Laura L. Maurer

A systematic review of evidence for silver nanoparticle-induced mitochondrial toxicity

Silver nanoparticles (AgNPs) are extensively used for their antibacterial properties in a diverse set of applications, ranging from the treatment of municipal wastewater to infection control in hospitals. However, the properties of AgNPs that render them conducive to bactericidal use in commerce may influence their potential toxicity to non-bacterial organisms. Based on the physiological and phylogenetic similarities between bacteria and mitochondria within eukaryotic cells, mitochondria are a likely intracellular target of AgNP toxicity. Mitochondria-specific outcomes of AgNP exposures have been identified in multiple cell types, including (but not limited to) loss of membrane potential, inhibition of enzymes involved in oxidative phosphorylation, and changes in calcium sequestration. However, the biological significance of mitochondrial toxicity due to AgNP exposure is currently incompletely understood. This review examines the existing evidence of mitochondrial toxicity induced by AgNP exposure, with discussions of the role of the physicochemical properties of the nanoparticles themselves in mitochondrial toxicity. The impacts of potentially differential cell- and tissue-specific significance of AgNP-induced mitochondrial dysfunction are also discussed.

Intracellular trafficking pathways in silver nanoparticle uptake and toxicity in Caenorhabditis elegans

Abstract We used the nematode Caenorhabditis elegans to study the roles of endocytosis and lysosomal function in uptake and subsequent toxicity of silver nanoparticles (AgNP) in vivo. To focus on AgNP uptake and effects rather than silver ion (AgNO3) effects, we used a minimally dissolvable AgNP, citrate-coated AgNPs (CIT-AgNPs). We found that the clathrin-mediated endocytosis inhibitor chlorpromazine reduced the toxicity of CIT-AgNPs but not AgNO3. We also tested the sensitivity of three endocytosis-deficient mutants (rme-1, rme-6 and rme-8) and two lysosomal function deficient mutants (cup-5 and glo-1) as compared to wild-type (N2 strain). One of the endocytosis-deficient mutants (rme-6) took up less silver and was resistant to the acute toxicity of CIT-AgNPs compared to N2s. None of those mutants showed altered sensitivity to AgNO3. Lysosome and lysosome-related organelle mutants were more sensitive to the growth-inhibiting effects of both CIT-AgNPs and AgNO3. Our study provides mechanistic evidence suggesting that early endosome formation is necessary for AgNP-induced toxicity in vivo, as rme-6 mutants were less sensitive to the toxic effects of AgNPs than C. elegans with mutations involved in later steps in the endocytic process.

From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion.

Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction

The nematode Caenorhabditis elegans is extensively utilized in toxicity studies. C. elegans offers a high degree of homology with higher organisms, and its ease of use and relatively inexpensive maintenance have made it an attractive complement to mammalian and ecotoxicological models. C. elegans provides multiple benefits, including the opportunity to perform relatively high‐throughput assays on whole organisms, a wide range of genetic tools permitting investigation of mechanisms and genetic sensitivity, and transparent bodies that facilitate toxicokinetic studies. This unit describes protocols for three nanotoxicity assays in C. elegans: lethality, growth, and reproduction. This unit focuses on how to use these well‐established assays with nanoparticles, which are being produced in ever‐increasing volume and exhibit physicochemical properties that require alteration of standard toxicity assays. These assays permit a broad phenotypic assessment of nanotoxicity in C. elegans, and, when used in combination with genetic tools and other assays, also permit mechanistic insight.

Materials and toxicological approaches to study metal and metal-oxide nanoparticles in the model organism Caenorhabditis elegans

Understanding the in vivo fate and transport of nanoparticles (NPs) is challenging, but critical. We review recent studies of metal and metal oxide NPs using the model organism Caenorhabditis elegans, summarizing major findings to date. In a joint transdisciplinary effort, we highlight underutilized opportunities offered by powerful techniques lying at the intersection of mechanistic toxicology and materials science,. To this end, we firstly summarize the influence of exposure conditions (media, duration, C. elegans lifestage) and NP physicochemical properties (size, coating, composition) on the response of C. elegans to NP treatment. Next, we focus on the techniques employed to study NP entrance route, uptake, biodistribution and fate, emphasizing the potential of extending the toolkit available with novel and powerful techniques. Next, we review findings on several NP-induced biological responses, namely transport routes and altered molecular pathways, and illustrate the molecular biology and genetic strategies applied, critically reviewing their strengths and weaknesses. Finally, we advocate the incorporation of a set of minimal materials and toxicological science experiments that will permit meta-analysis and synthesis of multiple studies in the future. We believe this review will facilitate coordinated integration of both well-established and underutilized approaches in mechanistic toxicology and materials science by the nanomaterials research community.

1,3-Dinitrobenzene neurotoxicity – Passage effect in immortalized astrocytes

Age-related disturbances in astrocytic mitochondrial function are linked to loss of neuroprotection and decrements in neurological function. The immortalized rat neocortical astrocyte-derived cell line, DI-TNC1, provides a convenient model for the examination of cellular aging processes that are difficult to study in primary cell isolates from aged brain. Successive passages in culture may serve as a surrogate of aging in which time-dependent adaptation to culture conditions may result in altered responses to xenobiotic challenge. To investigate the hypothesis that astrocytic mitochondrial homeostatic function is decreased with time in culture, low passage DI-TNC1 astrocytes (LP; #2-8) and high passage DI-TNC1 astrocytes (HP; #17-28) were exposed to the mitochondrial neurotoxicant 1,3-dinitrobenzene (DNB). Cells were exposed in either monoculture or in co-culture with primary cortical neurons. Astrocyte mitochondrial membrane potential, morphology, ATP production and proliferation were monitored in monoculture, and the ability of DI-TNC1 cells to buffer K(+)-induced neuronal depolarization was examined in co-cultures. In HP DI-TNC1 cells, DNB exposure decreased proliferation, reduced mitochondrial membrane potential and significantly decreased mitochondrial form factor. Low passage DI-TNC1 cells effectively attenuated K(+)-induced neuronal depolarization in the presence of DNB whereas HP counterparts were unable to buffer K(+) in DNB challenge. Following DNB challenge, LP DI-TNC1 cells exhibited greater viability in co-culture than HP. The data provide compelling evidence that there is an abrupt phenotypic change in DI-TNC1 cells between passage #9-16 that significantly diminishes the ability of DI-TNC1 cells to compensate for neurotoxic challenge and provide neuroprotective spatial buffering. Whether or not these functional changes have an in vivo analog in aging brain remains to be determined.

The mechanisms of neurotoxicity and the selective vulnerability of nervous system sites

The spatial heterogeneity of the structure, function, and cellular composition of the nervous system confers extraordinary complexity and a multiplicity of mechanisms of chemical neurotoxicity. Because of its relatively high metabolic demands and functional dependence on postmitotic neurons, the nervous system is vulnerable to a variety of xenobiotics that affect essential homeostatic mechanisms that support function. Despite protection from the neuroglia and blood-brain barrier, the central nervous system is prone to attack from lipophilic toxicants and those that hijack endogenous transport, receptor, metabolic, and other biochemical systems. The inherent predilection of chemicals for highly conserved biochemical systems confers selective vulnerability of the nervous system to neurotoxicants. This chapter discusses selective vulnerability of the nervous system in the context of neuron-specific decrements (axonopathy, myelinopathy, disruption of neurotransmission), and the degree to which neuronal damage is facilitated or ameliorated by surrounding nonneural cells in both the central and peripheral nervous systems.