Abstract
Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd2+) and lead (Pb2+). Leadmium Green has been used in various paradigms, such as tracking Cd2+ sequestration in plant cells, heavy metal export in protozoa, and Pb2+ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd2+, Pb2+, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd2+ (KD ~600 nM) and also Pb2+ (KD ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca2+, Co2+, Mn2+ and Ni2+. Leadmium Green also responded to Zn2+ with a KD of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd2+ or Zn2+ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn2+ was much less than that produced by Cd2+. When tested in a model of oxidant-induced liberation of endogenous Zn2+, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd2+, particularly in models where Cd2+ accumulates rapidly, and when concomitant fluctuations of intracellular Zn2+ are minimal.
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Aizenman E, Stout AK, Hartnett KA et al (2000) Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release. J Neurochem 75:1878–1888
Atar D, Backx PH, Appel MM et al (1995) Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J Biol Chem 270:2473–2477
Berglund M, Elinder CG, Nordberg G, Vahter M (1998) Health effects of cadmium exposure—a review of the literature and a risk estimate. Scand J Work Environ Health 24(suppl 1):1–51
Carter KP, Young AM, Palmer AE (2014) Fluorescent sensors for measuring metal ions in living systems. Chem Rev 114:4564–4601. doi:10.1021/cr400546e
Chiu T-Y, Chen P-H, Chang C-L, Yang D-M (2013) Live-cell dynamic sensing of Cd(2+) with a FRET-based indicator. PLoS One 8:e65853. doi:10.1371/journal.pone.0065853
Dalton TP, He L, Wang B et al (2005) Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis. Proc Natl Acad Sci USA 102:3401–3406. doi:10.1073/pnas.0406085102
Dineley KE, Malaiyandi LM, Reynolds IJ (2002) A reevaluation of neuronal zinc measurements: artifacts associated with high intracellular dye concentration. Mol Pharmacol 62:618–627
Dineley KE, Devinney MJ, Zeak JA et al (2008) Glutamate mobilizes [Zn2+] through Ca2+ -dependent reactive oxygen species accumulation. J Neurochem 106:2184–2193. doi:10.1111/j.1471-4159.2008.05536.x
Duncan FE, Que EL, Zhang N et al (2016) The zinc spark is an inorganic signature of human egg activation. Sci Rep 6:24737. doi:10.1038/srep24737
Gee KR, Zhou ZL, Ton-That D et al (2002) Measuring zinc in living cells: a new generation of sensitive and selective fluorescent probes. Cell. doi:10.1016/S0143-4160(02)00053-2
Haugland RP (1996) Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene
Hayes RB (1997) The carcinogenicity of metals in humans. Cancer Causes Control 8:371–385
Hinkle PM, Kinsella PA, Osterhoudt KC (1987) Cadmium uptake and toxicity via voltage-sensitive calcium channels. J Biol Chem 262:16333–16337
Hinkle PM, Shanshala ED, Nelson EJ (1992) Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake. J Biol Chem 267:25553–25559
Hoch E, Lin W, Chai J et al (2012) Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2 + over Cd2 + selectivity. Proc Natl Acad Sci USA 109:7202–7207. doi:10.1073/pnas.1200362109
Johri N, Jacquillet G, Unwin R (2010) Heavy metal poisoning: the effects of cadmium on the kidney. Biometals. doi:10.1007/s10534-010-9328-y
Klassen RB, Crenshaw K, Kozyraki R et al (2004) Megalin mediates renal uptake of heavy metal metallothionein complexes. Am J Physiol Renal Physiol 287:F393–F403. doi:10.1152/ajprenal.00233.2003
Knoch ME, Hartnett KA, Hara H et al (2008) Microglia induce neurotoxicity via intraneuronal Zn(2+) release and a K(+) current surge. Glia 56:89–96. doi:10.1002/glia.20592
Lofts S (2009) Speciation of pyrithione in freshwaters. NERC/Centre for Ecology and Hydrology, Lancaster
Lu L, Tian S, Yang X et al (2008) Enhanced root-to-shoot translocation of cadmium in the hyperaccumulating ecotype of Sedum alfredii. J Exp Bot 59:3203–3213. doi:10.1093/jxb/ern174
Malaiyandi LM, Dineley KE, Reynolds IJ (2004) Divergent consequences arise from metallothionein overexpression in astrocytes: zinc buffering and oxidant-induced zinc release. Glia 45:346–353. doi:10.1002/glia.10332
Manzano JI, Garcia-Hernandez R, Castanys S, Gamarro F (2013) A new ABC half-transporter in Leishmania major is involved in resistance to antimony. Antimicrob Agents Chemother 57:3719–3730. doi:10.1128/AAC.00211-13
Maret W, Vallee BL (1998) Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci USA 95:3478–3482
Miura S, Takahashi K, Imagawa T et al (2013) Involvement of TRPA1 activation in acute pain induced by cadmium in mice. Mol Pain 9:7. doi:10.1186/1744-8069-9-7
Moulis JM (2010) Cellular mechanisms of cadmium toxicity related to the homeostasis of essential metals. Biometals. doi:10.1007/s10534-010-9336-y
Müller L (1986) Consequences of cadmium toxicity in rat hepatocytes: effects of cadmium on the glutathione-peroxidase system. Toxicol Lett 30:259–265
Nordberg GF (2009) Historical perspectives on cadmium toxicology. Toxicol Appl Pharmacol. doi:10.1016/j.taap.2009.03.015
Park JD, Cherrington NJ, Klaassen CD (2002) Intestinal absorption of cadmium is associated with divalent metal transporter 1 in rats. Toxicol Sci 68:288–294
Prozialeck WC, Edwards JR (2012) Mechanisms of cadmium-induced proximal tubule injury: new insights with implications for biomonitoring and therapeutic interventions. J Pharmacol Exp. doi:10.1124/jpet.110.166769
Sensi SL, Canzoniero LM, Yu SP et al (1997) Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17:9554–9564
Squibb KS, Cousins RJ, Silbon BL, Levin S (1976) Liver and intestinal metallothionein: function in acute cadmium toxicity. Exp Mol Pathol 25:163–171
St Croix CM, Wasserloos KJ, Dineley KE et al (2002) Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am J Physiol Lung Cell Mol Physiol 282:L185–L192. doi:10.1152/ajplung.00267.2001
Stayner L, Smith R, Thun M et al (1992) A dose-response analysis and quantitative assessment of lung cancer risk and occupational cadmium exposure. Ann Epidemiol 2:177–194. doi:10.1016/1047-2797(92)90052-R
Thévenod F (2010) Catch me if you can! Novel aspects of cadmium transport in mammalian cells. Biometals 23:857–875. doi:10.1007/s10534-010-9309-1
Tsien RY (1999) Monitoring Cell Calcium. In: Carafoli E, Klee C (eds) Calcium as a cellular regulator. Oxford University Press, New York, pp 28–54
Vinkenborg JL, van Duijnhoven SMJ, Merkx M (2011) Reengineering of a fluorescent zinc sensor protein yields the first genetically encoded cadmium probe. Chem Commun 47:11879–11881. doi:10.1039/c1cc14944j
Yang CF, Shen HM, Shen Y et al (1997) Cadmium-induced oxidative cellular damage in human fetal lung fibroblasts (MRC-5 cells). Environ Health Perspect 105:712–716
Zhao J, Bertoglio BA, Devinney MJ et al (2009) The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green. Anal Biochem 384:34–41. doi:10.1016/j.ab.2008.09.019
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This work was supported by funds from Midwestern University.
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Malaiyandi, L.M., Sharthiya, H. & Dineley, K.E. Fluorescence detection of intracellular cadmium with Leadmium Green. Biometals 29, 625–635 (2016). https://doi.org/10.1007/s10534-016-9939-z
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DOI: https://doi.org/10.1007/s10534-016-9939-z