Rh-coated focusing mirrors; samples were mounted at 45° to the incident X-ray beam and were spatially rastered in the microbeam with a 5 μm step size and 0.6 s exposure per step (20-ID-B). The submicrometer focused beams of 250 (2-ID-D) or 500 nm (2-ID-E) diameter were generated by Fresnel zone plates (X-Radia, Pleasanton, CA). Samples were mounted at 90° to the incident X-ray beam to minimize scattering and were raster-scanned with a 0.5 μm step size (0.15 μm for Figure 3B) and 0.5 s exposure per step. XFI Data Analysis. Analysis of 20-ID-B data was performed as described previously.2 For high resolution mapping (2-ID-D and 2-IDE), full X-ray fluorescence spectra were acquired at each scan position and fitted, after estimation of background,43 with modified Gaussians using MAPS software 44 to accurately determine detected counts. Elemental content was calculated by comparison of normalized count rates to standard reference materials (NBS 1832 and 1833, NIST, Gaithersburg, MD, and RF8-200-S2453, AXO Dresden GmbH, Germany). Scanning Transmission X-ray Microscopy. Nitrogen K-edge spectromicroscopy data were collected using the Scanning Transmission X-ray Microscope (STXM) in the soft X-ray spectromicroscopy (SM, 10ID-1) beamline at the Canadian Light Source (CLS) using a zone plate with a 40 nm outer zone diameter; beamline details and capabilities are given elsewhere.45 The N K-edge images were recorded using the transmission detector. After mounting the samples into the microscope, the STXM chamber was evacuated and then backfilled with about 250 Torr of He. Beamline slit sizes were selected to achieve a spectral resolution of about 125 meV at the N K-edge region and to keep the flux on the sample within the linear response range of the detector (maximum 20 MHz).
Figure 6. X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM) maps of elemental and protein distributions in the pineal gland of the CH3HgCl-treated zebrafish larva. (A) Quantitative XFI maps of Zn, S, and Hg, and their overlay, in the pineal gland from 5 dpf zebrafish larva treated with 500 nM CH3HgCl for 48 h, compared with the differential interference contrast micrograph of the same section (unst) and with the H&E histological image of the adjacent section (H&E). The black box marks the pineal gland on the head section. (B) STXM difference map of the area marked by the red box (panel A, unst), calculated by subtracting transmitted X-ray intensity at (401 eV) and below (395 eV) the 1s → π*(amide) transition, to facilitate detection of dense protein deposits in the pineal gland and the colocalization of those deposits with the black spots in the unstained section.
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METHODS
Animal Care and Embryo Collection. Adult zebrafish were kept at 28 °C in carbon-filtered tap water with a photoperiod of 14 h. Embryos were collected and staged following standard procedures.42 After collection, embryos and larvae were reared in 25-ml Petri dishes with culture water changed daily. This work was approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines on the care and use of fish in research, teaching, and testing. Mercury Treatment Solution. Since methylmercury compounds and solutions are extremely toxic, appropriate precautions are required to prevent inhalation or skin contact. Methylmercury chloride aqueous solution (1000 ppm, Alfa Aesar) was diluted in triple-distilled water to give a 100 μM stock solution, which was further diluted by fish culture water (carbon-filtered tap water) for zebrafish treatment; all solutions were freshly made prior to each exposure. All exposures were at 28 °C for 48 h starting at 3 dpf. Larvae were placed in 25-ml Petri dishes containing 500 nM methylmercury chloride (1:200 dilution) or control dishes with no added mercury. After exposure, larvae were rinsed several times in fresh carbon-filtered water to remove any remaining mercury. Preparation of Sections. Larvae were fixed in 4% paraformaldehyde for 24 h at 4 °C immediately following exposure. Fixed larvae were dehydrated in a graded series (0, 25, 50, 75, and 100%) of ethanol in PBST buffer (30 mM PBS, 0.01% Tween 20) for 5 min each and stored in 100% ethanol at −20 °C until needed. For sectioning, the fixed and dehydrated larvae were rehydrated into PBST by 5 min washes in the reversed ethanol gradient. Selected larvae were properly oriented and embedded in 1% low melting point agarose gel. The blocks of gel containing the larvae were cut out and dehydrated in 100% ethanol by gentle shaking for 5 to 8 h at 4 °C. Following dehydration, the blocks were infiltrated overnight on a rotating stirrer at 4 °C with JB-4 catalyzed solution A (10 mL solution A/0.125 g catalyst; Polysciences Inc., Warrington, PA, U.S.A.). Infiltration using fresh solution continued on the following day for 5 to 6 h. Infiltrated samples were placed in embedding molds filled with a mixture of JB-4 solution B and fresh infiltration solution (1 mL solution B:25 mL infiltration solution) and left overnight at 4 °C to polymerize. Sections
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AUTHOR INFORMATION
Corresponding Author
*Phone 306-966-5722. E-mail:
[email protected]. Author Contributions
M.K., I.J.P., P.H.K., and G.N.G. designed research; M.K. B.L., C.K., S.-C.G. carried out synchrotron-based measurements; B.L. and S.V. calibrated X-ray fluorescence data; M.K., I.J.P., and G.N.G. wrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank K. Yuen for guidance in tissue processing and sectioning, N. Sylvain for assistance with zebrafish embryo collection and culture, and T. MacDonald and A. James for help with data collection. We also thank R. Gordon for assistance at the 20-ID-B beamline at the Advanced Photon Source. This work was supported by the Canadian Institutes of Health Research (G.N.G., I.J.P.), the Saskatchewan Health Research 2261
dx.doi.org/10.1021/cb4004805 | ACS Chem. Biol. 2013, 8, 2256−2263
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Foundation (G.N.G., I.J.P.), the University of Saskatchewan, Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (I.J.P., G.N.G., P.H.K.), and the Canada Foundation for Innovation (G.N.G., I.J.P.). G.N.G. and I.J.P. are Canada Research Chairs. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Canadian Light Source is supported by NSERC, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.
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