Drosophotoxicology: Elucidating Kinetic and Dynamic Pathways of Methylmercury Toxicity in a Drosophila Model

Abstract

The risks of methylmercury (MeHg) toxicity are greatest during early life where it has long been appreciated that the developing nervous system is an especially sensitive target. Yet, understanding the discrete mechanisms of MeHg toxicity have been obscured by the wide variation in the nature and severity of developmental outcomes that are typically seen across individuals in MeHg exposed populations. Some insight has come from studies aimed at identifying a role for genetic background as a modifier of MeHg toxicity, which have predominantly focused on factors influencing MeHg toxicokinetics, notably, polymorphisms in genes related to glutathione (GSH) metabolism. For example, variants in genes encoding the catalytic and modifier subunits of glutamyl-cysteine ligase (GCLc and GCLm), the rate limiting enzyme for GSH synthesis, have been reported to associate with Hg body burden (Hg levels in blood or hair) in humans. However, GSH can facilitate both toxicokinetics and toxicodynamics of MeHg by forming MeHg-GSH conjugates, which are readily transported and excreted, and by acting indirectly as an anti-oxidant. In this study, we refine a model to distinguish kinetic and dynamic traits of MeHg toxicity using a paradigm of Drosophotoxicolgy. First, we identify that the pupal stage is selectively sensitive to MeHg toxicity. Using a protocol of larval feeding, measurements of Hg body burden, and assays of development to adulthood (pupal eclosion), we identify strain-dependent variation in MeHg elimination as a potential kinetic determinant of differential tolerance to MeHg. We also find that global upregulation of GSH levels, with GCLc trans-gene expression, can induce MeHg tolerance and reduce Hg body burden. However, we demonstrate that MeHg tolerance can also be achieved independently of reducing Hg body burden, in both wild-derived strains and with targeted expression of GCLc in developing neuronal and muscle tissue, pointing to a robust toxicodynamic mechanism. Our findings have important implications for understanding variation in MeHg toxic potential on an individual basis and for informing the process of relating a measurement of Hg body burden to the potential for adverse developmental outcome.

Developmental Toxicology of Metal Mixtures in Drosophila: Unique Properties of Potency and Interactions of Mercury Isoforms

“…In the U.S., mercury levels in fish are estimated to range from 0.3-3 ppm [38]. However, in contrast to humans, the larva feed continually on the MeHg food and reach a much-elevated steady state, such that 5 µM feeding gives ~20 ppm (100 µM) body burden [22]. In human cases where MeHg poisoning had occurred, neurological effects were observable at levels equivalent to ~10 ppm in blood (~50 µM), only a 2-fold difference from that given by our lowest dose that showed delays in pupariation [39,40].…”

“…Canton S embryos were collected within a 12 h laying period from a mating population of 150-300 flies, then aged to larvae at 25 • C. First instar Canton S larvae were transferred to vials of food (Jazz Mix, Fisher Scientific, Waltham, MA, USA, #AS153) containing desired concentrations of MeHg (methylmercury(II) chloride, Sigma-Aldrich, St. Louis, MO, USA, #442534, prepared in DMSO at 50 mM), HgCl 2 (mercury(II) chloride, Sigma-Aldrich #215465), As (sodium (meta)arsenite, Sigma-Aldrich #S7400), and Pb (lead(II) acetate trihydrate, Sigma-Aldrich #467863), with four replicate vials of n = 50 larvae in each vial. Dose ranges for each metal were established empirically, based on the minimal and maximal effect seen across all the endpoints measured from prior Drosophila experiments [20][21][22]44]. Once per day, vials of developing flies were scored for pupa formation, pharate adult formation, and eclosion of adult fly for three of the four vials until these values in all vials were stagnant for 3 consecutive days, representing the endpoint.…”

“…The fruit fly has proven particularly effective in studying neurodegenerative diseases [18]. Use of Drosophila in toxicology has grown recently, largely because of the ability to implement controlled doses, the ease of scoring multiple developmental landmarks within a short life cycle, and the ability to study a great number of individuals at once [19][20][21][22][23]. Furthermore, the genetic tractability of this model is ideally suited for mechanistic studies.…”

“…Furthermore, the genetic tractability of this model is ideally suited for mechanistic studies. Applications of Drosophila to metal toxicology studies have also emerged as a powerful investigative tool [20,22,24,25]. However, only in limited cases has the model been leveraged to investigate metal mixtures in a systemic manner [26].…”

Motor dysfunction in Drosophila melanogaster as a biomarker for developmental neurotoxicity

“…Models such as zebra fish, C. elegans and Drosophila melanogaster are increasingly recognized for their suitability to test a larger number of candidate compounds, in addition to acquire "mode of action" type of information regarding neurotoxicity, and neurological disorders and diseases (Peterson et al, 2008). The fruit fly Drosophila melanogaster has got particular attention in the modern regimen of neurotoxicological testing due to similarities of their neurological and developmental pathways with those of vertebrates (Rand, 2010), emphasizing its unique attributes for assaying neurodevelopment and behavior (Affleck and Walker, 2019;Rand et al, 2019). Recent investigations have propagated several powerful assay methods with Drosophila in developmental and behavioral toxicology (Rand, 2010).…”