* Linus Pauling Institute and the Marine/Freshwater Biomedical Sciences Center, 571 Weniger Hall, Oregon State University, Corvallis, Oregon 97331; Environmental and Molecular Toxicology, the Environmental Health Sciences Center, 1007 Ag and Life Science Bldg, Oregon State University, Corvallis, Oregon 97331;
Linus Pauling Institute, Environmental and Molecular Toxicology, the Environmental Health Sciences Center and the Marine/Freshwater Biomedical Sciences Center, 435 Weniger Hall, Oregon State University, Corvallis, Oregon 97331; and
Environmental and Molecular Toxicology, the Environmental Health Sciences Center and the Marine/Freshwater Biomedical Sciences Center, 1007 Ag and Life Science Bldg, Oregon State University, Corvallis, Oregon 97331
Received April 29, 2004; accepted June 14, 2004
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ABSTRACT |
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Key Words: sodium metam; methyl isothiocyanate; zebrafish; developmental toxicity; notochord.
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INTRODUCTION |
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One of the most remarkable facts regarding NaM is that the mechanism(s) of its widespread biocidal activities is unknown. NaM degrades into multiple breakdown products following application, including methylisothiocyanate (MITC), carbon disulfide (CS2), and methylamine (Fig. 1) (Tomlin, 1997). In soils, the decomposition occurs rapidly with a half-life of as little as 30 min, but this has been shown to vary widely dependent on temperature, concentration, pH, and water content of the soil (Frick et al., 1998
; Gerstl et al., 1977
; Joris et al., 1970
; Saeed et al., 1996
; Turner and Corden, 1963
). In aqueous solutions exposed to light, NaM degrades into MITC with a half-life of less than 10 min (Draper and Wakeham, 1993
). Since MITC is thought to be the active biocide, it is important to consider the rate of degradation of NaM in a given system (Tomlin, 1997; Pruett et al., 2001
). However, it has been challenging to develop analytical methods of detection for NaM and MITC at ppb concentrations, which has made it difficult to ascertain the biological or ecological consequences of NaM (Yu et al., 2003
).
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Zebrafish (Danio rerio) share many cellular, anatomical, and physiological characteristics with other vertebrates. For developmental toxicology studies, zebrafish are especially useful in that they rapidly develop externally, their transparency allows observation during development, and hundreds of animals are easily obtained from the large clutch sizes. We have utilized zebrafish in order to characterize the toxicity and effects of NaM on the earliest stages of development. Our data suggest that both NaM and MITC are teratogenic to zebrafish embryos and cause notochord and muscle malformations when exposed during gastrulation and early somitogenesis. We have described the effects on the notochord and muscle using histological and molecular techniques. Because zebrafish are vertebrates, it will be critical to determine if other organisms, including mammalian vertebrates, are sensitive to NaM exposure prior to organogenesis. By utilizing the molecular and genetic tools available in the zebrafish, we will be able to investigate the mechanism of toxicity for NaM and possibly other dithiocarbamates, as well as whether NaM poses a significant health risk.
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MATERIALS AND METHODS |
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Stock solutions and exposure protocols. Sodium metam (NaM; CAS # 137-42-8) and methyl isothiocyanate (MITC; CAS # 556-61-6) were both purchased from Chem Service, Inc. (West Chester, PA). A sodium metam stock solution of 0.29 M in water at pH 9 was stored at 4°C until further dilution. Stock solutions of MITC were prepared at 40 mM in fish water and stored in aliquots at 20°C. Embryos were waterborne exposed in their chorions to varying concentrations of NaM or MITC in 20 ml glass vials sealed with Teflon®-lined lids (VWR International, West Chester, PA) to prevent loss by volatilization. Twenty embryos per vial were used as one replicate. At the end of the exposure period, the embryos were washed two times in excess fish water and allowed to grow until sampled.
In situ hybridization. Embryos were fixed overnight in 4% paraformaldehyde at specific hours or days post-fertilization (hpf or dpf). In situ hybridization was performed as described with minor modifications (Westerfield, 2000). Briefly, embryos were stored in 100% methanol at 20° until ready for use. The embryos were rehydrated in PBS + 0.1% Tween-20 (PBST) and treated with proteinase K at 2 µg/ml in PBST for varying lengths of time depending on the stage. The embryos were prehybridized in 50% formamide, 5X SSC, and 0.1% Tween (cheap hyb) for 1 h, and hybridized overnight at 70°C with digoxigenin-labeled antisense probe in cheap hyb + 500 µg/ml yeast RNA and 50 µg/ml heparin at pH 6.0. The embryos were first washed at 70°C in 2X SSC, 0.2X SSC, and 0.1X SSC, and then at 25°C in PBST. Digoxigenin was detected with an anti-DIG-AP Fab fragments antibody (Roche, Indianapolis, IN) in a blocking solution containing 1% DMSO, 2% sheep serum, and 2 mg/ml bovine serum albumin in PBST. Finally, the embryos were developed with 20 µl NBT/BCIP per ml (Roche) in color buffer containing 100 mM Tris-Cl, pH 9.5, 50 mM MgCl2, 100 mM NaCl, and 0.1% Tween-20. The collagen 2a1 and myoD antisense RNA probes have been described (Weinberg et al., 1996
; Yan et al., 1995
).
Histology. Embryos were fixed in 2% paraformaldehyde, 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 overnight. The embryos were embedded in 1% low temperature agarose, processed in epoxy resin on a Lynx EL automatic tissue processor, and sectioned a+ 1 µm. The sections were stained with paragon (basic fuschin-tolulidine blue in sodium borate) stain for 30 s (Bourne and St John, 1978).
Statistics. Data is illustrated as the mean with standard error of the mean (SEM). Sigmoidal regression analysis and LC50 estimation was completed using SigmaPlot 2001 for Windows (SPSS, Inc., Chicago, IL). For lowest observed adverse effect levels (LOAELs), ANOVA statistical analysis was performed using a p-value of 0.05 for significance. This was tabulated with SigmaStat Version 2.03 for Windows software (SPSS, Inc., Chicago, IL).
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RESULTS |
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DISCUSSION |
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Embryos that survive the initial exposure to NaM or MITC continue to develop despite notochord malformations. At 5 dpf, the embryos from both exposure groups appear indistinguishable, and are similarly unable to feed, abnormally respond to touch and exhibit decreased motor activity (data not shown). These results indicate that NaM and MITC are potent teratogens during early zebrafish development. This study is especially timely given that NaM is one of the proposed replacements for the agricultural biocide, MeBr, after the 2005 phase-out (EPA 2002a,b
, 2003
). Since no studies have yet been published examining the effects of NaM or MITC on similar stages of mammalian development, it is imperative not only that these studies be undertaken but also that the mechanism of action of these compounds be further elucidated.
Despite the fact that there are few studies regarding NaM toxicity, a number of different hypotheses have been proposed to explain its mechanism of action. We hypothesize that NaM or MITC may be targeting a specific reactive site, protein, or biological pathway. Because the effective concentration is very low, this suggests that the cellular targets are limited in number. The dose response curves for NaM and MITC are both very steep, which also indicates that there are molecular sites of action that become saturated. MITC has the capacity to form protein adducts, which may contribute to its toxicity (Valentine et al., 1995). MITC and NaM are both metabolized via glutathione conjugation (Lam et al., 1993
). It has been proposed that the MITC-glutathione conjugate may serve as a source of MITC elsewhere in the body at a later time point (Slatter et al., 1991
). Zebrafish exhibit functional glutathione-S-transferase activity throughout all of development (Wiegand et al., 2000
). Alternatively, S-methylation of NaM or MITC to S-methyl metam could potentially inhibit aldehyde dehydrogenase, thereby causing toxicity (Staub et al., 1995
). MITC has been shown to be cytotoxic and cause DNA strand breaks in cultured human hepatoma cells (Kassie et al., 2001
). These investigators suggest that the DNA damage may be due to the production of reactive oxygen species, which could damage other macromolecules in addition to DNA. Because dithiocarbamates are known to chelate metals, it is possible that they may act via sequestering bioactive metals away from proteins. However, since MITC is not a dithiocarbamate and is not known as a metal chelator, it is unlikely that its mechanism of toxicity is mediated by chelation (Tomlin, 1997
). Therefore, it is clear from the diversity of targets and hypotheses that the mechanism of action of NaM or MITC remains unknown.
The notochord is an axial structure common to the Chordata phylum. In lower chordates and in larval stages of lower vertebrates it plays an important role as a structural element required for locomotion and coordinated movement. More importantly, the notochord is required for proper differentiation of adjoining tissues such as the neurectoderm, muscle, and vertebral elements in all vertebrates. This is accomplished in part by release of signaling molecules such as sonic hedgehog (shh) from the notochord. In zebrafish, Shh induces the formation of slow muscle fibers in the medial somite, which then migrate laterally and complete their differentiation (Blagden et al., 1997). A Shh gradient is also thought to regulate the dorsal-ventral identity of neurons in the neural tube, such that neurons closest to the notochord form motoneurons, whilst neurons further away become interneurons or sensory neurons (for recent review see Jacob and Briscoe, 2003
). Therefore, the notochord is the primary axial structure upon which many other tissues depend for their proper formation and differentiation. Toxicants that disrupt normal morphogenesis and differentiation of the notochord may therefore result in permanent skeletal deformities, muscle abnormalities, and neurological dysfunction.
A search of the literature has revealed a few examples of severe notochord malformations following early life stage exposure to various toxicants, including dithiocarbamates. Zebrafish exposed to cadmium exhibit notochord and skeletal deformities (Hen Chow and Cheng, 2003). A study in rainbow trout embryos revealed that a number of dithiocarbamates are teratogenic, with the notochord being particularly sensitive; however, the teratogenic effects of NaM were not specifically examined (Van Leeuwen et al., 1986
). In this study, the notochord increased considerably in both length and diameter and therefore became twisted and distorted. The authors also observed ectopic osteogenesis. Among the animals that were raised to adulthood, compression and fusion of vertebrae and various twisted skeletal elements were observed. Xenopus laevis exposed to nabam (a dithiocarbamate) developed malformed notochords which contained larger and more numerous cells at concentrations greater or equal to 0.40 µg/l (Birch and Prahlad, 1986b
). Interestingly, embryos exposed to MITC and ethylene thiourea concurrently developed notochord malformations similar to nabam, while neither of these compounds alone resulted in malformations at any of the concentrations tested. Interactions between these and additional degradation products may be responsible for these results (Birch and Prahlad, 1986b
). North African catfish (Clarias gariepinus) exposed to 2.5 and 5 mg/l of the organophosphate malathion also develop remarkably similar notochord malformations (Lien et al., 1997
). Lien et al. suggest that the notochord may become malformed by overactive muscle spasms. Teraoka et al. have recently proposed a similar explanation for notochord waviness following a 24 h exposure to the dithiocarbamate thiuram (Teraoka et al., 2004
). In this study, the notochord malformations could be prevented by inhibition of spontaneous muscle contractions by co-exposure to the anesthetic MS-222.
The data from the present study suggests that the notochord is sensitive to NaM at a period in its differentiation prior to spontaneous muscle contractions. The end of the developmental window during which exposure to NaM or MITC causes notochord malformations (414 hpf) is 3 h prior to the onset of spontaneous movement (17 hpf). It is conceivable that the notochord could be compromised during early development and the consequence of this earlier exposure becomes evident once muscle activity initiates. Alternatively, degradation products deposited in the tissue early could exert their effects later in development. Since exposure later in development does not result in notochord defects, the active toxicant must either be present earlier or create a degradation product that exerts its effects later. However, we believe that the notochord is affected by NaM and MITC because MITC is the major degradation product, is relatively stable, and both cause similar effects during the same window of development. In contrast to the malathion study noted above (Lien et al., 1997), we have observed that zebrafish exhibit decreased touch responsiveness and movement following exposure to NaM or MITC. This indicates that either NaM and MITC exert similar effects by a different mechanism, or that notochord malformations result from alterations of other developmental processes besides muscle activity. Perhaps NaM and MITC affect notochord vacuolization and/or proliferation, which would distort the overall rigidity and axial length of the notochord, indirectly resulting in abnormal muscle structure. This concept is supported by studies examining ultrastructural changes in the notochord following dithiocarbamate exposure in Xenopus (Birch and Prahlad, 1986a
; Prahlad et al., 1974
). Embryos exposed to nabam or a combination of ethylenethiourea and MITC both exhibit disruption of collagen fibers in the notochord sheath and surrounding tissue. Nabam exposed embryos also showed an absence of plasmalemmal vesicles normally found adjacent to the intercellular space of notochord cells (Prahlad et al., 1974
). Studies examining the effects of another dithiocarbamate, thiram, on development have shown that it is teratogenic to mice when administered between days 5 and 17. These embryos exhibited an increase in resorption as well as a syndrome of skeletal malformations including cleft palate, wavy ribs, curved long bones and micrognathia (Fishbein, 1976
; Matthiaschk, 1973
; Roll, 1971
). These results suggest a conserved response and warrant more detailed examination of the effects of dithiocarbamates in general and NaM in particular on early mammalian development.
We believe that the zebrafish is a valuable system in which to elucidate the mechanism of action of these compounds specifically because of its molecular accessibility. For example, a zebrafish mutant, leviathan, has been identified in which a very similar notochord malformation has been noted (Stemple et al., 1996). Given the large number of notochord mutants that have been found, it is interesting that only one has a phenotype similar to NaM exposed embryos (Stemple et al., 1996
). The determination of the mutation responsible for this phenotype may allow the molecular dissection of a regulatory cascade whose dysfunction leads to specific notochord malformations. Advances in micro array technology will also be useful in the molecular characterization of NaM or MITC effects during specific periods of development. This study has provided an initial morphological and molecular characterization of the effects of NaM and MITC which will allow more mechanistic questions regarding the effects of these compounds on tissue morphogenesis and gene expression to be addressed.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be sent at Marine/Freshwater Biomedical Sciences Center, 435 Weniger Hall, Oregon State University, Corvallis, OR 97331. Fax: (541) 737-7966. E-mail: robert.tanguay{at}oregonstate.edu.
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